Host Parasite Interactions

239. Employing Polymorphic Gene Sequences as Tool for Investigating the Epidemiology of Trichophyton tonsurans Infection. Susan Abdel-Rahman, Andrea Gaedigk. Division of Pediatric Clinical Pharmacology, Children's Mercy Hospital. Kansas City, MO 64108.

Despite the fact that dermatophytoses represent the first human disease attributed to a microorganism, the dermatophyte genomes remain largely uncharacterized and little information is available exploring fungal carriage and infection on a molecular level. Using three gene loci, we have developed a genotyping algorithm which affords the ability to discriminate strains of T. tonsurans. All sequence variations were stable upon serial passage over 18 months suggesting that these loci provide a sufficiently sensitive means for longitudinal genotyping. With this approach, we have undertaken a large scale longitudinal epidemiologic study designed to 1) describe the genetic relatedness of strains that cause active infection vs. carriage and 2) determine whether the carrier state that has been previously described represents transient acquisition of different genetic strains or persistent colonization with a unique strain. Cultures are collected monthly from over 150 children attending an urban daycare center with over 12 months of data accumulated to date.

On average, 43% of individuals are positive every month for fungal growth consistent with T. tonsurans. A sufficient quantity of high quality DNA is available to confirm species and discriminate T. tonsurans strain type in 33% of the population with over 20 distinct strain types observed. Of interest, only 9% of the children sampled display signs or symptoms consistent with infection, thus illustrating the large number of fungal carriers in the pediatric population. More than 90% of the children who are culture positive on serial sampling display persistent carriage with the same strain(s). Although not wholly specific, unique sequence variations appear to segregate with strain types causing carriage versus infection.

240. Is a homolog of a host-selective toxin from Pyrenophora tritici-repentis at work in the Pyrenophora bromi-bromegrass interaction? Rachael M. Andrie and Dr. Lynda M. Ciuffetti. Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR.

Pyrenophora bromi is the causal agent of brown leaf spot of bromegrass (Bromus inermis), a widely cultivated hay and pasture grass. The presence of conspicuous chlorotic halos surrounding brown leaf spots suggests the involvement of a phytotoxin in disease. P. tritici-repentis, the species of Pyrenophora most closely related to P. bromi, requires the production of multiple host-selective toxins (HSTs) to cause the disease tan spot of wheat, including the proteinaceous HSTs Ptr ToxA and Ptr ToxB. Because of the relatedness of P. bromi and P. tritici-repentis, it is conceivable that P. bromi contains homologous sequences to ToxA and/or ToxB, the products of which may be involved in its interaction with bromegrass. Southern analysis revealed the presence of ToxB-like sequences in P. bromi. Polymerase chain reaction (PCR) was used to clone multiple PbToxBs from a number of P. bromi isolates. Comparisons of the putative open reading frames of the PbToxB sequences of P. bromi and ToxB of P. tritici-repentis clearly indicate a high level of relatedness, though at the inferred amino acid level the PbToxB proteins are more similar to each other than with the ToxB protein. At least one copy of PbToxB for each of three P. bromi isolates is transcribed in culture and in planta as shown by reverse transcriptase (RT)-PCR. To assess the role of PbToxBs in the interaction between P. bromi and bromegrass, the corresponding PbToxB loci were heterologously expressed in Pichia pastoris and the resultant PbToxB proteins infiltrated into bromegrass and wheat. Infiltration of PbToxBs into bromegrass did not result in obvious disease symptoms; however, infiltration into wheat resulted in chlorosis.

241. A regulator of G-protein signaling in Ustilago maydis promotes sporulation in planta and suppresses hyphal growth on limited nutrients. Lori G. Baker1, Scott Gold2, and Sarah F. Covert3. 1Department of Genetics, 2Department of Plant Pathology, 3Warnell School of Forest Resources. The University of Georgia, Athens, GA, U.S.A.

Ustilago maydis only produces teliospores when growing within in its host plant, Zea mays. The signaling pathway(s) that U. maydis uses to switch from vegetative growth to reproductive growth in planta are not well characterized, but one G-alpha subunit, Gpa3, has been shown to be a negative regulator of sporulation. Based on data from other fungi, we hypothesized that U. maydis uses a regulator of G-protein signaling, Rgs1, to deactivate Gpa3 and thus promote sporulation in planta. Deletion of rgs1 reduced U. maydis in planta sporulation dramatically and over-expression of rgs1 caused filamentous growth in liquid medium. The latter phenotype partially mimics gpa3 deletion, thus both lines of evidence suggest that Rgs1 negatively regulates Gpa3 as predicted. However, crosses between rgs1 deletion strains and gpa3 constitutively active (gpa3Q206L) mutants were less virulent than gpa3Q206L x gpa3Q206L crosses, raising the possibility that Rgs1 regulates more than one G-alpha involved in pathogenicity. We found that the individual deletions of gpa1 and gpa4 caused precocious sporulation in planta, indicating that both Gpa1 and Gpa4 are likely to be negative regulators of sporulation in U. maydis. Furthermore, deletion of rgs1 induced filamentous growth on minimal medium, while deletion of gpa1 or gpa4 suppressed hyphal growth on low nutrients. Preliminary evidence from double deletion mutants, suggests that functional Gpa1 or Gpa4 is essential for the filamentous phenotype of the rgs1 deletion mutant. Therefore, our emerging model is that at least three pathways suppress sporulation in U. maydis and that Rgs1 down-regulates this suppression on multiple fronts.

242. Wheat leaf rust, Puccinia triticina, ESTs and functional analysis in a surrogate system; a rust MAPK functions in Ustilago maydis. Guus Bakkeren, Guanggan Hu*, Rob Linning, Andrena Kamp. Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Highway 97, Summerland, BC, V0H 1Z0, Canada; *Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

Genomics approaches to advance the study of rust fungi are beginning to bear fruit. We are developing a 40,000-read EST database, currently at 15,000 and 4,200 unigenes, representing cDNA libraries from several developmental stages of P. triticina including host infection. We discovered a MAPK with homology to kinases known to be involved in pathogenicity in other fungi. It is similar to the U. maydis MAPK, UBC3/KPP2, but has a longer N-terminal extension of 43 aa with identities to U. maydis KPP6, a homolog of UBC3/KPP2 with a 170 aa extension. UBC3/KPP2 is involved in mating (and subsequent pathogenic development) whereas KPP6 functions during invasive growth in corn tissue. PtMAPK, expressed from a Ustilago-specific promoter, was able to complement a ubc3/kpp2 mutant and restore mating. It also substantially increased virulence on corn, measured as tumor formation, of a kpp6 mutant. Moreover, this construct restored to near-full pathogenicity a ubc3/kpp2 kpp6 non-pathogenic double mutant. Complementation with the complete PtMAPK gene indicated that the rust promoter might be recognized in U. maydis. Phylogenetically, these basidiomycete plant pathogens are relatively close. This is corroborated by our Pt-EST analyses in which most sequences matching fungal genes best, are from U. maydis. This system opens up avenues along which rust research can be advanced, currently frustrated by the lack of transformation protocols.

243. Characterization of yps a putative Aspergillus nidulans adhesin. S. Venkatramen, A.M. Dranginis and D.C. Bartelt, Dept. of Biological Sciences, St. John's University, Queens NY 11439

In a screen of an A. nidulans lamda cDNA library for genes encoding calcium-binding proteins, we identified a homolog of Histoplasma capsulatum yps -3. H. capsulatum is a dimorphic fungus in which only the yeast phase is pathogenic. YPS-3p is expressed on the cell surface in a yeast phase specific manner [Weaver, C.H., K.C. Sheehan and E.J. Keath, (1996) Infect. Immun. 64:3048-3054]. Antibodies to YPS-3p have been found in the serum of patients with Histoplasmosis but not in normal sera. The A. nidulans cDNA encodes a 118 residue protein with a predicted N terminal signal sequence, and an EGF homology calcium-binding domain. A cDNA containing the ORF and a transcription termination sequence from S. cerevisiae was cloned into a yeast shuttle vector pRS426 containing a PGK promoter, and URA3 selectable marker. The resulting plasmid, pYPSA, was used to transform S. cerevisiae Sigma1278b flo11-. FLO11p, a cell surface flocculin, is required for adhesion of yeast to the extracellular matrix proteins, fibronectin, lamin, and collagen. The S. cerevisiae Sigma1278b flo11- is unable to bind to fibronectin coated polystyrene in a microtiter plate assay. Of the seven yeast transformants isolated, the same four consistently displayed binding to fibronectin coated wells of a microtiter plate. These data support the characterization of YPSAp as an A. nidulans adhesin. Supported by NIAID R15AI062801-01.

244. Mitochondrial virulence determinants in the maize pathogen Ustilago maydis. Kathrin Stelter, Miriam Bortfeld, Christine Vogler, Kathrin Auffarth, Regine Kahmann and Christoph W. Basse. Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Straße, 35043 Marburg, Germany

The basidiomycete fungus Ustilago maydis is a facultative biotrophic pathogen causing smut disease in maize plants. The U. maydis mrb1 gene encodes a mitochondrial protein of the p32 family. Members of this family are known from different eukaryotic organisms and have diverse regulatory functions. In U. maydis mrb1 is necessary for pathogenic development of dikaryotic hyphae originating from a1 and a2 strains, while solopathogenic, haploid a1 mrb1 mutant strains are not affected in pathogenicity. Deletion analysis showed that the a2 mating type locus genes lga2 and rga2 account for the loss of pathogenicity in the absence of mrb1 with lga2 being the major contributor. We show that the lga2 and rga2 products are localized in mitochondria and provide evidence for a complex involving Mrb1, Lga2 and Rga2. Conditional overexpression of lga2 in haploid U. maydis cells severely affected growth, triggered mitochondrial fragmentation as well as loss of mitochondrial DNA, and compromised respiratory activity. We provide evidence that Lga2 interferes with mitochondrial fusion and that Mrb1 is required to control this activity. lga2 represents a direct target of the bE/bW complex and thus its expression is linked to pathogenic development. To get further insight into the consequences of lga2 expression a microarray analysis was performed. This revealed marked transcriptional alterations related to metabolism, transport and the induction of stress responses. We suggest that U. maydis has developed a system that allows the regulation of mitochondrial morphology under control of the mating type loci a and b and possibly serves to cope with special stress conditions during biotrophic growth.

245. Whole genome mutagenesis in Magnaporthe grisea, insertional mutant analysis and recovery of DNA regions flanking the insert. Melania Betts1, Sara L. Tucker1, Natalia Galadima1, Lei Li4, Yang Meng2, Gayatri Patel2, Nicole Donofrio3, Jin-Rong Xu4, Thomas Mitchell3, Mark Farman2, Ralph Dean3, Marc J. Orbach1. 1University of Arizona, Tucson, AZ 85721. 2University of Kentucky, Lexington, KY 40546. 3North Carolina State University, Raleigh, NC 27695, 4Purdue University, West Lafayette, IN 47907.

M. grisea is an ascomycete and the causal agent of rice blast disease. Our goal is to identify the specific genes involved in all stages of the infection cycle from attachment and colonization through lesion development and sporulation. An insertional mutagenesis approach to saturate the Magnaporthe genome was chosen as the strategy to reach this goal. Over 50,000 DNA insertion lines of M. grisea strain 70-15 have been generated mostly through Agrobacterium tumefaciens-mediated transformation. The library has been screened for defects in pathogenicity, morphology, metabolism, conidiation and growth rate. Data on the characterization of strains with interesting phenotypes is currently being generated and will be presented. A second stage of the project involves the recovery of sequences flanking the sites of insertion to look at the randomness of insertion. We are also recovering sequences from mutants that have pathogenicity defects in order to idenfity novel genes involved in the infection process.

246. Resistance and pathogenicity interactions between Arabidopsis and Hyaloperonospora parasitica. Jim Beynon, Rebecca Allen, Laura Baxter, Peter Bittner-Eddy, Mary Coates, Sharon Hall, Julia Meitz and Anne Rehmany. Warwick HRI, University of Warwick, Wellesbourne, Warwick, CV35 9EF, UK

The oomycetes represent a distinct group of organisms that cause diseases in plants, fish and humans. H. parasitica represents an excellent choice in which to study pathogenicity in these organisms as it infects the model plant Arabidopsis. We have used subtractive and map based cloning methods to clone two pathogen genes (ATR1 and ATR13) that trigger a host resistance response when the plant contains specific resistance genes. Both ATR genes reveal very high levels of allelic diversity and suggest an "arms race" between host and pathogen. Highly variable alleles of ATR13 are recognised by the same resistance gene allele. In contrast, different alleles of ATR1 trigger different combinations of resistance genes. We have used naturally occurring allele variants to define regions within these pathogen gene products that are responsible for specificity in their recognition by the resistance genes.

247. Investigation of fungal morphological differentiation in apple scab infection. Bowen JK1,2, Rees-George J2, Hill GN1,2, Hahn M3, Kemen E4, Kucheryava N3, Templeton MD2, Plummer KM1,2. 1University of Auckland, PB 92019, Auckland, NZ. 2HortResearch, Mt Albert Research Centre, PB 92169, Auckland, NZ. 3University of Kaiserslautern, Postbox 3049, 67653 Kaiserslautern, Germany. 4Konstanz University, Germany.

The hemi-biotrophic fungus Venturia inaequalis causes apple scab disease. The fungus grows between the cuticle and epidermal cells during its parasitic phase. It undergoes a dramatic morphological change during infection since a stroma, which resembles laterally dividing cells rather than hyphal filaments, is formed. A similar structure is formed within cellophane in vitro. Fungal growth reverts to tubular hyphal growth habit on emergence from the cellophane. We are using the growth of V. inaequalis on cellophane as a model for stroma formation during infection of apple leaves. Fungal genes up-regulated on cellophane and during infection, compared with growth on nutrient media, are being sought as these may be involved in cellular differentiation and may also be potentially important for infection. One such gene, CIN1 has such an expression profile, supporting the hypothesis that cellophane-grown V. inaequalis mimics infection processes. CIN1 is predicted to be a relatively large (~460 amino acid), highly cysteine rich protein, with repetitive amino acid motifs and a predicted signal peptide, suggesting that it is located in the cell wall or secreted externally. CIN1 is being expressed in Pichia to determine the structure/function of the protein and to verify peptide sequence. Suppression subtractive hybridisation is being employed to construct a cDNA library enriched for cellophane-induced genes. We plan to use gene silencing in V. inaequalis to facilitate the functional characterisation of genes of interest.

248. Gain of fertility mutants lost their pathogenicity in Magnaporthe grisea. Myoung-Hwan Chi, Sook-Young Park, Soonok Kim and Yong-Hwan Lee. School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea

Magnaporthe grisea, the causal agent of rice blast, is a heterothallic filamentous ascomycete. The fungus is considered as a model organism for studying fungal pathogen-plant interactions not only due to the great economic importance involved, but also due to the molecular genetic tractabilities. Two gain of female fertility (GFF) mutants were obtained from T-DNA mutant library of a Korean field strain M. grisea KJ-201, a female sterile MAT1-2. Genetic crosses of two GFFs with M. grisea strain 70-6, a female fertile MAT1-1, formed two lines of perithecia containing viable ascospores. Backcrosses of F1 progenies with strain 70-6 confirmed gain of female fertility. Comparison of x2 distribution at the expected 1:1 ratio indicated single gene inheritance of a marker gene, hygromycin phosphotransferase (HPH) in random ascospore analysis. T-DNA insertion sites in the chromosome were identified by TAIL-PCR and sequencing. Inactivation of corresponding genes was confirmed by RT-PCR analysis. Both GFF mutants lost their pathogenicity on susceptible rice cultivar, Nagdongbyeo. Functional complementation of these mutants is in progress. This research might uncover the relationship between female fertility and pathogenicity of M. grisea.

249. Fungal polyketide synthase is involved in cercosporin biosynthesis and fungal virulence of Cercospora nicotianae. Mathias Choquer, Katherine A. Dekkers, Hui-Qin Chen, Lihua Cao, and Kuang-Ren Chung. Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, U.S.A.

Cercosporin is a photosensitizing perylenequinone toxin produced by the plant pathogenic Cercospora species. A polyketide synthase gene (CTB1) was cloned and functionally characterized to involve in cercosporin biosynthesis in C. nicotianae. The CTB1 gene product contains a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and has high levels of similarity to many fungal type I polyketide synthases. Targeted disruption of CTB1 resulted in mutants completely devoid of both CTB1 transcript and cercosporin biosynthesis. The ctb1-null mutants caused fewer necrotic lesions on inoculated tobacco leaves compared with wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have conclusively demonstrated that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in Cercospora spp. Chromosome walking and sequence analysis of the surrounding DNA fragments of the CTB1 gene, suggesting that the clustering of cercosporin biosynthetic genes, similar to many fungal secondary metabolites, also occurs in Cercospora spp.

250. Molecular cloning and characterization of a putative phophatidyl inositol-specific phospholipase C (PLC) gene from Cryphonectria parasitica. Hea-Jong Chung, Ae-Young Mo, Seung-Moon Park, Moon-Sik Yang and Dae-Hyuk Kim. Division of Biological Sciences, Basic Science Research Institute, Chonbuk National University, Dukjindong 664-14, Chonju,Chonbuk 561-756, Korea

The hypovirus is known to downregulate the fungal laccase1 (lac1), the modulation of which is tightly governed by the inositol triphosphate (IP3) and calcium second messenger system in a virus-free strain. We cloned the gene cplc1 encoding a phosphatidyl inositol-specific phospholipase C (PLC), in order to better characterize the fungal gene regulation by hypovirus. Sequence analysis of the cplc1 gene indicated that it contained both the X and Y domains, which are the two conserved regions found in all known PLCs, with a 133 amino acid extension between the 2nd beta-strand and the alpha-helix in the X domain. In addition, the gene organization appeared to be highly similar to that of a delta type PLC. Disruption of the cplc1 gene resulted in slow growth and downregulation of lac1 expression. However, temperature sensitivity, osmosensitivity, virulence, and other hypovirulence-associated characteristics did not differ from the wild-type strain. Functional complementation of the cplc1-null mutant with the PLC1 gene from S. cerevisiae restored lac1 expression, which suggests that the cloned gene encodes PLC activity. The present study indicates that the cplc1 gene is required for proper mycelial growth and that it regulates the lac1 expression, which is also modulated by the hypovirus.

251. Molecular cloning and characterization of a gene, lac3, encoding an inducible laccase from Cryphonectria parasitica. Hea-Jong Chung, Eun-Sil Choi, Min-Jae Kim, Seung-Moon Park, Tae-Ho Kwon and Dae-Hyuk Kim. Division of Biological Sciences, Basic Science Research Institute, Chonbuk National University, Dukjindong 664-14, Jeonju, Chonbuk 561-756, Korea

Although the laccase activity has shown a correlation to the hypovirulence and has been considered as one of major virulence factors, molecular characterization of its biological function has been hampered due to the functional redundancy. At least three laccases have been suggested in Cryphonectria parasitica: one intracellular and two extracellular forms. We cloned a novel laccase gene lac3. The deduced amino acid sequence appeared to contain a putative leader peptide of 18 amino acids long and characteristic four copper binding regions. Northern blot analysis of the lac3 gene revealed that the lac3 was specifically induced by tannic acid, which is abundant in the bark of chestnut tree where the primary infection occurred. Moreover, the lac3 gene was specifically down-regulated by the presence of hypovirus, CHV1.

252. Nondegradative tolerance to the phytoalexin, pisatin, in the fungal pathogen Nectria haematococca MPVI. Jeffrey J. Coleman and Hans D. VanEtten. University of Arizona

In plants, a major resistance mechanism is the production of antimicrobial compounds (phytoalexins) in response to the attack of pathogenic microorganisms. To avoid this host response, pathogens have evolved tolerance mechanisms that make them resistant to their host's phytoalexin. The phytopathogenic fungus, Nectria haematococca MPVI, has at least two known mechanisms to tolerate the phytoalexin pisatin, produced by its host, garden pea. The most extensively characterized mechanism of tolerance to pisatin in N. haematococca involves a one-step demethylation catalyzed by an enzyme called pisatin demethylase (pda). However, this pathogen also has a "nondegradative tolerance" (NDT) mechanism that is inducible and specific for pisatin. Preliminary results suggest that this mechanism may be the result of an efflux pump, or more specifically, an ABC transporter. ABC transporters that might be responsible for NDT have been identified using RT-PCR and degenerate primers biased to other fungal transporters on mRNA from pisatin treated mycelia of N. haematococca. Three putative ABC transporters have high amino acid similarity to Gpabc1 from Gibberella pulicaris, ABC1 from Magnaporthe grisea, or BcatrB from Botrytis cinerea. These three ABC transporters are involved in antimicrobial tolerance and/or pathogenesis. Two of these transporters were significantly up-regulated in pisatin treated mycelia as measured by real time PCR, and complementation in Neurospora crassa suggests at these ABC-transporters may be involved in NDT.

253. Functional analysis of the Magnaporthe grisea ACE1 locus involved in avirulence toward Pi33 resistant rice cultivars. Jérôme Collemare1, Heidi U. Böhnert1, Mikaël Pianfetti1, Isabelle Fudal1, Didier Tharreau2 and Marc-Henri Lebrun1. 1FRE 2579 CNRS-Bayer Cropscience, Physiologie des plantes et des champignons lors de l'infection, Bayer Cropscience, 14-20 rue P Baizet, 69263 Lyon Cedex 09, France. 2UMR BGPI, CIRAD-INRA-ENSAM, TA41/K, 34398 Montpellier Cedex 05, France

Interactions between resistant rice cultivars and the rice blast fungus, Magnaporthe grisea, are often governed by gene-for-gene relationships. The avirulent isolate Guy11 carries the avirulence gene ACE1 controlling the production of a signal specifically recognized by rice cultivars carrying the resistance gene Pi33. ACE1 encodes a polyketide synthase fused to a non-ribosomal peptide, an enzyme involved in the biosynthesis of a microbial secondary metabolite. ACE1 is specifically expressed in mature appressoria during the penetration of the fungus into host plant leaves, and the resulting enzyme is retained in the cytoplasm of appressoria. Deletion analysis of ACE1 promoter led to the identification of a 102 bp region required for its transcriptional regulation. This region contains a putative binding site of fungal binuclear zinc finger transcription factors. Site-directed mutagenesis of this putative binding site will be used to assess its role in the regulation of ACE1 expression. Ace1-ks0, a non-functional ACE1 allele obtained by site-directed mutagenesis of an essential amino acid of the polyketide synthase KS domain, is unable to confer avirulence. This result suggests that the avirulence signal recognized by Pi33 resistant rice is not the Ace1 protein, but is likely to be the secondary metabolite synthesized by Ace1. In order to characterize this metabolite, we are performing a metabolic profiling of M. grisea appressoria by LC-MS-MS, using onion epidermis infected with virulent or avirulent strains. The ACE1 locus is 70kb long and carries 15 genes predicted to encode enzymes involved in secondary metabolism, including two enoyl-reductases, one MFS-transporter and a binuclear zinc-finger transcription factor. All these genes have the same penetration-specific expression pattern as ACE1, thus defining a cluster. The inactivation of these genes in an avirulent isolate is underway to evaluate their role in the biosynthesis of the avirulence signal recognized by Pi33 resistant rice cultivars.

254. The transcription-associated proteins of Fusarium graminearum identified by sequence clustering and profile analyses. Richard Coulson1, Martin Urban2, John Antoniw2, and Kim Hammond-Kosack2. 1European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, CB10 1SD, UK and 2Rothamsted Research, Herts, AL5 2JQ, UK

The trichothecene mycotoxin producing Ascomycete fungus Fusarium graminearum causes ear blight disease of small grain cereals. Infections lower grain quality and safety, and are of increasing global concern. In 2003, its genome was sequenced to ~10 × coverage by the Broad Institute ( As part of the global initiative to complete the manual annotation of the genome, we have explored in depth F. graminearum sequences involved with the transcriptional process. Eukaryotic transcription is a highly regulated process involving interactions between large numbers of proteins, exhibiting a high degree of taxon-specificity. To identify transcription-associated proteins (TAPs), the genome was queried with a reference set of TAPs, extracted from the protein sequence databases via keyword searches (Coulson & Ouzounis (2003) Nucleic Acids Research 31, 653-660). The TRIBE-MCL algorithm was employed to detect TAP families in F. graminearum, in addition to those present in six model organism species: Schizosaccharomyces pombe, Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus. Four plant pathogens Ashbya gossypii, Magnaporthe grisea, Aspergillus oryzae, Ustilago maydis, two human pathogens, Candida albicans and Aspergillus fumigatus and two free living saprophytes, Aspergillus nidulans and Neurospora crassa were also included in the study. The findings from this TAP-TRIBE analysis, and a second complementary approach utilising profile-hidden Markov models of domains present in well-characterised transcriptional regulators, will be presented. Currently, we are exploring the physical distribution of each TAP gene amongst the four F. graminearum chromosomes.

255. Analysis of Expressed Sequence Tags from Alternaria brassicicola infected Brassica oleracea var. capitata. R.A. Cramer Jr1, Y. Cho 2, T.K.Mitchell3, K.D. Craven3, M. Thon4, Christopher B. Lawrence2. 1 Current Address Duke University Medical Center, Durham, NC. 2 Virginia Bioinformatics Institute, Blacksburg, VA. 3 North Carolina State University, Raleigh, NC. 4 Texas A&M University, College Station, TX.

Alternaria brassicicola (AB) is a necrotrophic fungus that causes black spot disease on economically important Brassicas such as cabbage and is a model pathogen for studies with Arabidopsis. The purpose of this study was to identify genes involved in the interaction between AB and Brassica oleracea var. capitata (BO). A functional genomics approach was used to identify candidate pathogenicity genes by creating a suppression subtractive hybridization (SSH) library enriched for AB and BO genes expressed during pathogenesis. A total of 4224 expressed sequence tags (ESTs) were sequenced and assembled into a 3112 unisequence set using the assembly program CAP3. This unisequence set contained 608 contigs and 2504 singletons. The library had an estimated redundancy rate of 26%. BLAST algorithms were used to search publicly available databases to gain putative identities of the ESTs. BLAST searches identified 312 of the sequences as AB, 1614 as BO, and 1186 as unknown. A similar analysis was conducted on a cDNA library created from AB nitrogen starved (NS) mycelia with an approximate 40% redundancy rate. This full length cDNA library contained 1660 unisequences (253 contigs and 1407 singletons). NS sequences proved useful in identifying unknown sequences in the BO library. Many ESTs from the infected BO and NS libraries had homology with known fungal pathogenicity factors. RT-PCR was used to confirm the differential expression of 10 putative fungal pathogenicity factors and 10 host genes in planta. Results of these experiments will be presented.

256. Cloning and Characterization of a Cyanide Hydratase from the Necrotropic Fungal Pathogen Alternaria brassicicola. R.A. Cramer Jr1, Juan Wang, Josh Davis2, Christopher B. Lawrence2. 1 Current Address Duke University Medical Center, Durham, NC. 2 Current Address Virginia Bioinformatics Institute, Blacksburg, VA.

In order to colonize and infect their hosts, plant pathogenic fungi must overcome potentially toxic compounds produced by plants. Previously a cDNA clone with homology to a cyanide hydratase enzyme was identified in a suppression subtracted library of Alternaria brassicicola infected Arabidopsis. In this study, random amplification of cDNA ends was used to clone the full length cyanide hydratase cDNA sequence which was named ACH (Alternaria Cyanide Hydratase), This sequence was used to disrupt the genomic cyanide hydratase locus using a single homologous recombination event. Mutants with a disrupted ACH locus displayed normal vegetative growth in vitro, normal conidiation, and increased sensitivity in vitro to KCN. Lesion diameters, in developed lesions, on infected Brassica oleraceae var. capitata leaves were similar for wild type A. brassicicola and ach mutants. However, there was a significant difference in the number of lesions that developed between wild type and ach mutants suggesting a possible role in fungal pathogenicity for ACH. Bioinformatic analysis of available fungal genomes revealed that cyanide hydratase homologs are found in fungi with diverse life styles including saprophytes and pathogens. Thus, the exact role in fungal biology that cyanide hydratase enzymes play is still not clear.

 257. Signal Transduction and Hydrophobin Gene Expression in the Maize Pathogen Cochliobolus heterostrophus. Ofir Degani1, Sophie Lev1, Mark S. Rose2 and Benjamin A. Horwitz1. 1Department of Biology, Technion–Israel Institute of Technology, Haifa 32000, Israel; 2Syngenta Biotechnology Inc., Research Triangle Park, NC 27709.

Filamentous fungi produce hydrophobins, small proteins that are localized on the outer surface of their cell walls and are involved in growth and development. Nutrient availability and light regulate hydrophobin gene expression. Recently it was demonstrated that signal transduction components such as a MAPK in Magnaporthe grisea and a G-protein alpha subunit in Cryphonectria parasitica are also involved. Here we conducted a comparative study on the effect of loss of function mutations in different signaling components on hydrophobin gene expression in Cochliobolus heterostrophus. Furthermore, a mutant deficient in both G-protein alpha and beta subunits (Cgab1) was constructed. This mutant has an albino phenotype, lacked conidia but still formed abortive pseudothecia in a backcross, and was unable to infect maize. Loss of the G-alpha subunit led, in some conditions, to increased hydrophobin expression, which may be involved in the spore aggregation phenotype of this mutant. Mutations in the G-beta subunit led to increased hydrophobin class II expression on liquid media, while MAPK mutants or cgab1 double mutants showed reduced expression of hydrophobins and was found to have a wettable colony phenotype in their early growth stage. Together, these findings suggest an important role for MAPK and for G-protein alpha and beta subunits in mediating hydrophobin gene expression.

258. Identification of Arabidopsis genes that support parasitic symbiosis by Peronospora parasitica. Terrence P. Delaney1 and Nicole M. Donofrio.2 1The University of Vermont, Department of Botany, Burlington VT, USA. 2North Carolina State University, Fungal Genomics Laboratory, Raleigh NC, USA.

Pathogens that establish intimate, non-lethal relationships with their hosts may employ strategies for host immune system evasion, and may manipulate host metabolism or physiology to benefit the parasite without destroying host tissue. We are examining infection of Arabidopsis thaliana by the oomycete Peronospora parasitica to search for these parasite activities, which may constitute essential yet poorly recognized virulence functions. Using gene expression profiling methods, we identified plant genes whose expression is altered specifically by virulent strains of P. parasitica, but are not linked to defense responses. Such genes are candidates to be those manipulated by the pathogen for its own benefit. We obtained T-DNA insertion mutations in some of these genes, and are now performing functional tests to determine whether the genes play a role in supporting growth of this compatible biotrophic pathogen.

In a companion study, we performed mutant screens in Arabidopsis nim1-1 mutants, which are compromised in expression of systemic acquired resistance and thus highly susceptible to a virulent strain of P. parasitica. Several mutants were recovered that supported less growth of the pathogen without the induction of known defense-related genes, suggesting that these plants may lack gene products important for pathogen vitality.

259. Fpr1, a member of the pathogenesis-related (PR) protein superfamily is required for virulence of Fusarium oxysporum on mammals. Rafael C. Prados-Rosales1, Raquel Roldán-Rodríguez1, Montserrat Ortoneda2, Josep Guarro2, and Antonio Di Pietro1

1Departamento de Genetica, Universidad de Cordoba, Campus de Rabanales Ed. C5, 14071 Cordoba, Spain. 2Unitat de Microbiologia, Facultat de Medicina i Ciencies de la Salut, Universitat Rovira i Virgili, 43201 Reus, Tarragona, Spain

The pathogenesis-related (PR) protein superfamily is widely distributed in eukaryotic organisms. While their precise biological activity remains elusive, PR proteins have been implicated in a variety of processes, including the plant defense and the mammalian immune response. We have examined the role of Fpr1, a PR-like protein of the soilborne fungus Fusarium oxysporum, the causal agent of vascular wilt disease in plants and an emerging opportunistic pathogen of humans. The fpr1 gene was expressed at high levels in actively growing hyphae of F. oxysporum. The presence of a predicted N-terminal signal peptide suggests that FPR1 is co-translationally translocated to the ER, although GFP-tagged Fpr1 protein was found to accumulate intracellularly in spherical organelles. Targeted knockout mutants showed no detectable changes in vegetative growth or virulence on tomato plants. In contrast, the fpr1 knockout mutants exhibited markedly reduced virulence in a disseminated immunodepressed mouse model. Virulence was fully restored by re-introduction of a wild type fpr1 allele, but not of a fpr1mut allele in which two conserved residues of the predicted active site of the PR-1 protein family, His170 and Gln177, were changed to Leu and Ala, respectively. Our current efforts are directed towards the elucidation of the biochemical function of Fpr1.

260. Genomic architecture of ftf1 and ftf2 genes in F. oxysporum. B. Ramos, M.A. García-Sánchez, N. Martín-Rodrigues, A.P. Eslava and J.M. Díaz-Mínguez. Área de Genética. Centro Hispano-Luso de investigaciones Agrarias (CIALE). Universidad de Salamanca. 37007, Salamanca, Spain.

We have previously reported the isolation of ftf1 (Fopta1a) and ftf2 (Fopta1b), two genes coding for highly homologous transcription factors differentially involved in pathogenicity in F. oxysporum f. sp. phaseoli (Kendrick & Snyder). Both transcription factors contain a Zn(II)-Cys6 binuclear cluster DNA-binding motif and a fungal transcription factor domain. The structural region of both genes share a 80-85% of homology at the nucleotide level; this homology falls to a 70% in the promoter region. The ftf1 gene is present only in highly virulent strains and show a dramatic peak of expression 24 to 48 h after infection of common bean plants (Phaseolus vulgaris L.). On the contrary, ftf2 is present in all tested strains (pathogenic and non-pathogenic) and shows a very low level of expression in vivo and in vitro.

We have detected up to four copies of ftf1 in highly virulent strains, while ftf2 is a single copy gene in all the strains tested. Genomic architecture is also different in both genes. Some of the copies of ftf1 show some restriction fragment length polymorphisms, but all of them are closely linked to the only copies of transposon marsu detected in F. oxysporum f.sp. phaseoli. The single copy of ftf2 is located downstream of an homologue of bimB3, a gene involved in coupling DNA replication with mitosis in Aspergillus nidulans.

261. A Botrytis cinerea gene induced in planta encodes a protein with homology with cyclins. Benito Pescador, D., Martín-Domínguez, R., Díaz-Mínguez, J.M., Eslava A. P. and Benito E.P. Area de Genética. Centro Hispano-Luso de Investigaciones Agrarias. Universidad de Salamanca. Edificio Departamental. Campus Unamuno, s/n. 37007 Salamanca. SPAIN.

The causal agent of grey mold, Botrytis cinerea Pers. (teleomorph: Botrytinia fuckeliana (de Bary) Whetzel) is a filamentous fungus with a broad host range and responsible of important economic losses. A large number of research groups have been working intensively on the characterization of the fungal pathogenicity mechanisms and on the design of control strategies, either chemical or biological.

In order to understand the molecular mechanisms involved in the infection process of B. cinerea, an experimental approach based on the analysis of differential gene expression during the plant/fungus interaction was applied. This analysis allowed us to detect different cDNA fragments derived from B. cinerea genes whose expression is induced in planta. The gene encoding one of these fragments, named Bde2, is being characterized in detail.

Using as a probe the cDNA fragment initially detected, it was shown by Northern blot hybridization analysis that gene Bde2 is expressed only during late stages of the infection process, specifically during colonization and maceration of the infected tissue. Southern analysis demonstrated it is a single copy gene. The full-length genomic copy of Bde2 was cloned from a genomic library and its structure and sequence was determined. Sequence analysis and public databases searches revealed an ORF translation of which generates a protein with significant homology to cyclins.

To get deeper insights into the role of the Bde2 gene, and of the encoded protein, in the infection process of B. cinerea, a functional characterization approach based on the isolation and characterization of mutants specifically altered in gene Bde2 was designed. Transformation experiments are being carried out in order to obtain Bde2 gene replacement mutants using a plasmid carrying a mutant allele in which the coding region of the wild type allele has been replaced by a gene fusion expressing the bacterial gene conferring hygromicin resistance under the control of fungal promoter and terminator sequences. Transformants lacking gene Bde2 will be analyzed for alterations during growth in planta and during saprophytic growth.

262. The melanin biosynthetic gene PKS1 is dispensable for virulence of the banana pathogen Mycosphaerella fijiensis. Bruno Giuliano Garisto Donzelli and Alice C.L. Churchill, Department of Plant Pathology, Cornell University, Ithaca, NY, USA.

Black leaf streak disease (BLSD) or black Sigatoka, caused by Mycosphaerella fijiensis, is the most destructive disease of bananas and plantains (Musa sp.) worldwide. Shunt metabolites from the DHN-melanin pathway, such as juglone and 2,4,8-trihydroxytetralone, have been hypothesized to play a role in the disease as phytotoxins causing leaf chlorosis and necrosis. We cloned a polyketide synthase gene (PKS1) responsible for the first step of the DHN-melanin pathway in M. fijiensis and generated melanin-deficient mutants of the fungus by both targeted gene knockout (KO) and RNA-mediated silencing. Targeted gene KO was accomplished using a construct in which the Hygromycin resistance cassette was flanked by ca. 1 and 3 Kb of the PKS1 gene. RNA-mediated gene silencing was achieved by expressing an inverted repeat fragment of the PKS1 gene. Both constructs were delivered to a wild type virulent strain by Agrobacterium tumefaciens-mediated transformation. Both the PKS1-KO isolates and the PKS1-silenced isolates displayed similar reduced pigmentation phenotypes. Pathogenicity assays on the susceptible banana cultivar Grand Naine indicated no detectable alteration in virulence of two KO mutants. In contrast, several RNA-silenced mutants showed reductions in both melanization and virulence. Chemical analyses of the virulent PKS1-KO isolates indicated that production of melanin-DHN pathway shunt metabolites was abolished in the mutants, suggesting that these metabolites are dispensable for M. fijiensis virulence. This is a first report of gene disruption and silencing in a Mycosphaerella pathogen of bananas and the first genetic evidence to suggest that melanin shunt metabolites do not play roles in BLSD.

263. Towards the development of a large–scale transposon insertional mutagenesis across the Fusarium graminearum genome.

Marie Dufresne, Sarrah Ben M'barek, Gert H.J. Kema and Marie-Josée Daboussi. Institut de Génétique et Microbiologie, Université Paris-Sud, 91405 Orsay Cedex, France

The mimp1 element is a miniature inverted-repeat transposable element (MITE) previously identified in Fusarium oxysporum and shown to be mobilized by the transposase of a Tc1-mariner member, impala. Analysis of mimp1 distribution in Fusarium strains revealed that it is present in all F. oxysporum strains and in most of related species such as F. redolens and F. hostae. On the contrary, this element is absent from more distant species such as F. culmorum and F. graminearum.

Here, we show that the double component system mimp1/impala transposase is fully functional in F. graminearum. Transposition characteristics (TA target site, duplicated upon mimp1 insertion, excision footprints) were found to be the same as in the original host species. Moreover, as already observed in F. oxysporum, mimp1 reinserted very frequently (around 95%) allowing the generation of an efficient collection of revertants.

Taking advantage of the availability of the F. graminearum genome sequence and using TAIL-PCR, we started to recover sequences flanking mimp1 reinsertions in a collection of revertants obtained from several initial transformants. Preliminary results obtained from a set of 25 revertants indicated that mimp1 tends to reinsert in 5'-non coding regions, 40% of the insertion events taking place less than 1000 bp upstream the initiation codon of a predicted ORF.

Such results are very promising for the development of this novel double component system as a powerful mutagenesis tool on a high throughput scale in F. graminearum as well as other ascomycete fungi.

264. Determining the role of reactive oxygen species generation in Magnaporthe grisea. Martin Egan and N Talbot. University of Exeter, Devon, UK

NADPH oxidases (Nox) are enzymes used to generate reactive oxygen species (ROS). Here we investigate the role of NADPH oxidase-generated ROS in the infection related development of the phytopathogenic fungus Magnaporthe grisea. M.grisea parasitizes more than fifty species of grasses, but is best known as the causal agent of rice blast disease. The formation of a specialised cell known as the appressorium allows the fungus to breach the host cuticle and subsequently cause infection. Using the super oxide detector dihydrofluorescein diacetate, we have monitored the production of ROS during conidial germination and report the occurence of an oxidative burst during appressorium formation. We also report the accumulation of ROS in hyphal tips. We demonstrate that appressorial development is inhibited by DPI, an NADPH oxidase inhibitor.

We have also characterised NOX1, one of two genes encoding NADPH oxidases in the M. grisea genome. Through targeted gene replacement, we have shown that NOX1 cannot cause disease and is required for pathogenicity. Deltanox1 mutants also exhibit aberrant germ tube morphology and frequently produce multiple appressoria. Strikingly normal hyphal growth appears unaffected in deltanox1 mutants indicating that ROS generation is associated with cellular differentiation.

265. Characterization of the MST7 MAP kinase kinase gene in Magnaporthe grisea. Xinhua Zhao, Yangseon Kim and Jin-Rong Xu. Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907

Many fungal pathogens invade plants using specialized infection structures called appressoria. In the rice blast fungus Magnaporthe grisea, the PMK1 MAP kinase is essential for appressorium formation and invasive growth. We functionally characterized the MST7 and MST11 genes of M. grisea that are homologous to the yeast MAP kinases kinase STE7 and MAP kinases kinase kinase STE11. Similar to the pmk1 mutant, the mst7 and mst11 deletion mutants were non-pathogenic and failed to form appressoria. When a dominant allele MST7S212D T216E was introduced into a mst11 mutant, the resulting transformants formed appressoria but failed to penetrate and infect rice leaves, indicating that constitutively active MST7 only partially rescued the defects of the mst11 mutant. Although the interaction between Mst7 and Mst11 is weak in yeast two-hybrid assays, both of them directly interact with Mst50, a putative adapter protein for the Pmk1 MAPK cascade. Interesting, a putative MAPK docking site is well-conserved in MST7 and its homologs. The MST712-20 allele failed to complement the mst7 mutant, suggesting that this docking site may be essential for the interaction between Mst7 and Pmk1. The role of this MAPK docking site and the interaction of Mst7 with Pmk1, Mst11, and Mst50 will be further characterized.

266. A Precocious Sporulation mutant of Leptosphaeria maculans has altered pathogenicity on Brassica napus. Candace Elliott and Barbara J. Howlett, The School of Botany, University of Melbourne, VIC Australia

Blackleg disease, caused by the ascomycete, Leptosphaeria maculans, is the most serious disease of canola (Brassica napus) worldwide. Knowledge of both plant defence mechanisms and fungal disease processes (pathogenicity) will allow development of novel control strategies. I have used an Agrobacterium-mediated random mutagenesis approach to generate isolates with a reduced ability to infect B. napus. Of 353 mutants screened on B. napus cotyledons and stems, 4 showed reduced lesion size. Sequencing of flanking regions revealed one isolate with an insertion in a hypothetical protein of unknown function. Three other isolates contained single T-DNA insertions in genes encoding a zinc-finger protein, a histone H4 gene and an alcohol dehydrogenase (ADH)-like gene. A detailed analysis of the ADH-like mutant revealed the T-DNA insertion did not disrupt expression of the ADH-like gene, but caused constitutive expression of the ADH-like gene and the adjacent thiolase gene. This has resulted in a mutant that attains competence for sporulation at an earlier stage than wild type but is unable to produce lesions with the same frequency as wild type on canola cotyledons and stems. Experiments including complementation, over-expression and gene knockout are currently underway to elucidate whether the ADH-like or the thiolase gene is responsible for this alteration in sporulation and pathogenicity.

267. The location of Sphaeropsis sapinea, the causal agent of pine tip blight, in latently infected versus symptomatic Austrian pine shoots. Jennifer L. Flowers, John R. Hartman, and Lisa J. Vaillancourt; Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546

Sphaeropsis tip blight is a common disease that affects many conifers worldwide. Symptoms of Sphaeropsis sapinea infection include stunted shoots with necrotic, stunted needles, resinous cankers, and a general decline of the tree. These symptoms lead to significant economic losses of pines in managed plantings. Latent infections of S. sapinea are common in current-year symptomless Austrian pine trees, as well as within apparently healthy branches on diseased pines. Dissection and culturing of latently infected pine shoots suggested that S. sapinea was located in the region between the inner bark and vascular cambium. In contrast, the fungus could be recovered from all tissue types in shoots with symptoms of tip blight. Microscopy was conducted to further define the location of the pathogen in latently infected shoots, and to compare and contrast latent versus symptomatic infections. Diseased, latently infected, and uninfected Austrian pine shoots were differentiated using a combination of visible symptoms and a S. sapinea-specific nested-polymerase chain reaction protocol. The shoot samples were embedded in Spur's resin, and 1 micron thick sections were cut, stained with toluidine blue, and observed under the microscope. Large numbers of fungal hyphae were observed throughout the tissues of diseased pine shoots, while no hyphae were observed in uninfected pine shoots. In some of the latently infected shoots, fungal hyphae were observed in small pockets of collapsed periderm cells. A common assumption in tree pathology is that latent infections can transform into pathogenic ones when the host is physiologically stressed. We are continuing our observations as part of our effort to understand the nature of this transformation from latent to pathogenic infection, with the hope that this may lead to more effective management of this devastating disease.

268. Impaired purine biosynthesis affects pathogenicity of Fusarium oxysporum f. sp. melonis. Youlia Denisov1,2, Oded Yarden1 and Stanley Freeman2. 1Department of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Rehovot 76100; 2Department of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

The vascular wilt pathogen Fusarium oxysporum f.sp. melonis causes worldwide yield losses of muskmelon. In this study we characterized a UV-induced non-pathogenic mutant (strain 4/4) of F. oxysporum f.sp. melonis, previously identified as a potential biological control agent. During comparative analysis of vegetative growth parameters using different carbon sources, strain 4/4 showed a delay in development and secretion of extracellular enzymes, compared to the wild type strain. Amendments of the growth medium with yeast extract, adenine or hypoxanthine, but not guanine, complemented the growth defect of strain 4/4, as well as secretion and partial activity of cellulases and endopolygalacturonases, indicating that the strain is an adenine auxotroph. Incubation of strain 4/4 conidia in adenine solution, prior to inoculation of muskmelon plants, partially restored pathogenicity to the mutant strain. As part of the characterization of pathogenicity factors of Fusarium wilt, a collection of approx. 2000 Agrobacterium-transformed mutants were generated and screened for pathogenicity on melon plants. At this stage, five putative impaired pathogenicity mutants are being characterized.

269. A defect in a NirA-like transcription factor confers morphological abnormalities and lack of pathogenicity in Colletotrichum acutatum. Sigal Horowitz 1,2, Stanley Freeman 2, Aida Zveibil 2, and Oded Yarden 1. 1 Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100; 2 Department of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel.

A nonpathogenic REMI mutant of Colletotrichum acutatum, designated Ca-5, was isolated whereby in the absence of an external nitrogen source exhibited extended germ tube growth prior to appressoria formation on solid surface and strawberry leaf. Ca-5 exhibited restricted hyphal growth and did not cause lesions on plants but grew necrotrophically when inoculated directly onto wounded sites. The deduced amino acid sequence of the REMI-impaired gene product, designated Nir1, is highly similar to the Aspergillus nidulans NirA protein, a transcriptional regulator of nitrogen metabolism. Inoculation of leaves with wild type or Ca-5 conidia in the presence of a nitrogen source resulted in massive epiphytic hyphal production, appressoria formation and rapid symptom development. The nutritional status of C. acutatum at an early stage of colonization and appressoria formation was assessed by following the expression of nitrate reductase (NR) and glutamine synthetase (GS) in different media. Under all growth conditions there was no effect on GS, however, NR was induced by nitrate and repressed by a rich medium. In addition, NR transcription increased at the appressoria stage, indicating that nitrogen starvation constitutes a cue for regulation of appresoria development. Our results suggest that nitrogen starvation stimulates synchronous preinfection development which is lacking in Ca-5.

270. Characterisation of a P-type ATPase in Magnaporthe grisea defines an exocytotic pathway required for fungal pathogenicity. Martin J.Gilbert, Christopher R.Thornton, Nicholas J.Talbot. School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, EX4 4QG, United Kingdom.

The ability to secrete protein products into host cells is a fundamental characteristic of pathogenic organisms. In bacterial pathogens, the type III secretion system is utilised to deliver these effector proteins into host cells during infection. It is not known, however, how fungal pathogens achieve delivery of effector proteins during disease establishment or whether they possess specialised secretion systems that have evolved for this purpose.Here we report that a mutant of the rice blast fungus Magnaporthe grisea, deltaMgapt2, that is affected in exocytosis, is also compromised in its ability to cause disease. MgAPT2 encodes an aminophospholipid translocase (APT), one of four putative APTs present in the M. grisea genome. This family of APTs includes the previously characterised PDE1 gene, a virulence factor for rice blast disease (Balhaldere & Talbot 2000. Plant Cell, 13, 1987-2004). deltaMgapt2 mutants accumulate abnormal Golgi cisternae. These appear as double-membrane ring structures similar to Berkeley bodies. deltaMgapt2 mutants grow, sporulate and form appressoria normally but are unable to grow on several carbon sources including starch and glycogen. We have shown that the ability to secrete a subset of extracellular enzymes is impaired in deltaMgapt2 mutants. The product of MgAPT2 is found in the Golgi network indicating a role in vesicle docking during exocytosis. We discuss the implications of this finding for our knowledge of fungal pathogenesis.

271. Identification of Novel Pathogenicity Genes by Macroarray Analysis of the Botrytis-arabidopsis Interaction. Anastasia Gioti, Adeline Simon, Muriel Viaud, Jean-Marc Pradier,Caroline Levis. Unité de Phytopathologie et Méthodologies de la Détéction (P.M.D.V), I.N.R.A de Versailles, Route de St-Cyr, 78026, Versailles Cedex, France.

Our group is investigating the molecular mechanisms of pathogenicity of Botrytis cinerea, an ascomycete with a broad spectrum of plant-hosts. Here we present results of the expression profiling of 3.032 fungal genes, spotted onto macroarray filters, during the interaction of Botrytis with Arabidopsis thaliana.

In order to follow the kinetics of fungal gene expression in planta , we hybridized the macroarrays with RNAs extracted from the infected plant leaves at three different stages of infection. As "reference" probes we used RNAs of Botrytis grown in vitro, as well as the RNAs of the uninfected plant. The resulting data were treated statistically (GeneAnova, P.C.A) and clustered (Genesis). 29 genes were shown to be systematically induced at specific infection stages and over-expressed during infection compared to the in vitro growth reference.

From the above, eight genes were chosen for a reverse genetic analysis aiming to confirm their role in the pathogenicity of Botrytis. Knock-out mutants were obtained for two of these genes so far. Their phenotype during pathogenicity tests on different plants will be presented. In overall, our results show that the transcriptomic study of the fungus in planta is possible and permits us to identify new genes involved in the infection process.

272. Cell surface mannan and the Candida-host interaction. Gow, N.A.R., Bain, J.M, Bates, S., Bertram, G., Hobson, R.P., Hughes, H.B., Munro, C.A., MacCallum, D., Odds, F.C., Brown, A.J.P. Aberdeen Fungal Group, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.

The outer layer of the cell wall of Candida albicans is heavily enriched in glycosylated proteins that play critical roles in cell adherence, and act as major antigens and in the immunoregulation of the host. A null mutant in the Golgi manganese transporter gene PMR1 was viable in vivo, had greatly reduced N- and phosphomannan and was attenuated in virulence. Therefore normal mannosylation is required for pathogenesis. The C. albicans O-linked mannan consists of a pentasaccharide in which Mnt1p and Mnt2p participate as partially functionally redundant enzymes in the assembly of the terminal two a-1,2-mannose residues. Deletion of either MNT1, MNT2 or both MNT1 and MNT2 resulted in strains with reduced adherence to epithelia and attenuation of virulence. This suggests that O-mannan functions as a ligand in interactions with host surfaces. Mutants with deletions in the MNN4 gene are almost devoid in phosphomannan, which has been implicated in recognition of C. albicans by macrophages, but were phagocytosed normally. Deletion of the OCH1 resulted in elimination of the outer N-mannan chains, induction of the cell wall salvage pathway and loss of virulence. Analysis of glycosylation mutants deomonstrates that the carbohydrate epitopes of mannoproteins play key roles in pathogenesis of C. albicans.

273. Siderophore biosynthesis but not reductive iron assimilation is essential for Aspergillus fumigatus virulence. Markus Schrettl1, Elaine Bignell2, Claudia Kragl1, Chistoph Jöchl1, Tom Rogers2, Herbert N. Arst Jr2, Ken Haynes2 and Hubertus Haas1. 1Department of Molecular Biology, Medical University Innsbruck, Austria; 2Department of Infectious Diseases, Imperial College London, UK.

The ability to acquire iron in vivo is essential for most microbial pathogens. Here we show that Aspergillus fumigatus does not have specific mechanisms for the utilization of host iron sources. However, it does have functional siderophore-assisted iron mobilization and reductive iron assimilation systems, both of which are induced upon iron deprivation. Abrogation of reductive iron assimilation, by inactivation of the high-affinity iron permease (FtrA), has no effect on virulence in a murine model of invasive aspergillosis. In striking contrast, A. fumigatus L-ornithine-N5-monooxygenase (SidA), which catalyses the first committed step of hydroxamate-type siderophore biosynthesis, is absolutely essential for virulence. Thus, A. fumigatus SidA is an essential virulence attribute. Combined with the absence of a sidA ortholog - and the fungal siderophore system in general - in mammals, these data demonstrate that the siderophore biosynthetic pathway represents a promising new target for the development of antifungal therapies.

This work was supported by the Austrian Science Foundation), the Chronic Granulomatous Disorder Research Trust and the Biotechnological and Biological Sciences Research Council.

274. GzGRR1 encoding a putative F-box protein is involved in pathogenesis and sexual development by Gibberella zeae. You-Kyoung Han1, Sung-Hwan Yun2, and Yin-Won Lee1. 1School of Agricultural Biotechnology, Seoul National University, Seoul 151-921; 2Division of Life Sciences, Soonchunhyang University, Asan, 336-745, Korea

Gibberella zeae is an important pathogen of cereal crops in many areas of the world, causing head blight of small grains including corn, wheat, barley, and rice. In addition, this fungus produces mycotoxins such as trichothecenes and zearalenone on diseased crops and has been a potential threat to human and animal health. To identify pathogenesis-related genes, we selected several G. zeae mutants defective for the traits involved in disease development. The mutant ZH436, generated by restriction enzyme-mediated integration, showed significantly reduced virulence toward host plants along with other pleiotropic phenotypes such as reduced hyphal growth on nutrient rich conditions and no sexual development. In addition, this mutant produced incomplete tetrads with aberrant morphology when outcrossed to a mat1-2 deletion strain. Molecular characterization revealed that vector insertion point was located within the ORF, designated GzGRR1 showing a high similarity to GRR1, a regulator for glucose repression in Sacharomyces cerevisiae; the translation product of GzGRR1 carries both a putative F-box and a leucine-rich repeats (LRR) domain. Northern blot analysis showed that GzGRR1 was constitutively expressed but the transcript was highly produced during the perithecial stage. These results suggest that GzGRR1, as other F-box proteins, may be involved in degradation of proteins by ubiquitination, specifically those required for virulence or sexual development in G. zeae.

275. Oomycete zoospores secrete adhesins containing thrombospondin type-1 repeats. Andrea V. Robold and Adrienne R. Hardham. Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, Canberra, ACT 2601, Australia.

Adhesion is a key aspect of the establishment of disease by pathogens of animals and plants. Adhesion anchors the parasite to the host surface and is often a prerequisite for the differentiation of specialized infection structures and host invasion. A number of adhesin molecules produced by microbial pathogens infecting animals have been characterised, however, to date molecular details of adhesins of plant pathogens, especially fungi, are largely restricted to general descriptions of the nature of heterogeneous secreted materials. Many devastating plant diseases are caused by oomycetes, fungus-like organisms that include Phytophthora infestans, responsible for late blight of potato. In this presentation we describe the cloning and characterisation of a gene encoding a Phytophthora adhesin and the discovery that the adhesin protein contains 47 copies of the thrombospondin type-1 repeat, a motif found in adhesin proteins in animal cells and apicomplexan (e.g. malarial) parasites but not in plants, green algae or true fungi. Our results give the first detailed information on an adhesin of a fungal or fungus-like plant pathogen, and highlight intriguing similarities in structural and molecular features of host attachment in oomycete and apicomplexan parasites.

276. Identification of AM-toxin biosynthesis gene cluster in the apple pathotype of Alternaria alternata. Yoshiaki Harimoto1, Rieko Hatta1, Motoichiro Kodama2, Mikihiro Yamamoto3, Hiroshi Otani3 and Takashi Tsuge1. 1Graduate School of Bioagricultural Science, Nagoya University, 2Faculty of Agriculture, Tottori University, 3 Faculty of Agriculture, Okayama University, Japan.

The apple pathotype of Alternaria alternata produces host-specific AM-toxin and causes Alternaria blotch of apple. We previously isolated two genes, AMT and AMT2, involved in AM-toxin biosynthesis and found that these genes are encoded by conditionally dispensable (CD) chromosomes of the apple pathotype strains. In this study, we conducted the expressed sequence tag (EST) analysis of the 1.4-Mb chromosome encoding AMT genes in strain IFO08984. A cDNA library (41,000 clones) of IFO08984 was screened with the 1.4-Mb chromosome probe, and 270 clones were isolated. Sequence analysis showed that 270 clones are derived from 148 unique genes. Database homology search detected 22 candidate genes, which are possibly involved in AM-toxin biosynthesis. To identify AM-toxin biosynthesis gene cluster, BAC clones encoding the AMT genes and the candidate genes were isolated. Structural analysis of a 118-kb insert of a BAC clone (AM-BAC1) detected AMT, AMT2, 16 candidate genes, and additional 19 putative genes. Expression analysis of these genes by real-time RT-PCR showed that AMT, AMT2, and eight genes were specifically up-regulated in AM-toxin production medium. All the up-regulated genes are present within the 62-kb region in AM-BAC1, suggesting that this region is responsible for AM-toxin biosynthesis.

277. Partial structure of the conditionally dispensable (CD) chromosome controlling AF-toxin biosynthesis and pathogenicity in the strawberry pathotype of Alternaria alternata. Rieko Hatta1, Akihisa Shinjo1, Yoshiaki Harimoto1, Mikihiro Yamamoto2, Kazuya Akimitsu3, and Takashi Tsuge1. 1Graduate School of Bioagricultural Sciences, Nagoya University, 2Faculty of Agriculture, Okayama University, 3Faculty of Agriculture, Kagawa University, Japan.

The strawberry pathotype of Alternaria alternata produces host-specific AF-toxin and causes black spot of strawberry. We previously isolated AF-toxin biosynthesis genes (AFT genes) from strain NAF8. The AFT genes were found to be clustered on a 1.05-Mb CD chromosome of NAF8, which is dispensable for saprophytic growth in culture. We conducted expressed sequence tag (EST) analysis of the 1.05-Mb chromosome. A cDNA library of NAF8 was screened with the 1.05-Mb chromosome probe, and 342 clones were isolated. Sequence analysis showed that 342 clones are derived from 97 unique genes, and that 11 genes correspond to known AFT genes. We analyzed structure of the 1.05-Mb chromosome. An 8X whole chromosome shotgun sequence assembly of the 1.05-Mb chromosome was obtained, and 150-kb and 420-kb contigs were identified. The 150-kb contig contains 19 AFT genes and 11 transposon-like sequences. In contrast, the 420-kb contig contains no AFT genes and encodes 94 putative ORFs. Database homology search of these ORFs detected no housekeeping gene homologs. These results suggest that the 420-kb region is not essential for AF-toxin biosynthesis and saprophytic growth.


278. Distinct signaling pathways regulate plant cell death induced by INF1, CRN2 and PiNPP1.1 of Phytophthora infestans. Edgar Huitema, Cahid Cakir, Thirumala-Devi Kanneganti, Natalia Norero and Sophien Kamoun. Department of Plant Pathology, The Ohio State University-OARDC, Wooster, OH 44691

Phytophthora infestans, a plant pathogenic oomycete, causes late blight on potato and tomato. Most plant species display active defense responses upon P. infestans infection and are fully resistant (nonhost resistance). The mechanism that underlies nonhost resistance is hypothesized to involve recognition of P. infestans effectors. Perception of these proteins initiates a series of discrete signaling steps, resulting in cell death and defense responses. Previously, P. infestans INF1, CRN2 and PiNPP1.1 were identified as necrosis inducing proteins. To expand our understanding of resistance, we investigated various aspects of INF1, CRN2 and PiNPP1.1- induced cell death. First, we used virus induced gene silencing (VIGS) to silence 35 signaling genes in N. benthamiana and measure their impact on elicitor induced necrosis. Second, we tested whether AVRPTOB, a suppressor of AVRPTO-induced cell death, suppresses the activity of INF1, CRN2 and PiNPP1.1. Third, we applied combinations of INF1, CRN2 and PiNPP1.1 in agroinfiltration assays to test whether cross-talk occurs between signaling pathways. Our results point to at least two distinct cell death pathways. This work is helping us dissect nonhost resistance to this economically important pathogen.

279. EST analysis during conidiation of the plant pathogenic fungus Fusarium oxysporum. Yuichiro Iida, Toshiaki Ohara, and Takashi Tsuge. Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

Fusarium oxysporum is a soil-borne facultative parasite that causes economically important losses on a wide variety of crops. F. oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. Falcate macroconidia are generally formed from terminal phialides on conidiophores. Ellipsoidal microconidia are formed from intercalary phialides on hyphae. Globose chlamydospores with thick walls are developed by the modification of hyphal and conidial cells. Here we describe expression sequence tag (EST) analysis during conidiation of F. oxysporum. F. oxysporum produces macroconidia and microconidia in carboxymethyl cellulose liquid medium (CMC) but not in complete liquid medium (CM). The cDNA libraries were constructed using mRNAs from fungal tissues grown in CM and CMC. The 5f ends of 1,352 and 1,289 cDNA clones from CM (vegetative growth) and CMC (conidiation) libraries, respectively, were sequenced, and cDNAs derived from 543 and 542 unique genes were identified. Number of common genes in both libraries was only 112 (20%), suggesting that the vegetative growth and conidiation libraries had different patterns of gene expression. A total of 430 genes specific for the conidiation library were subjected to macroarray analysis, and 173 genes were identified to be up-regulated during conidiation. Expression analysis of these genes by real-time RT-PCR detected 54 genes that are markedly up-regulated during conidiation.

280. Managing durable resistance to stem canker of oilseed rape: molecular tools and mathematical modelling . Neal Evans (1), Thierry Rouxel (2), Hortense Brun (3), Peter Gladders (4), Xavier Pinochet (5), Malgorzata Jedryczka (6), Ingrid Happstadius (7), Michel Renard (8). (1) Rothamsted Research, Harpenden, UK (2) INRA, PMDV, Versailles, France (3) INRA UMR BiO3P, Le Rheu, France (4) ADAS Boxworth, Cambridge, UK (5) CETIOM, Thiverval-Grignon, France. (6) Institute of Plant Genetics PAS, Poznan, Poland. (7) Svalöf Weibull AB, Svalöv, Sweden. (8) UMR APBV, Le Rheu, France

Stem canker (blackleg) of crucifers, caused by Leptosphaeria maculans, is a damaging disease of oilseed rape worldwide and is responsible for substantial annual crop losses in Europe. In 2003, the European Commission funded the SECURE multinational project (StEm Canker of oilseed rape: molecular tools and mathematical modelling to deploy dUrable REsistance: QLK5-CT-2002-01813) which aims to deliver a model for deployment of cultivars with durable resistance. Project work includes the development of a L. maculans life-cycle model, genomic analysis and fitness studies using virulent and avirulent races of the pathogen, studies of the effects of plant genetic background and environmental factors on durability of resistance under controlled conditions and at field sites across Europe. Data generated during the project will be used to develop a mathematical model that can be used to produce recommendations for the sustainable deployment of durable resistance against stem canker of oilseed rape.

281. Comparative genomics and synteny studies revealing the reservoir of secreted proteins in Phytophthora. Rays H.Y. Jiang, Brett Tyler* and Francine Govers. Plant Sciences Group, Laboratory of Phytopathology, Wageningen University, and Graduate School Experimental Plant Sciences, The Netherlands.* Virginia Bioinformatics Institute, Virginia Polytechnic and State University, Blacksburg VA, USA

Pathogenic fungi and oomycetes possess a wide range of molecules to interact with their hosts. Proteins secreted by plant pathogens are of ultimate interest because these proteins might be effector molecules that play important roles in pathogenesis. The presence of signal peptides and transmembrane domains was analyzed on all annotated genes in two sequenced Phytophthora genomes revealing the whole reservoir of secreted proteins. A total of 1570 and 1256 putative secreted protein genes from P. sojae and P. ramorum, respectively, were investigated for their sequence diversity, expansion of family members and genome organization. More than 80% of the secreted protein genes form gene families, and many of the families are clustered in the genome. Differences in expansion of gene families in different Phytophthora spp. were observed, and these expansion patterns may explain the difference in their pathogenicity. Some genes are located in genomic regions having many re-arrangements and insertions/deletions and these "hotspots" are particular interesting to explore.

282. Developing a lichen model system. Suzanne Joneson1 and François Lutzoni1. 1Duke University, Durham NC, USA.

Lichens are the mutualistic association of a fungus and an alga. This successful and terrestrially ubiquitous lifestyle is shared by 40% of all higher fungi. Multiple origins of lichen forming fungi share a common ancestor with pathogenic fungi. Both types of fungi make contact with bacterial, plant (including green algae) and/or animal cell walls, and must be able to recognize foreign cells as acceptable or unacceptable symbionts. With this as our primary hypothesis, we would like to explore to what extent lichen-forming fungi share common recognition, and cell signaling pathways with other mutualistic fungi. Using Cladonia grayi, we are developing a system in which we can detect differential gene expression between the symbiotic and aposymbiotic states, identifying candidate genes through homology searches. Additionally we are trying to develop a transformational system in which we can make mutants and ultimately targeted transformants. Our current goal of developing a model lichen-forming fungus will lay the groundwork for future dissection of the fungal-algal symbiosis, as well as the evolution of varying symbioses from a common ancestor.

283. Elucidating the Role of the F-box Protein Frp-1 in Pathogenesis of Fusarium oxysporum. W. Jonkers, R. G. E. Duyvesteijn, B. J. C. Cornelissen and M. Rep. University of Amsterdam, Swammerdam Institute for Life Sciences, Plant Pathology, Amsterdam, the Netherlands.

During evolution, plants have developed effective ways to defend themselves against microbial invasion. A microbial pathogen has to break through these defences in order to colonize the plant. To investigate the genetic basis of this ability of pathogens, we use the interaction between the soil-borne fungus Fusarium oxysporum f.sp. lycopersici and its host tomato as a model system. Using insertional mutagenesis of an F-box protein called Frp1 was found to be required for pathogenicity. It was shown that this protein, like other F-box proteins, binds to Skp1, a subunit of the E3 complex. This complex is involved in the ubiquitination of proteins recruited by F-box proteins.

One approach towards determination of the function of Frp1 is to find interacting proteins. To do so, a yeast two-hybrid screening using Frp1L226S will be carried out. This Frp1 mutant has lost the ability to bind Skp1, but is still able to bind other interactors. For yeast two-hybrid screening, a genomic Fol Y2H library is being constructed.

The second approach to determine the function of Frp1 is to study the phenotype of the mutant. Microscopic studies of GFP-labelled wild type Fol and an ?frp1 mutant showed that the mutant has lost its ability to colonise the roots. The mutant also showed less growth on agar plates with alcohol, root-exudate sugars, organic acids or cell wall components as the sole carbon source. Probably, the mutant has a defect in assimilation of certain carbon sources which might be related to loss of pathogenicity. To investigate this further, transcript levels of genes involved in carbon source assimilation will be determined.

284. Regulatory cascades during pathogenic development of Ustilago maydis. J. Kämper, M. Scherer, M. Vranes, C. Pothiratana. MPI for terrestrial Microbiology, Marburg, Germany

In the plant pathogen Ustilago maydis, the change from saprophytic growth to the biotrophic stage is controlled by a unique genetic switch, namely the bE and bW homeodomain proteins encoded by the b-mating type locus. Our aim is to understand the processes that lead to the establishment of the biotrophic stage. To this end, we have employed genome-wide DNA arrays for U. maydis. The arrays were used to depict the gene expression profiles of U. maydis cells in response to the activation of the bE/bW heterodimer in axenic culture, but also at early and late plant infection stages. For detailed analysis of the differentially regulated genes, we have focused so far on genes encoding proteins with potential regulatory functions. By this means we were able to identify three novel pathogenicity factors for U. maydis. The expression profiles and phenotypes of the respective mutants revealed that they are part of a network regulating pathogenicity and filamentous growth. One of the genes, rbf1, encodes a zink finger transcription factor that is required (and sufficient) for the expression of the majority of b-regulated genes, by that assigning a central role within the network. Among the rbf1 dependent genes are hdp1 and riz1, encoding a homeobox protein and a potential zink finger transcription factor. While hdp1 can be linked to filamentation, riz1 is required during the early infection stages.

285. Signalling cascades in Ustilago maydis : The use of functional genomics for the identification of downstream genes. R. Kahmann, T. Brefort, H. Eichhorn, F. Lessing, B. Winterberg, P. Mueller and A. Mendoza, Max Planck Institute for terrestrial Microbiology, Marburg, Germany

Ustilago maydis is a dimorphic fungus that switches from a yeast-like haploid stage to a filamentous dikaryon after mating. In nature it is the dikaryon that is able to differentiate infection structures and cause disease on corn plants. In this system cAMP signaling as well as two MAP kinase modules regulate discrete steps during pathogenesis. To identify downstream targets of these pathways we have applied molecular tools for functional genome analysis. In particular we have constructed strains where the individual pathways can be genetically activated and have followed changes in gene expression pattern using Affymetrix arrays. This has allowed us isolate novel genes whose products play crucial roles during pathogenesis. I will describe the identification of gene clusters involved in iron uptake and demonstrate that the reductive iron uptake system affects virulence. In addition, I will describe a novel HMG-domain transcriptional regulator of prf1 and how it fits into the regulatory network controlling signalling.

286. Molecular and Cellular Biology of Biotrophic Interactions in Rice Blast Disease. Prasanna Kankanala and Barbara Valent. Department of Plant Pathology, Kansas State University, Manhattan, KS 66503 USA.

The filamentous ascomycete fungus Magnaporthe grisea is the hemi-biotrophic pathogen that causes rice blast disease. We hypothesize that the fungal effectors play a dual role in establishing biotrophy and in R-gene mediated resistance. To identify candidate fungal effectors we are using the Zeiss Positioning Ablation Laser capture Microdissection (PALM) system to collect biotrophic invasive hyphae from the infected tissues. RNA extracted from these samples will be used to do microarray experiments and analyze the gene expression patterns in both resistant and susceptible interactions. To study the cellular changes during the biotrophic infection process we developed a transgenic fungal strain expressing EYFP constitutively under control of the fungal ribosomal promoter RP27. The initial plasmolysis experiments indicate that the host membrane is intact when the fungus grows inside the plant cell. To study the host membrane-pathogen interface in the first invaded plant cell, we are doing live-cell confocal microscopy with the FM4-64 fluorescent endocytotic tracer that labels the membranes. Our preliminary imaging indicates that the invasive fungal hyphae inside the host cells are surrounded by a very prominent membrane. Experiments are underway to determine the source and nature of this membrane.

287. The role of Botrytis cinerea endopolygalacturonases in pathogenesis: BcPG2 is the most important virulence factor. Ilona Kars, Lia Wagemakers, Geja Krooshof, Rob Joosten, Jac Benen and Jan van Kan. Lab. of Phytopathology, Wageningen University, The Netherlands

During infection of its host plants, B. cinerea secretes a considerable number of cell wall-degrading enzymes, among which are six polygalacturonases and two pectin methylesterases. The presence of multiple genes encoding CWDEs raises numerous questions about the exact function of such a set of enzymes. Several approaches were taken to understand how these pectinases are involved.

Mutants were generated in several BcPG and BcPME encoding genes by targeted mutagenesis. Virulence assays showed that elimination of Bcpg2 caused a reduction of virulence on different hosts, whereas elimination of Bcpg3, Bcpg5, Bcpme1 and Bcpme2 did not have an effect on the virulence of strain B05.10.

Five BcPGs were produced in P. pastoris, purified and characterized. Infiltration of BcPGs into plant tissue caused different symptoms. Especially BcPG2 was extremely potent in causing necrosis in a range of host plants. Other isozymes did not cause such severe symptoms. Symptoms differed per enzyme and per plant species. In broad bean BcPG2 very rapidly ( To distinguish cell death by maceration and plasmolysis from cell death by protein recognition, we conducted two types of experiments: 1) infiltration of inactive mutant protein (mutated in active site) produced in P. pastoris and 2) A. tumefaciens-mediated transient expression of active as well as inactive BcPGs. Results of this thesis work will be presented.

288. Characterization of the promoter region of hypoviral regulated fungal hydrophobin gene from Cryphonectria parasitica. Myoung-Ju Kim, Min-Jae Kim, Seung-Moon Park, Moon-Sik Yang and Dae-Hyuk Kim. Division of Biological Sciences, Basic Science Research Institute, Chonbuk National University, Dukjindong 664-14, Jeonju, Chonbuk 561-756, Korea

Cryparin is a cell wall associated fungal hydrophobin of chestnut blight fungus, Cryphonectria parasitica. Although the cryparin is encoded by a single copy gene (crp1), its expression is so strong that the amount of transcript can reach up to 22% of total mRNA. In addition, it is one of well-known examples of the transcriptional down-regulation of a fungal gene by hypovirus. To identify regulatory elements in crp1 promoter, transcriptional fusions of EGFP to various lengths of crp1 promoter were used to transform C. parasitica and EGFP activity of resulting transformants was compared in the presence and absence of hypovirus. Deletion of the promoter region between -1280 to -907 resulted in a drastic decrease of the promoter activity and the presence of corresponding fragment was required for the hypovirus-mediated down-regulation, which suggests that the 376 bp region is necessary for the high level expression of cryparin and hypoviral regulation. The mobility shift assay using the corresponding 367 bp region revealed the presence of cellular factor(s) of C. parasitica suggesting that it contains the cis-acting regulatory elements involved in the cryparin expression and hypovirus regulation.

289. Lipid-induced filamentation in Ustilago maydis. J. Klose and J.W. Kronstad. The Michael Smith Laboratories, Dept. of Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada

The transition from a yeast-like to a filamentous morphology in Ustilago maydis is regulated by conserved cAMP and MAPK signaling pathways and is correlated with virulence because only the filamentous cells can invade host plant and cause disease. We showed recently that lipids induce filamentation in U. maydis (Mol. Microbiol. 2004. 52:823-835). This may be relevant to infection of the plant because the components of the signaling networks are required for the dimorphic transition in response to lipids, and the morphological features of the lipid-induced filaments formed in vitro resemble those observed in planta. We are exploring the potential metabolic and signaling roles of lipids to further characterize the morphological transition. On the metabolic side, we have disrupted the mfe2 gene encoding the multifunctional enzyme involved in beta-oxidation of fatty acids. The strains defective in mfe2 did not grow on medium with oleic acid as the sole carbon source. Preliminary results indicate that the mutants respond morphologically to saturated but not to unsaturated fatty acids. The observations suggest possibility to separate utilization of fatty acids as a carbon source from the filamentation response and perhaps the saturation state of fatty acids may influence the dimorphic switch. The mfe2 gene was disrupted in strains of compatible mating type and the resulting mutants were able to mate. Subsequent inoculation of the compatible mutants into corn seedlings revealed that they were attenuated for virulence although weak disease symptoms were observed. The defect in virulence may result from a metabolic deficiency that prevents proper utilization of host nutrients and blocks extensive proliferation. Additional characterization of the mfe2 mutants is underway including examination of the fungal morphology during growth in planta.

290. Withdrawn.

291. Pathogenicity-specific domains in Ubc2, a pheromone-responsive adapter protein. Steven J. Klosterman, Alfredo D. Martinez-Espinoza, Jeffrey R. Seay, David L. Andrews, Scott E. Gold. Plant Pathology, University of Georgia, Athens.

The plant pathogenic fungus Ustilago maydis alternates between a haploid budding form and a dikaryotic filamentous form. Genes involved in the cAMP and MAPK pathways that control mating and morphogenesis have been identified previously by complementation of mutants that suppress a constitutively filamentous uac1 (Ustilago adenylate cyclase) haploid mutant. These genes were named ubc for Ustilago bypass of cyclase. The protein encoded by ubc2 is essential for virulence and possesses distinct protein interaction domains (N-terminal SAM, RA and two C-terminal SH3 domains). Ubc2, like other basidiomycete database orthologs, has two C-terminal SH3 domains while related ascomycete proteins are truncated and lack this region. Site-directed mutagenesis indicated that the N-terminal SAM and RA domains are necessary for mating and filamentous growth. Interestingly, C-terminal deletion mutants lacking the SH3 domains are still capable of mating and filamentous growth like the wild type but are nonpathogenic. The C-terminal domains of Ubc2 thus act as a basidiomycete-specific pathogenicity determinant. Yeast two-hybrid screens identified a number of proteins interacting with the various Ubc2 domains, including SAM-SAM heterodimer formation between Ubc2 and the pheromone-responsive Ubc4/Kpp4 MAPKK kinase. The other candidate proteins identified will be discussed.

292. Differentially expressed genes and host specifity of ectomycorrhizal fungi genus Tricholoma. Katrin Krause and Erika Kothe. Friedrich Schiller university, Jena Germany

RNA fingerprinting was carried out to identify genes showing differential expression in ectomycorrhiza between basidiomycete Tricholoma vaccinum and compatible host spruce Picea abies using ectomycorrhizal roots of different stages, pure roots and cultures of the fungus. Resulting PCR fragments were verified and the clones origin and expression pattern were checked. Of the fungal fragments with mycorrhiza-specific expression sequence analyses were performed to identify the nature of the encoded protein. Among them were genes with function in plant pathogen response, signal transduction, nutrient exchange, growth in plant and stress answer. Two genes an aldehyde dehydrogenase and a retrotransposon were investigated more intensively. Expression studies were performed with quantitative RT-PCR testing fungal mRNA of several aldehyde dehydrogenase substrates containing media, of stress inducible conditions, of different mycorrhizal stages, tissues and low compatible symbiotic interaction with pine Pinus sylvestris. The spreeding of the retrotransposon in genus Tricholoma was examined in 10 Tricholoma species also using ITS sequences, because it has a bit higher amino acid identity to RTase and RNase domains of retrotransposons of phytopathogenic ascomycetes than to MarYI of Tricholoma matsutake.

293. Identification and mapping of Pyrenophora teres f. teres genes conferring avirulence on barley. Lai, Z., Steffenson, B.J., Faris, J.D., Cartwright, R.D., Webster, R.K., Weiland, J.J., and Friesen, T.L. Plant Pathology, North Dakota State University

Pyrenophora teres f. teres is an ascomycetous fungus that causes net blotch of barley, a serious foliar disease throughout the world. A P. teres f. teres cross between parental isolates 0-1 and 15A yielded 78 single ascospore progeny. A molecular map consisting of 108 AFLP markers and 15 linkage groups was constructed. The linkage groups spanned a total genetic distance of approximately 909 cM. Electrophoretic karyotype analysis revealed a minimum of six chromosomes ranging from 1.8 Mb to 6.0 Mb with a minimum estimated genome size of 23 Mb. Net blotch infection response data were generated for this population by performing conidial inoculations of each progeny isolate on the four barley lines Canadian Lake Shore, Ming, Tifang, and Prato, which showed a differential response to parental isolates. The molecular map was used in conjunction with the phenotypic data to identify linkage groups containing loci associated with avirulence. Phenotype data from each of the four barley lines indicate that avirulence is controlled by major genes in this population and that these avirulence/virulence genes are linked.

294. Fungal tetraspanins: key players in host plant invasion. Lambou K., Fargeix C., Catusse J., Gourgues M., Cottier F. and Lebrun M.H.. FRE2579 CNRS / Bayer cropscience Physiologie des plantes et des champignons. 14-20 rue Pierre Baizet 69263 Lyon cedex 09 (France)

The non-pathogenic mutant punchless from the rice blast fungus Magnaporthe grisea is unable to invade its host leaves. This invasion process requires the differentiation of a fungal cell, the appressorium, specialized in the penetration of the pathogen into host leaves. The mutant punchless differentiates appressoria that are non-functional, as they cannot direct the penetration of the fungus into host leaves. The gene inactivated in this mutant encodes a putative integral membrane protein of 225 AA (Pls1) that exhibits classical features of animal tetraspanins. Genes orthologous to PLS1 were identified in other fungi such as Botrytis cinerea, Colletotrichum lindemuthianum, Fusarium graminearum and Neurospora crassa defining a new tetraspanin family with highly conserved domains. Deletion of PLS1 orthologs in the plant pathogenic fungi B. cinerea (C. Levis, A. Simon, PMDV, INRA, France) and C. lindemuthianum (C. Veneault, D. Parisot, R. Lauge, T. Langin, IBP, CNRS-UPS-INRA, France) leads to non-pathogenic mutants. These mutants have the same defect as M. grisea PLS1 deletion mutant, as they differentiate appressoria unable to direct fungal penetration into host plants. These results suggest that fungal tetraspanins control a conserved appressorial function essential for the penetration of fungi into host leaves. Amino acids from domains conserved among fungal tetraspanins were modified by site-directed mutagenesis in M. grisea Pls1. The functionality of these mutant proteins was assessed by complementation of the M. grisea non-pathogenic PLS1 deletion mutant. The four cysteines from Pls1 ECL2 and the C-terminus tail are required for Pls1 function. Pls1 is only expressed in appressoria and its Pls1-GFP fusion protein is mainly localized in the vacuoles of this fungal cell. Comparison of genome wide expression profiles of wild type and PLS1 deletion mutant appressoria are currently used to identify the downstream targets of Pls1.

295. Involvement of autophagy in the appressorium functionality of Colletotrichum lindemuthianum. Eugénie Bard, Richard Laugé et Thierry Langin. Phytopathologie Moléculaire, Institut de Biotechnologie des Plantes (UMR-CNRS 8618), Bât 630, Université Paris-Sud, 91405, Orsay, France.

Colletotrichum lindemuthianum is the causal agent of the anthracnose disease on common bean, Phaseolus vulgaris. This fungus attacks all green parts of the plant ending in necrosis of infected tissues. Previous analyses of non pathogenic mutants obtained through insertional mutagenesis allowed the identification of a mutant that differentiates mature appressoria but is unable to penetrate the plant tissue. A mutation in the clk1 gene (Dufresne et al., MPMI (1998), 11, 99-108), that encodes a Sérine/Threonine kinase, is responsible for the observed phenotype. clk1 is the orthologous gene of the Saccharomyces cerevisiae atg1 gene, that triggers the autophagy process under starvation conditions. Functional homology between clk1 and atg1 was demonstrated via the complementation of yeast atg1- mutants. In order to demonstrate the involvement of autophagy in the functionality of C. lindemuthianum appressoria, clapg2 (C. lindemuthianum autophagy-2) the orthologue of the yeast atg8 gene, that is compulsory for the formation of the yeast autophagosomes, was cloned. The clapg2 gene is induced in vitro under nitrogen starvation as expected. Furthermore, upon plant inoculation, clapg2 is also transiently induced during appressoria development. Taken together, these results suggest a role for autophagy in the mobilization of cell ressources for appressorium functionality. Nul mutants (clapg2::hph) are currently under construction to validate this hypothesis.

296. Transcriptional regulation during the infection process of Colletotrichum lindemuthianum. Marcos Soares, Eugénie Bard, Anne-Laure Pellier, Richard Laugé et Thierry Langin. Phytopathologie Moléculaire, Institut de Biotechnologie des Plantes (UMR-CNRS 8618), Bât 630, Université Paris-Sud, 91405, Orsay, France.

Colletotrichum lindemuthianum is a hemibiotrophic fungus that causes anthracnose disease on common bean. It develops along its infection cycle a strict succession of specialized cell structures: appressorium, infection vesicle, primary hyphae, and secondary hyphae that coincidate with the succession of the penetration, biotrophy and necrotrophy phases. Such a life cycle certainly requires precise genetic programs to be fulfilled in order to accomplish developmental and metabolic modifications/adjustments. Two alternative strategies are developed in order to identify transcriptional activators involved in the control of this complex process. The molecular analysis of non-pathogenic C. lindemuthianum mutants led to the identification of clta1 a Zn finger transcriptional activator that controls the transition between biotrophy and necrotrophy (Dufresne et al., Plant Cell, 2000). Putative targets giving clues about its involvement in development (hyphal transition) and/or metabolism (shift of trophic mode) are currently searched directly by substractive hybridization. clnr1, the areA/NIT-2 orthologue, was cloned and demonstrated to be the global nitrogen regulator of C. lindemuthianum. clnr1- mutants are non pathogenic but surprisingly impaired at the beginning of necrotrophy (Pellier et al., Molecular Microbiology, 2003). We have also cloned the orthologue of the pacC gene. Its role in vitro is currently studied, and the construction of null mutants is on the way in order to evaluate the role of pH regulation during the infection. In parallel, an EST project provided us with many genes exhibiting typical domains of transcriptional activators. We plan to test more broadly a number of them for involvement in the infection process of the fungus.

297. Virulence gene discovery in the Brassica pathogen, Alternaria brassicicola using functional genomics. Yangrae Cho 1, Josh Davis 1, Carlos Mauricio La Rota 1, Robert Cramer2, Christopher Lawrence1. 1 Virginia Bioinformatics Institute, Blacksburg, VA. 2 Duke University Medical Center, Durham, NC.

Mitogen-activated protein (MAP) kinases have been shown to be involved in signal transduction cascades required for virulence in several pathogenic fungi. We cloned the yeast fus3 homolog (AMK1) in A. brassicicola, a necrotrophic fungal pathogen of cultivated Brassicas. Knock out (KO) mutants showed a complete loss of pathogenicity on three Brassica species and two Arabidopsis ecotypes. The mutants demonstrated partially restored pathogenicity, as determined by measuring lesion diameters, compared to the wild type (WT) fungus only when leaves were wounded prior to inoculation. Transcription patterns were compared between WT and mutants for seven genes encoding secreted hydrolytic enzymes and three putative toxic proteins using semi-quantitative RT-PCR. Mutants showed elevated expression of many of the secreted hydrolytic enzyme and toxin genes compared to WT on wounded leaves, but were expressed at low levels compared to WT on intact leaves. This suggests that signals derived from wounded tissues may activate downstream components of the pathway with important roles in pathogenicity. To find additional infection-related fungal genes, ESTs from a full-length cDNA library constructed from RNA isolated from infected rapeseed were characterized. We found numerous candidate pathogenicity genes from sequencing over 3,000 ESTs thus far. We have KOÕd three secreted hydrolytic enzyme genes, one zinc finger transcription factor, and two signaling pathway genes and observed a reduction in pathogenicity in several cases. We will present gene annotation of sequences derived from the infected rapeseed library and phenotype information for each of the six initial KO mutants.

298. Two putative isocitrate lyase genes (GzICL1 and GzICL2) are required for virulence and sexual development in Gibberella zeae. Seung-Ho Lee1, Sung-Hwan Yun2, and Yin-Won Lee1. 1School of Agricultural Biotechnology, Seoul National University, Seoul 151-921; 2Division of Life Sciences, Soonchunhyang University, Asan, 336-745, Korea

Isocitrate lyase (ICL) is one of two enzymes consisting of the glyoxylate pathway that are involved in the metabolism of two-carbon compounds such as acetate. Recent studies on Leptosphaeria maculans and Magnaporthe grisea revealed that the ICL genes were essential for disease development by these phytopathogenic fungi. To elucidate the function of ICL in the cereal head blight fungus Gibberella zeae, we identified two orthologs of the ICL gene, designated GzICL1 and GzICL2 from the G. zeae genome database. Transgenic strains of G. zeae deleted for either of two GzICL (designated delGzICL1 and delGzICL2, respectively), or for both (delGzdIl) were generated using a gene replacement strategy. Transgenic delGzICL1 strains were normal compared with its wild-type progenitor except ascospore formation; they produced fewer perithecia, when selfed. In contrast, delGzICL2 produced fertile perithecia as many as wild-type, but were slower in hyphal growth on medium containing 0.25% glucose or C12 fatty acid (0.25% Tween 20). For virulence on barley heads, both delGzICL1 and delGzICL2 caused disease symptoms as severe as wild-type. Interestingly, the delGzdIl mutants showed significantly reduced virulence on host plant; they produced no perithecia on mating plates. These results strongly suggest that both GzICL1 and GzICL2 genes are required for virulence as well as sexual development in G. zeae.

299. Fungal peroxisomes in the Stagonospora nodorum / wheat interaction. Robert Lee, Kasia Rybak, Peter Solomon and Richard Oliver. Australian Centre for Necrotrophic Fungal Pathogens, Murdoch University, Perth, WA, Australia.

Peroxisomes contain biochemical pathways that are important for the growth and development of fungi. Enzymes for methanol oxidation and peroxide dissimilation, the fatty acid beta-oxidation pathway and the glyoxylate cycle are located in peroxisomes. Peroxisomes have been recently reported to be essential for appressorium development and spore germination in fungal phytopathogens and this paper describes our recent work to further investigate the roles of peroxisomes in fungal pathogenesis. A S. nodorum strain with peroxisomally-targeted GFP was prepared by transformation with a GFP expression construct encoding a COOH-terminal peroxisomal targeting signal. Peroxisome proliferation and increased GFP fluorescence were observed with addition of methanol or oleate to culture media and peroxisomes were observed throughout surface hyphae in infected wheat leaves but not in hyphae growing inside leaf tissue. GFP-containing peroxisomes in a malate synthase-deficient mutant (mls1-8) exhibited a 30-fold increase in fluorescence. A functional analysis study of genes encoding peroxisomal proteins is also underway. A cDNA library prepared from oleic acid-grown S. nodorum was screened with a SSH probe enriched in genes expressed in oleate media. Several genes that encode enzymes from known pathways were identified, along with others that may have novel functions in fungal peroxisomes. Progress in the characterization of a number of these genes will be discussed.

300. QTLs for pathogenicity on Pinus silvestris located in a Heterobasidion annosum genetic linkage map. Mårten Lind, Åke Olson and Jan Stenlid. Swedish University of Agricultural Sciences, Uppsala, Sweden

The basidiomycete Heterobasidion annosum is the most devastating fungal pathogen on conifers in the world. Its intersterility groups S and P are named after host preference (spruce and pine). Using a mapping population of 102 single spore isolates, originating from a compatible mating between North American isolates of the P and S groups, a genetic linkage map of the Heterobasidion annosum genome was constructed. The map consists of 39 linkage groups and spans 2252 cM in total. The average distance between two markers is 6.0 cM.

In order to map QTLs for pathogenicity, two methods were used. First, 29 two weeks old Pinus silvestris seedlings were grown in homogenized mycelia for 25 days. Every third day the number of dead seedlings were estimated. The virulence was determined as the regression value of the disease increase rate for each isolate. The data suggested a QTL on linkage group 11 with a LOD of 3.09, explaining 16.4% of the variation in virulence.

Second, ten plants of one year old P. silvestris was infected with a fungal infested wooden plug in a wound in the cambium. After four weeks the necrosis was measured upstem and downstem from the cambial wound. The virulence was determined as mean necrosis length for each isolate. The data suggested one QTL on linkage groups 15 and one on group 20, with peak LOD values of 3.29 and 4.24, explaining 15.8% and 18.2% of the variation in virulence, respectively.

These QTLs will be identified and characterised in future studies.

This project is funded by The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, FORMAS.

301. The role of alternative carbon metabolism in Candida-phagocyte interactions. M. Ramirez1, J. Bender2, G. Fink2, and M. Lorenz1. 1 Dept. of Microbiology, Univ. of Texas Health Science Center, Houston 2 Whitehead Inst., Cambridge, MA

Candida albicans occupies a unique niche as both a ubiquitous commensal and common pathogen of humans. Because the most serious infections result from defects in innate immunity, we have studied the interaction between C. albicans and phagocytes using genomic microarrays. Phagocytosis of C. albicans by murine macrophages stimulates a massive response involving nearly 10% of the genome, including altered carbon utilization, translation repression, and stress responses. We are focusing on the role of alternative carbon utilization and have found that the glyoxylate cycle, used to assimilate simple carbon compounds like acetate, is upregulated upon phagocytosis and that it is required for full virulence in a mouse model. Preliminary evidence suggests that fatty acid beta-oxidation (the product of which enters the glyoxylate cycle) is absolutely required in vivo as fox2 mutants are completely avirulent. In addition, the regulation of beta-oxidation and the glyoxylate cycle appears to be linked in C. albicans, unlike in S. cerevisiae, suggesting a tight functional link in the pathogen. The importance of this pathway is further reinforced by the induction by macrophage contact of several genes implicated in acetate homeostasis. Taken together, these results indicate that the ability to recognize and adapt to carbon sources that are poor in both quantity and quality is a key component of the in vivo success of C. albicans.

302. Functional complementation and comparative expression analysis of CPS1, a common virulence determinant in filamentous ascomycetes. Shun-Wen Lu and B. Gillian Turgeon, Department of Plant Pathology, Cornell University, Ithaca, NY 14853.

Ascomycetes share a highly conserved acyl-AMP ligase-like protein (CPS1), originally identified in the maize pathogen, Cochliobolus heterostrophus (Chet). We have previously demonstrated that CPS1 is required by diverse plant pathogens for virulence on their hosts. Towards further understanding of the biological and biochemical nature of CPS1, we have: 1) Complemented a reduced virulence, Chet CPS1-deletion strain (deltaChcps1), with a cloned native ChCPS1 gene. 2) Expressed ChCPS1 orthologs from the wheat scab fungus, Gibberella zeae (GzCPS1) and the saprobe, Neurospora crassa (NcCPS1) in deltaChcps1. 3) Examined ChCPS1, GzCPS1, and NcCPS1 in vitro expression patterns. Preliminary results indicate that: 1) The native ChCPS1, and the heterologous GzCPS1 and NcCPS1 genes all restore wild-type virulence capability to the deltaChcps1 strain. 2) CPS1 is expressed in a species- and culture condition-dependent manner, and there appears to be more than one type of transcript. One transcript is expressed differently in the pathogen G. zeae compared to the closely related saprobe, N. crassa. These results suggest that CPS1 function is likely the same in saprobes and pathogens and that this function is also required for the pathogenicity niche. Although the CPS1-mediated virulence mechanism is unclear, morphological abnormalities, observed when cps1- mutants of C. heterostrophus colonize the host, and CPS1 expression data suggest that it might be important for adaptation to stress conditions, including those derived from host defense during fungal infection.

303. Ptr ToxA: Hitting where it hurts. Viola A. Manning1, Ganapathy N. Sarma2, P. Andrew Karplus2, Lynda M. Ciuffetti1. 1Department of Botany and Plant Pathology, 2Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA.

Ptr ToxA, the first proteinaceous, host-selective toxin isolated, is produced by the fungus Pyrenophora tritici-repentis and induces necrosis in sensitive but not insensitive wheat cultivars. Understanding the site-of-action of ToxA and how its structure impacts symptom development will provide insight to formulating controls for tan spot of wheat. Several lines of evidence including immunolocalization, GFP-ToxA localization and immunoprecipitation suggest that the difference between ToxA-insensitive and -sensitive cultivars lies in the ability of the toxin to be internalized. Results suggest that toxin is internalized only in sensitive cultivars. Biolistics indicate that if toxin could be internalized in insensitive wheat, cell death would result. ToxA appears to localize to the cytoplasm and chloroplast following internalization. A single clone isolated from a yeast two-hybrid screen of a library produced from a ToxA-sensitive wheat cultivar supports chloroplast localization. Crystal structure analysis of ToxA has confirmed a putative protein-protein interaction interface, a vitronectin-like sequence that contains an RGD cell attachment motif, previously identified via site-directed mutagenesis. Whether this domain is necessary for internalization, for protein-protein interactions after toxin enters the cell, or both, is currently under investigation.

304. Chromosomal location and expression of ftf1 and ftf2 genes in pathogenic strains of F. oxysporum. N. Martín-Rodrigues, B. Núñez-Corcuera, B. Ramos, M.A. García-Sánchez, , A.P. Eslava and J.M. Díaz-Mínguez. Área de Genética. Centro Hispano-Luso de investigaciones Agrarias (CIALE). Universidad de Salamanca. 37007, Salamanca, Spain.

We have described two novel Fusarium transcription factors: ftf1 and ftf2, wich show a high nucleotide and aminoacidic homology but very different genomic architecture and expression. Both genes contain an ORF of 3240 nt including a 49 bp intron. The predicted 1080 amino acid protein contains a Zn(II)-Cys6 binuclear cluster DNA-binding motif with homology to various fungal regulatory proteins. The gene ftf1 is specifically expressed on early stages of infection, while ftf2 shows a very low level of expression in vivo and in vitro.

We have analyzed the presence and expression of both genes in 14 formae speciales of F. oxysporum. The results mostly confirm those previously obtained in F. oxysporum f.sp. phaseoli. Chromosome hibridizations show the presence of ftf2 in all the formae speciales and several copies of ftf1 in different chromosomes. We have also analyzed the presence of transposon marsu and the homologue of bimB3 (Aspergillus nidulans). RT-PCR analyses of expression using primers specific for each gene show that ftf2 is expressed in vitro in all the formae speciales.

305. Spatial and Temporal In Planta Expression of a Bioprotective Metabolite Gene from Epichloë festucae using the GUS Reporter Gene. Kimberley May, Michelle Bryant, Xiuwen Zhang, Barbara Ambrose and Barry Scott. Center for Functional Genomics, IMBS, Massey University, Palmerston North, New Zealand

Fungal endophytic species of the genera Epichloë and Neotyphodium form mutualistic symbioses with cool season grasses of the family Pooideae. Isolates of E. festucae and N. lolii provide protection to their host against grazing animals through the production of lolitrem alkaloids. A cluster of genes involved in the biosynthesis of lolitrems (ltm genes) have been isolated and characterised from N. lolii and E. festucae. To gain an understanding of where and when these genes are expressed in planta, several PltmM-uidA constructs were prepared and transformed into E. festucae. Associations between perennial ryegrass (Lolium perenne) and these transformants were established and GUS expression analysed. Expression of GUS was observed in vegetative tillers but not in the roots. In the spikelets, expression was initially observed in the rachis, glumes, lemma, palea and rachilla but not in the male or female reproductive organs. However, in later stages, GUS expression appeared to occur largely in the female reproductive organs with little expression observed in the lemma, palea or the male reproductive organs. Future work will involve an analysis of GUS expression patterns in germinating seed and young seedlings.

306. Genetic and functional analyses of a 96 kb region harboring a putative avirulence locus from the fungal wheat pathogen Mycosphaerella graminicola. Rahim Mehrabi, Odette Mendes, Cees Waalwijk, Theo van der Lee and Gert H. J. Kema. Wageningen University and Research Centre, Plant Research International B.V., P. O. Box 16, 6700 AA Wageningen, The Netherlands.

Segregation analyses in a mapping population of a cross between two Dutch field isolates, IPO323 (avirulent) and IPO94269 (virulent) of the wheat pathogen Mycosphaerella graminicola demonstrated that avirulence in IPO323 is controlled by a single locus. We generated BAC libraries from both isolates and constructed BAC contigs of each isolate that include all markers cosegregating with avirulence. Sequence analysis of this 96 kb region predicted over 100 ORFs from which we selected two candidate avirulence genes for further analyses. The first candidate, designated ORF43, was a small cystine rich gene expressed in planta, with no homology to the public DNA databases and was partly deleted in the virulent parent. Targeted disruption of ORF43 in the avirulent isolate did not affect the phenotype indicating this gene is not an avirulence factor. The second candidate is located about 10 kb downstream of ORF43 and has strong homology to polyketide synthases. Disruption of this gene in both isolates did not affect the phenotype indicating that this gene is neither an avirulence nor a virulence factor. To identify the avirulence gene in this region we are currently swapping larger BAC derived DNA fragments between both IPO323 and IPO94269 and expect to be more successful with this non biased approach.

307. Mlt2, a MAP kinase of Mycosphaerella graminicola involved in cell wall integrity, is dispensable for penetration but is essential for invasive growth. Rahim Mehrabi, Cees Waalwijk, Theo van der Lee, and Gert H. J. Kema. Wageningen University and Research Centre, Plant Research International B.V., P. O. Box 16, 6700 AA Wageningen, The Netherlands.

Through analyses of EST libraries of Mycosphaerella graminicola, we identified a full-length cDNA clone with high homology to a mitogen-activated protein (MAP kinase), Slt2 in Saccharomyces cerevisiae. This MAP kinase, designated mlt2, possesses a 1242 bp open reading frame, and encodes a 414aa protein. We generated knock-outs of this gene and identified several altered phenotypes in vitro as well as in planta. On yeast glucose broth, upon aging, mlt2 disruptants show a defect in polarized growth in the tip cells causing an enlarged tip and a swollen cell shape with two or four nuclei showing that nuclear division occurs without de novo cell wall synthesis. Mlt2 mutants are hypersensitive to glucanase, indicating that the composition of the cell wall is affected by disruption of mlt2. Unlike the wild type, mutants do not produce aerial mycelium and do not melanize on PDA. The mlt2 disruptants are reduced in virulence and cannot develop regular septoria tritici symptoms as they are unable to produce pycnidia. Cytological analysis of the disruptants in planta shows normal penetration of the germ tubes but subsequent branching and invasive growth are significantly hampered.

308. Distinct regulation and function of the thioredoxin proteins in Cryptococcus neoformans for oxidative and nitrosative stress or virulence. Tricia A. Missall1 and Jennifer K. Lodge1,2. 1Department of Biochemistry and Molecular Biology and 2Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine

The thioredoxin system, consisting of thioredoxin, thioredoxin reductase, and NADPH, is known to protect cells against oxidative stress. This disulfide reducing system is present in Cryptococcus neoformans and consists of two small, dithiol thioredoxin proteins and one thioredoxin reductase. In this study, we describe the thioredoxin proteins, Trx1 and Trx2, and present their importance not only to stress resistance, but also to the virulence of C. neoformans. Using real time PCR, we show the induction of TRX1 during oxidative stress and both thioredoxin genes during nitrosative stress. We show through deletion studies that the trx1delta mutant has a severe growth defect and is sensitive to oxidative and nitrosative stress, while the trx2delta mutant is only sensitive to nitric oxide stress. Using gene replacement studies, we demonstrate that the thioredoxin protein products are redundant in function and there is differential gene regulation which is important to nitrosative stress resistance. We have also identified two putative transcription factors, Atf1 and Yap4, which appear to differentially regulate the thioredoxin system under different conditions. Atf1 is necessary for oxidative stress induction and Yap4 is necessary for nitrosative stress induction of the thioredoxin genes in C. neoformans.

309. New functional genomic resource for Candida albicans. Aaron P. Mitchell, Qi Zhao, Frank J. Smith, and William C. Nierman. Microbiology Department, Columbia University, NYC, NY, and The Institute for Genomic Research, Rockville, MD

C. albicans, the most common human fungal pathogen, causes infections from oral and vaginal candidiasis to life-threatening invasive disease. We are using the recently published genomic sequence to identify virulence-related traits through insertional mutagenesis. We constructed a genomic C. albicans library, mutagenized the library with a Tn7-UAU1 transposon, and sequenced insertion sites to identify insertions within annotated ORFs. Among 32448 sequenced insertion sites, flanking sequences from 22199 clones have been mapped to 380 Candida albicans genomic contigs (Stanford release 19, with a total of 413 contigs) based on 85% length match, at least 85% identity, and the clone itself being at least 300 bp long. Within this group, we find insertions in 4170 (or 65.5%) of the 6362 unique ORFs defined by the NRCC annotation. A searchable insertion site database is available at

 We have examined a small group of insertion-bearing plasmids in detail to assess the feasibility of large-scale mutagenesis. We verified the insertion site sequences and determined endpoints of genomic DNA clones. We are currently transforming the pilot group of insertions into C. albicans to create heterozygotes, and will carry out UAU1-based selection to create homozygous insertion mutants.

 We view this project as an important contribution to the C. albicans scientific community. In a broader sense, it serves as one model for cost-effective post-genomic functional analysis.

310. Matrix-Assisted Laser Desorption/Ionization Time-of Flight (MALDI-TOF)-based cloning of a MEKK like protein, CBCK1, from Cryphonectria parasitica. Ae-Young Mo, Eun-Sil Choi, Myoung-Ju Kim, Jie-Hye Kim, Seung-Moon Park, and Dae-Hyuk Kim. Division of Biological Sciences, Basic Science Research Institute, Chonbuk National University, Dukjindong 664-14, Jeonju,Chonbuk 561-756, Korea

On the foundation of a database of genome sequences and protein analyses, the ability to clone a gene based on a peptide analysis is becoming more feasible and effective for identifying a specific gene and its protein product of interest. As such, the current study conducted a protein analysis using 2-D PAGE followed by MALDI-TOF and ESI-MS to identify a highly expressed and phosphorylated protein spot. A highly phosphorylated protein spot with a molecular size of 44 kDa was selected and MALDI-TOF MS analysis was conducted. A homology search indicated that the protein appeared to be a fungal Bck1, which is a MEKK. Meanwhile, multiple alignments of fungal MEKK revealed a conserved amino acid sequence, from which degenerated primers were designed. Using the degenerate primers, we amplified and cloned a DNA fragment with a size of 369 bp. Sequence analysis of the clone revealed that it has the highest homology to the Bck1 like MEKK gene of Podospora anserina. Molecular characteristics of a gene encoding Bck1, cbck1, suggest that the cbck1 encodes the Bck1 homologue of C. parasitica and the functional analysis of the cbck1 is under performing.

311. Characterization of a yap1-related gene in Ustilago maydis. Lázaro Molina and Regine Kahmann. Max-Planck-Institut für terrestrische Mikrobiologie, 35043-Marburg, Germany

One of the most effective plant defense responses to pathogen attack is the production of reactive oxygen species (ROS). However, it is presently unknown how plant pathogenic fungi cope with these responses during a successful infection. Therefore, we became interested in studying ROS signalling in Ustilago maydis. In Saccharomyces cerevisiae has been extensively studied this signalling pathway and it has been shown that YAP1 serves as central regulator for ROS signalling. U. maydis contains one yap1 related gene. To analyse its role during pathogenic development, knockout mutants were generated in such way that the yap1 ORF was substituted by the eGFP. In addition, yap1:eGFP fusions were constructed to localize the Yap1 protein during U. maydis development. The obtained mutants were significantly more sensitive to H2O2 than respective wild type strains and produced a dark pigment whose nature is currently unknown. This could indicate that under conditions where yap1-regulated genes are no longer expressed U. maydis is able to switch on the synthesis of other protectants. Compatible yap1 knockout strains were able to mate, produced dikaryotic hyphae, invaded plants and induced disease symptoms. However, compared to the respective wild type strains virulence, of the yap1 mutant strains was significantly attenuated. This was reflected by reduced number of tumors as well as reduced tumor size. These results indicate that yap1 controls genes which allow U. maydis to cope efficiently with the plant environment. We are currently establishing which genes are regulated by yap1 by microarray analysis. We will present this data and relate this to when and where Yap1 is active.

312. Mycotoxin production and fungal gene expression during infection of crowns and heads of wheat by Fusarium graminearum. A.M. Mudge, R. Dill-Macky*, Yan Hong*, I.Wilson and J.M. Manners. CSIRO Plant Industry, Queensland Bioscience Precinct, Brisbane 4072 and Canberra 2601, Australia; *Department of Plant Pathology, University of Minnesota, St. Paul, USA.

Fusarium graminearum (Fg) causes both Fusarium Head Blight (FHB) and Crown Rot (CR) of wheat in Australia. The mycotoxin deoxynivalenol (DON) has been reported to be important as a pathogenicity factor and grain contaminant in FHB but its role in CR is unknown. Following crown inoculation, hygromycin-resistant fungus was recovered from 100% of crowns, 85% of flag leaf nodes (FLN) and 55% of heads. Mycotoxin assays detected DON levels of 275 ppm in crowns, 14 ppm in FLN and 7 ppm in heads. Expression of Tri5 was strongly induced during crown infection. Results indicate that crown rot of wheat is associated with systemic fungal colonisation and DON production. To compare Fg gene expression in FHB and CR, a micro-array was printed that included 2300 clones from a subtracted FHB cDNA library and comparisons of gene expression were made between infected versus uninfected crowns and heads as well as to axenic cultured mycelium. The expression (normalised to fungal rRNA) of 48 selected infection-related fungal genes was then tested using qRT-PCR and 33 of these were validated as up-regulated during infection relative to their expression in cultured mycelium. Most were up-regulated in both diseases but 1 and 4 transcripts were only up-regulated in FHB and CR respectively. Most infection-related fungal genes were of unknown function.

313. Biological control of canegrubs, a proteomic approach. Helena Nevalainen1,2, Nirupama Bhikballapur1,2, Kathy Braithwaite3, Stevens Brumbley3, Peter Samson4 and Junior Te'o1,2. 1Department of Biological Sciences, Macquarie University, Sydney, NSW, Australia. 2Macquarie University Biotechnology Research Institute, Sydney, NSW, Australia. 3BSES Limited, Indooroopilly, Qld, Australia. 4BSES Limited, Mackay, Qld, Australia

The insect pathogenic fungus Metarhizium anisopliae is being developed as a biocontrol agent. However, attempts to discern the determinants of the infection process and produce a rational plan for strain improvement have been thwarted by the complexity of host-target related signals. Therefore, broad strategies are needed to pursue the functional genome of M. anisopliae. This study focuses on the identification of key proteins during infection of sugarcane whitegrub, the most serious insect pests of sugarcane in Australia by M. anisopliae var. anisopliae.

The experimental setup is designed to mimic the infection process in the field. Proteins were extracted from samples representing healthy and infected canegrubs, healthy and infected insect cuticles and fungal mycelia. The extracted proteins were separated by 1DE and 2DE and samples assessed by comparative analysis in order to identify the key proteins involved in pathogenicity. As an example, the protein profile from infected insect cuticles displayed a notable absence of higher molecular weight proteins present in non-infected cuticles. Further comparative analysis of the protein profiles will be presented with some identifications of unique "signatory" proteins for each condition.

314. Alpha cells predominate in the mammalian central nervous system during Cryptococcus neoformans mixed mating type infections. Kirsten Nielsen and Joseph Heitman. Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC

Cryptococcus neoformans is an opportunistic human pathogen that infects the central nervous system to cause meningitis that is uniformly fatal if untreated. This basidiomycete has evolved over the past 40 million years into three distinct varieties or sibling species (grubii, gattii, and neoformans). The three Cryptococcus varieties have different disease epidemiologies with var. grubii producing 95% of human disease. C. neoformans is a heterothallic fungus with two mating types - a and alpha. Interestingly, the vast majority of clinical isolates are alpha mating type with a strains accounting for only a limited proportion of isolates. It is unclear why alpha strains predominate in clinical samples because our studies with var. grubii congenic strains showed that a and alpha strains have equivalent virulence in cellular (macrophage), heterologous host (amoeba, nematode), and mammalian (mouse, rabbit) models of cryptococcosis. Humans are thought to be exposed to C. neoformans via inhalation of small yeast cells or spores - resulting in an initial pulmonary infection that hematogenously disseminates to the central nervous system in immunocompromised individuals. We have shown that a and alpha strains cause disease in a similar fashion when infected individually. In contrast, coinfection studies with the a and alpha congenic strains revealed equivalent levels of a and alpha cells in the lungs but a significantly higher proportion of alpha cells infecting the brain. Thus, the alpha strain out-competes the a strain during a mixed infection of the central nervous system. These results provide an explanation for why alpha strains are the predominant clinical isolates from human cerebrospinal fluid, lay the foundation for detailed studies on the interaction of a and alpha strains in vivo, and provide a mechanism to define virulence characteristics important for central nervous system infection.

315. Linkage between Mitochondrial Hypovirulence and Viral Hypovirulence in the Chestnut Blight Fungus, Cryphonectria parasitica, revealed by cDNA Microarray Analysis. Donald L. Nuss and Todd D. Allen, Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD 20742.

The phenomenon of transmissible hypovirulence (virulence attenuation) associated with biological control of natural populations of the chestnut blight fungus Cryphonectria parasitica can be experimentally reproduced by infection with hypovirus cDNA clones (viral hypovirulence) or by mutation of mitochondrial (mt) DNA in the absence of virus infection (mitochondrial hypovirulence). We now report the use of an established C. parasitica cDNA microarray to monitor nuclear transcriptional responses to a mtDNA mutation of C. parasitica strain EP155, designated EP155/mit2, previously shown to induce elevated alternative oxidase activity and hypovirulence [MonterioVitorello, C. B., Bell, J. A., Fulbright, D. W. & Bertrand, H.A. (1995) Proc. Natl. Acad. Sci. 92,5935-5939]. Approximately 10% of the 2,200 genes represented on the microarray exhibited altered transcript accumulation as a result of the mit2 mtDNA mutation. While genes involved in mitochondrial function were clearly represented in the EP155/mit2 responsive gene list, direct parallels to the well-characterized Saccharomyces cerevisiae retrograde response to mitochondrial dysfunction were not observed. Remarkably, 47% of the genes that were differentially expressed following infection of strain EP155 by the prototypic hypovirus CHV1-EP713 were similarly changed in transcript accumulation in virus-free EP155/mit2. These results establish a linkage between viral and mitochondrial hypovirulence and raise questions regarding the relationship between hypovirus infection and mitochondrial dysfunction. The results are discussed in terms of mitochondria-to-nuclear communications in the context of hypovirus infection and fungal pathogenesis.

316. Metabolic requirements for pathogenicity. RP Oliver, R Lowe, PS Solomon, R Trengove, R Lee, O Waters, J Rechberger. Australian Centre for Necrotrophic Fungal Pathogens

The ability of a fungal pathogen to acquire nutrients from its plant host is a key factor in the success of its lifecycle. An understanding of these processes may help us design new strategies to combat pathogens. Our current knowledge of this subject is limited and in many cases established dogma has remained unchallenged.

We study the wheat pathogen Stagonospora nodorum, by combining a study of specific gene knockouts with metabolomic analyses. GC-MS analyses detect 100-200 individual compounds on both in vitro and in planta samples.

Knockouts of malate synthase (MS), glyoxalase, a peptide transporter and mannitol 1-phosphate dehydrogenase (MPD)have been studied. Deletion of the peptide transporter had no obvious phenotypic consequences indicating that peptides do not constitute a significant nutrient source in planta. Likewise, deletion of the glyoxalase gave no pathophenotype but did result in strains that were less tolerant of methylglyoxal, the presumed substrate. Deletion of the MPD gene gave strains that were still capable of producing lesions but had reduced ability to sporulate in planta. This indicate that mannitol metabolism may have a role in ameliorating the stress produced by senescing leaves and thereby promoting sporulation. MS mutants were completely non-pathogenic as they were incapable of germinating on the leaf (although glucose could restore this), indicating that gluconeogenesis is critical in the early phases of infection.

This work is supported by the Australian Grains Research and Development Corporation

317. Effects of a Plant-Pathogen Interaction on the Fungal Endophyte Community. Jean J. Pan1 and Georgiana May1,2. 1Department of Ecology, Evolution, & Behavior, 2Department of Plant Biology, University of Minnesota, Saint Paul, MN 55108, USA

Humans have been modifying plant traits directly through breeding or indirectly through changes in the environment. Changes in plant traits affect not only plant fitness but can also affect microbes that interact with the plants. We investigated the effects of host plant variation for pathogen resistance and the effects of pathogen infection on the fungal endophyte community in the Zea mays-Ustilago maydis (corn-corn smut) system. We used a culture-independent approach to assess the fungal endophyte community on three recombinant inbred lines (RILs) of corn resistant to corn smut and three RILs susceptible to corn smut. We found that neither host trait variation or smut infection had consistent effects on endophyte species richness and species diversity. Instead, effects of smut infection seemed to vary with RIL, with three RILs having greater endophyte species richness and species diversity when infected by corn smut. Data on the community composition of fungal endophytes in relation to host trait variation, smut infection, and corn line will also be examined.

318. A proteomics-based approach for identification of the ToxD gene. Iovanna Pandelova and Lynda M. Ciuffetti. Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, USA.

Several host-selective toxins (HSTs) are produced by the wheat pathogen Pyrenophora tritici-repentis, including Ptr ToxA, Ptr ToxB and Ptr ToxC. The race structure of P. tritici-repentis is defined by the production of one or a combination of HSTs by a particular isolate and its interaction with a specific set of wheat differentials. Ptr ToxD, a recently isolated proteinaceous toxin, exhibits the same specificity as Ptr ToxA on the currently designated set of wheat differentials. The race 9 isolate, which produces Ptr ToxD and not Ptr ToxA, was identified only when polymerase chain reaction (PCR) and Southern blot analysis revealed the lack of the ToxA gene. As isolate-by-isolate protein purification promises to be tedious and time consuming, screening isolates for the presence of the ToxD gene first followed by confirmation of Ptr ToxD production provides a more efficient approach. In order to identify and clone the ToxD gene, we used a proteomics-based approach. Ptr ToxD activity was purified to a single 10 kDa protein band as determined by SDS-PAGE and confirmed by MALDI mass spectrometry. Purified protein was digested with trypsin. The resulting peptides were separated with a Waters CapLC system and subsequently analyzed with an electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) Global system mass spectrometer. Degenerate primers were designed based on peptide sequences and used in reverse-transcriptase PCR to amplify a partial cDNA clone from a race 9 isolate. This amplification product will be used to isolate and clone the gene.

319. The signal peptidase of Colletotrichum graminicola, and its role in pathogenicity to maize stalks and leaves. E. Park and L. Vaillancourt. University of Kentucky.

The fungus Colletotrichum graminicola causes anthracnose disease on maize. The anthracnose syndrome includes a leaf blight phase, as well as a more damaging and less understood stalk rot. A C. graminicola mutant was identified in our laboratory that colonized normally as a biotroph in leaves, but failed to make the transition to necrotrophic growth. The mutant was also significantly reduced in its ability to colonize and rot living stalks. The predicted mutant protein, CPR1, has 37% similarity to pSPC3, one of four subunits of the signal peptidase of yeast. Our current model is that the mutant is unable to secrete necessary proteins to sustain necrotrophic growth. Our goal was to test the hypothesis that CPR1 is a component of the C. graminicola signal peptidase. The Cpr1 cDNA was cloned and transformed into two different yeast spc3 mutants. However, CPR1 did not complement either mutant yeast strain, perhaps because CPR1 is too divergent from pSPC3. To obtain indirect evidence for the function of CPR1, a gene encoding a putative pSEC11 homologue of C. graminicola (Cgs11) was cloned and characterized. pSEC11 is one of four subunits of the signal peptidase in yeast, and has been shown to physically interact with pSPC3. The predicted protein encoded by the Cgs11 cDNA was 67 % similar to pSEC11. The Cgs11 cDNA complemented two different yeast sec11 mutants. Co-immunoprecipitation and co-immunolocalization experiments are currently in progress to test for an interaction between CGS11 and CPR1.

320. Mechanisms of differential spore germination and growth of Fusarium oxysporum in the root of Arabidopsis thaliana. Sook-Young Park1, Yin-Won Lee 2, Jim Tumlinson 3, Jurgen Engelberth 3, Seogchan Kang1. 1 Department of Plant Pathology and 3 Department of Entomology. The Pennsylvania State University, University Park, PA 16802. 2 School of Agricultural Biotechnology, Seoul National University, 151-742, Korea.


The soil-borne fungus Fusarium oxysporum causes vascular wilt in a wide variety of plant species, including Arabidopsis thaliana. We studied the germination of Fusarium oxysporum spores in the rhizosphere of various Arabidopsis ecotypes and the subsequent colonization of the root system using a fluorescently labeled strain. Two Arabidopsis ecotypes, Cape Verde (CV) and Greenville (GRE), differentially responded to O-685, a cabbage isolate. Seedlings of GRE inoculated with O-685 showed no external signs of disease, whereas CV seedlings were severely stunted and showed typical Fusarium wilt symptoms. Spores poorly germinated in the rhizosphere of GRE, whereas the germination rate in the rhizosphere of CV was high. Plant root exudates initiate and manipulate interactions with soil microbes, including F. oxysporum, through the secretion of various primary and secondary metabolites. We hypothesize that the root exudates from these ecotypes are significantly different in their composition. Metabolite analysis to identify chemical basis of differential spore germination is in progress.

321. Proteins involved in the apple scab disease interaction. Plummer KM1, Fitzgerald AM1,2, van Kan, JAL3, Greenwood DR2, Cui, W2, Templeton MD2. 1University of Auckland, PB 92019, NZ. 2HortResearch, Mt Albert Research Centre, PB 92169, NZ. 3Plant Sciences, Wageningen University, Netherlands.

The biotrophic fungus Venturia inaequalis causes apple scab disease. Resistance to V. inaequalis conforms to a gene-for-gene model and is due to the presence of major resistance genes in the Malus host and avirulence genes in the fungus. It is hypothesised that secreted fungal proteins are involved in the maintenance and specificity of scab disease. We are using genomics and proteomics approaches to identify avirulence and pathogenicity proteins secreted by the fungus, and plant proteins whose expression is influenced by the pathogen. Proteins isolated from infected apple leaf tissues are being analysed using ESI-MS/MS. Peptide fragment data and sequence information is then being screened against an EST (expressed sequence tag) database from V. inaequalis-infected leaf material, and from in vitro grown V. inaequalis. Genes and proteins of interest including: proteins identified from infected leaf tissue; proteins that elicit specific host resistance responses; genes differentially expressed on cellophane and in planta compared with growth on nutrient media; and ESTs with secretory peptides, are being screened in functional assays and using real-time PCR. We have developed gene silencing in V. inaequalis to facilitate the functional characterisation of genes of interest.

322. Tissue acidification and ROS accumulation during pathogenicity of Penicillium expansum. Yoav Hadas1, Israel Goldberg2, Ophry Pines2 and Dov Prusky1. 1Department of Postharvest Science of Fresh Produce in the Institute of Agricultural Products, ARO, Volcani center, Bet Dagan 50250 and 2Department of Molecular Biology, Hebrew University, Medical School, Jerusalem 91120, Israel.

The phytopathogenic fungus Penicillium expansum acidifies the host tissue and consequently enhanced pepg1 expression, a gene encoding for polygalacturonase, involved in host maceration and fungal attack of Penicillium expansum. The acidification is achieved by secretion of organic acids mainly gluconic acid. P. expansum isolates with increased pathogenicity, accumulated higher amounts of gluconic acid and reduced the apple tissue pH to lower values than isolates with reduced pathogenicity. Glucose oxidase activity, involved in gluconic acid production, was detected in P. expansum decayed tissue but not in the healthy tissue of the same fruit. Glucose oxidase activity and accumulation of gluconic acid were strongly affected by environmental pH conditions. Reactive oxygen species resulting from glucose oxidase activity were easily detected in the decayed apple tissue and specifically in the hyphae. It is suggested that acidification of host tissue by organic acids is an important factor involved in host maceration and fungal attack of P. expansum.

323. Withdrawn

324. Regulation of gene expression on Colletotrichum gloeosporioides by alkalinization. Itay Miyara1, Amir Sherman2 and Dov Prusky1. 1Department of Postharvest Science of Fresh Produce and 2Department of Genomics, Agricultural Research Organziation, Volcani Center, Bet Dagan 50250

C. gloeosporioides is an important pathogen of tropical and subtropical fruits. During the colonization the pathogen alkalinize the host tissue by secreting significant amount of ammonia. The alkalinization of the host tissue enhanced pelB expression, a gene encoding for pectate lyase that affect colonization of fruits and possible other genes that were not identified. As a first stage for identification of the genes expressed during the ammonification process and their contribution to fungal pathogenicity we prepared cDNA libraries of C. gloeosporioides grown at inducing pH in vitro and in semi in vivo on avocado fruits. The cDNA libraries were printed on nylon membranes in a macroarray format. The macroarrays were used to determine the differential expression of genes under inducing and non inducing conditions of colonization. Results from the macroarray experiments will be shown. We intend to use these results for gene knockouts and identification of new non-pathogenic mutants

325 Isolation of a Novel DNA Sequence Required for Pathogenicity of Colletotrichum Species. Regina S. Redman and Rusty Rodriguez. University of Washington, Biology, Seattle, WA. Montana State University, Bozeman MT. U.S. Geological Survey, Seattle, WA.

 We have isolated a unique DNA sequence (designated fsl-1, fungal symbiotic lifestyle) from ,Colletotrichum magna, a cucurbit pathogen, that is required for pathogenesis and is conserved in the genus Colletotrichum. The fsl-1 sequence is not currently represented in public sequence databases and was used to generate a targeted gene disruption vector (pGD13). Transformation studies involving six Colletotrichum species resulted in the conversion of virulent pathogens to non-pathogenic endophytic mutualists. The lifestyle altered transformants asymptomatically colonized plants and conferred disease resistance against the virulent wildtype isolates. Southern blot analysis revealed that pGD13 disrupted fsl-1 by homologous integration. Expression studies indicated that fsl-1 is expressed in culture in non-pathogenic transformants but not in virulent wildtype isolates. Collectively, these results indicate that fsl-1 is required for the expression of pathogenic lifestyles in Colletotrichum species. Studies are currently underway to determine the expression of fsl-1 inplanta and determine the ubiquity of fsl-1 in other fungal genera.

326. Genes Expressed During Early Infection of Arabidopsis thaliana by Ustilago maydis. Cristina G. Reynaga-Peña1 , Lucila Méndez-Morán1,2 , Barbara Jablonska3 , Patricia S. Springer3 and José Ruiz-Herrera1. 1Centro de Investigación y de Estudios Avanzados del I.P.N., Unidad Irapuato, MÉXICO, 2 Depto. de Ecología CUCBA, Universidad de Guadalajara, MEXICO and 3 University of California, Riverside, Riverside, CA, U.S.A.

We used two approaches to identify A. thaliana up-regulated genes during the early stages of infection by U. maydis: 1) screen of enhancer/genetrap lines from A. thaliana, and 2) preparation of an expression subtractive library. Through the screen of 1219 Arabidopsis lines from the UCR trap collection, we identified lines that showed an alteration in GUS reporter gene expression in response to fungal infection. TAIL-PCR was used to identify the genes tagged in these lines. We found insertions into genes encoding known and unknown proteins: a putative sulfate transporter, a predicted transcription factor, chloroplast-related genes, and others. A comparison of these results with screens made by colleagues on the same UCR lines allowed us to dissect general biotic from abiotic stress responses, and those that were specific for fungal infection. In a parallel approach, a subtractive library was obtained from RNA expressed after 24 or 96 h post-infection, and so far we have identified two up-regulated genes, one encoding the CP12 protein from chloroplast, and a serine protease inhibitor. Additionally, we searched on available databases to find if some of the A. thaliana genes up-regulated during U. maydis infection were also regulated during infection by other plant pathogens, including bacteria and fungi. This work is important for the identification of A. thaliana genes that may play an important role either in plant defense against U. maydis infection, or involved in the changes in plant growth that occur during infection. We are currently investigating these phenomena using different approaches.

327. Triacylglycerol Lipase 1 Gene of Magnaporthe grisea is Essential for Appressoria Turgor and Pathogenic Growth in Host Plant Cells. Hee-Sool Rho1, Soonok Kim1, ChangHyun Khang2, Seochan Kang2, and Yong-Hwan Lee1. 1School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea. 2Department of Plant Pathology, The Pennsylvania University, University Park, PA 16802, USA.

Magnaporthe grisea elaborates a specialized infection structure, called an appressorium, which is used to rupture the plant cuticle by mechanical force. Appressoria generate turgor pressure by accumulating high concentration of glycerol. Triacylglycerol lipase activity increased during appressoria maturation. To investigate the molecular genetic roles of triacylglycerol lipase on appressorium formation and turgor generation, we characterized the function of triacylglycerol lipase 1 gene (LIP1) by a gene disruption strategy. The lip1 mutants showed impaired properties on conidial germination and adhesion. Appressorium formation of lip1 mutants was retarded and reduced, which seemed to be associated with turgor pressure generation. lip1 mutants also showed reduced virulence on host plant. Defects on conidial germination and appressorium formation of lip1 mutants were restored by exogenous addition of diacylglycerol. These suggest that LIP1 plays important roles on infection-related morphogenesis and turgor generation in M. grisea. These results will provide further understanding of the relationship between lipid metabolism and turgor generation for fungal pathogenesis

328. Identification of cyst surface proteins from the fish pathogen Saprolegnia parasitica. Emma Robertson and Pieter van West. The Aberdeen Oomycete Group, College of Life Sciences and Medicine, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, Scotland, UK.

Fish pathogens, such as the oomycete Saprolegnia parasitica, cause devastating damage and loss of profit in the fish farming industry worldwide. These pathogens cause Saprolegniosis, a disease where filamentous mycelium grows into the fins and body of freshwater fish. The disease results in slow and sluggish movement of infected fish. Severe infection may result in death of the host. Very little is known of the molecular biology of S. parasitica, and pathogenicity of the oomycete is undetermined. It has been proposed that spines present on the surface of secondary zoospores hook onto the scales of the fish, and aid in the initial host-pathogen interaction. This would then permit an opportunity for further invasion and colonisation.

Here we present a proteomic approach to identify hook proteins and other surface proteins that may play a role in pathogenicity.

Furthermore, we are optimising an RNA interference (RNAi) protocol for S. parasitica. Using RNAi, it should be possible to perform functional analysis of genes in S. parasitica. By having a better understanding of the proteins involved in the pathogenicity of S. parasitica, it may be possible to identify potential drug targets and develop a new route of controlling this devastating, and economically important disease.

329. Construction of a physical and fingerprint fragment restriction map of a conditionally dispensable chromosome in Nectria haematococca. Marianela Rodriguez, Dai Tsuchiya, and Hans D. VanEtten. Division of Plant Pathology and Microbiology, Department of Plant Sciences, University of Arizona.

Nectria haematococca has a wide range of habitats, although individual isolates have a limited habitat range. Part of this habitat diversity is due to the presence of supernumerary chromosomes that contain unique habitat defining genes. Since these chromosomes are not needed for axenic growth but are important for habitat specificity, they have been called "Conditionally Dispensable" (CD) chromosomes. One of these CD chromosomes in N. haematococca contains a cluster of genes for pea pathogenicity called the PEP cluster. It has been suggested that the genes in the PEP cluster, as well as the CD chromosome, are of foreign origin, due to differences in GC content and codon usage when compared to the genes from other chromosomes of N. haematococca.

In the current study, we present a partial physical map of the 1.6 Mb CD chromosome of N. haematococca. The fingerprint data for the map were obtained by restriction enzyme digestion of cosmid clones from a CD-specific library. Contigs were generated by analyzing overlapping clones using the Arizona Genomics Institute's FPC software. Contig arrangements are being identified by fiber fluorescence in situ hybridizations. This CD chromosome contig map will be used for studies of syntony and recombination events, which could lead to a better understanding of the origins of CD chromosomal DNA.

330. Claviceps purpurea – Signalling in Directed Growth. Yvonne Rolke, Jan Scheffer and Paul Tudzynski. Institute of Botany, Westfaelische Wilhelms-Universitaet Muenster, Schlossgarten 3, 48149 Muenster, Germany

An interesting model to study directed growth of fungal hyphae in a pathogen host interaction is represended by the biothrophic ascomycete Claviceps purpurea on rye.

Two approaches will reveal new aspects for the understanding of directed growth mechanisms in this system.

In a forward genetics approach the creation of an insertional mutant library based on the Agrobacterium-mediated T-DNA-transfer is in progress. Mutants with impaired growth can be identified easily in an in vitro system for cultivation and infection of rye ovaries.

In a reverse genetics approach we are interested in the analysis of signal chain components. It was shown by J. Scheffer that cpcdc42, a small GTPase, is a prerequisite for directed growth of C. purpurea in the infection process. In yeast Cdc42 activates among other proteins PAK kinases. PAK kinases are known to influence hyphal growth in several fungi. Deletion of the PAK kinase gene cpcla4 revealed a striking phenotype.Cpcla4 deletion mutants grow in a corral like shape with shortened, blistered cells which contain big vacouls. A closer look to Cpcdc42 and Cpcla4 downstram acting elements in C. purpurea, via a macroarray approach which contains nearly 10.000 sequenced and spotted genes is underway. The analysis will contribute to the understanding of elements which are involved in the unique system of directed growth and polarisation during the infection process of C. purpurea.

331. Multidrug resistance in Mycosphaerella graminicola involves the novel major facilitator superfamily (MFS) transporter gene MgMfs1. Ramin Roohparvar1, Lute-Harm Zwiers1, Gert H.J. Kema2, and Maarten A. De Waard1. 1Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands. 2Businessunit Biointeractions and Plant Health, Plant Research International, Wageningen, The Netherlands

Screening of EST libraries from the wheat pathogen Mycosphaerella graminicola led to the identification of MgMfs1, a full length Major Facilitator Superfamily (MFS) gene with high homology to putative toxin transporters involved in virulence. Complementation of a Saccharomyces cerevisiae strain carrying multiple non-functional drug transporter genes, with MgMfs1 resulted in an impressive decrease in sensitivity of S. cerevisiae to a broad range of synthetic and natural toxic compounds. MgMfs1 disruptants of M. graminicola generated by Agrobacterium tumefaciens-mediated transformation showed an increased sensitivity to strobilurin fungicides and to the mycotoxin cercosporin, whereas no reduction in virulence on wheat seedlings could be observed. The results indicate that the encoded protein MgMfs1 is a true multidrug transporter that can function as a determinant of the pathogen in sensitivity and resistance to fungicides, and that might play a role in secretion of mycotoxins.

332. Lost in the middle of nowhere: the avirulence gene AvrLm1 of Leptosphaeria maculans. Lilian Gout1, Simon Ross1, Marie-Line Kuhn1, Françoise Blaise1, Laurence Cattolico2, Thierry Rouxel1, Marie-Hélène Balesdent1. 1 INRA, PMDV, F-78026 Versailles Cedex, France; 2Genoscope, Centre National de Séquençage, 91057 Evry cedex, France.

The stem canker fungus, Leptosphaeria maculans, develops gene-for-gene interactions with its Brassica hosts, and avirulence genes appear to be genetically clustered in the fungal genome. Map-based cloning of AvrLm1 was initiated and currently represents one of the longest walks performed in fungi. It encountered all possible troubles, including under representation in libraries and hundreds-of-kb stretches of repeats. AvrLm1 was located within one 630 kb NotI restriction fragment, fully covered by a BAC contig comprising 11 clones. The region was bordered by ORF-rich clusters, and encompassed one 35-kb ORF-rich region. The rest of the region consisted of large stretches of A+T-rich composite and degenerated repeats, and the candidate AvrLm1 was a solo gene surrounded by 175 kb of repeats on its 5' side and 90 kb of repeats on its 3' side. Functional complementation of a virulent isolate with the ORF and its putative promoter region fully restored the avirulent phenotype on a range of Rlm1 oilseed rape genotypes. Analysis of natural populations historically submitted to various levels of Rlm1 selection pressure indicated one highly predominant mechanism for loss of avirulence, i.e. deletion of an average 180 kb of the region encompassing AvrLm1.

333. Microarray transcription profiling provides insights into metabolic regulation and stress adaptation during infection of wheat by the Septoria leaf blotch pathogen, Mycosphaerella graminicola. Jason Rudd*, John Keon, John Antoniw, Wendy Skinner, John Hargreaves and Kim Hammond-Kosack*. Wheat Pathogenesis Programme, Plant-Pathogen Interactions Division, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK

Septoria leaf blotch disease is amongst the most important fungal diseases of cultivated wheat and is caused by the ascomycete fungus Septoria tritici (anamorph Mycosphaerella gramincola). Studies on host-pathogen interactions at the transcriptome level have predominantly been restricted to investigations of host responses. Here we present one of few examples of a global transcriptome analysis of a fungal pathogen performed during a natural encounter with its susceptible host. A Mycosphaerella graminicola EST-based microarray has been developed and used to compare the nutrition and development of the fungus in vitro under nutrient rich and nutrient limiting conditions and in vivo at a late stage of host infection. The data obtained have provided insights into; (1) the direction and utilisation of primary carbon flux in the three environments; (2) high and low nutrient transcriptional activation and / or repression events indicative of the nutrient sources available in planta; (3) elevated stress adaptation / tolerance in the host environment; (4) differential expression of fungal virulence / pathogenicity factor homologues. We are also able to conclude that with respect to primary metabolism and nutrient availability, late stage fungal growth in planta more closely resembles that in a nutrient rich, as opposed to a nutrient limiting, environment.

334. Novel genes specifically expressed at early infection stage of rice blast fungus. K. Saitoh1, F. Ishii1, M. Kanamori1, T. Yamashita1, T. Arie1, T. Kamakura2, and T. Teraoka1. 1Tokyo Univ. of Agric. & Techn., Fuchu, Tokyo, Japan. 2Tokyo Univ. of Science, Noda, Chiba, Japan.

The conidial germ tube of the rice blast fungus, Magnaporthe grisea, differentiates an appressorium, essentially required for penetration into the host plant. In our differential cDNA library of M. grisea strongly expressed during appressorium formation, unique and distinct clones were contained. From the library we have found and reported the novel CBP1 gene involved in sensing physically the surface on which the conidia attached. Additionally we found novel genes, B19, B48 and B59, specifically expressed in appressorium formation. Bioinformatic analyses suggested that these genes could encode sucrase/ferredoxin-, dual specificity phosphatase- and glycoside hydrolase (GH61)-like protein, respectively. These genes were disrupted by homologous recombination to elucidate the functions of the genes. The null mutants tested normally grew on YG medium, and sporulated on oatmeal medium. But the B19 and B48 mutants significantly decreased the ability to form the appressorium on polycarbonate plates, although the conidia normally germinated. Chemical inducers to trigger the appressorium formation, 1,16-hexadecandiol as a cutin monomer, cAMP and IBMX (an inhibitor of degradation of cAMP), restored the ability of the appressorium formation on the plate. Both B19 and B48 mutants decreased the ability of penetration and infectious growth in host cells, although they did not completely lose the ability to develop lesions in spray and punch-wounded inoculation. These results suggest that the genes set required for appressorium formation, penetration, and initial infectious growth by establishment of colonization in host cells may be quite different from the genes set after the establishment to develop the visible lesions, and that B19 and B48 genes may be involved in the earlier infection steps. Function(s) of B59 was also elucidated by the similar ways.

335. The Gpmk1 MAP kinase regulates the secreted lipase FGL1, a novel virulence factor of Fusarium graminearum. Attila Gacser, Christian A. Voigt, Nicole J. Jenczmionka, Siegfried Salomon, and Wilhelm Schaefer. Department of Molecular Phytopathology and Genetics, University of Hamburg, Germany.

Mitogen activated protein (MAP) kinases play important roles during different developmental processes including pathogenic stages of many filamentous fungi. It has been reported that Gpmk1 MAP kinase disruption mutants of Fusarium graminearum are apathogenic and cannot infect wheat spikes. At this time it is not possible to explain the complete apathogenicity of the MAP kinase deletion mutants, because the Gpmk1 MAP kinase affects several processes in the cells.

An effective fungal pathogen must overcome physical and chemical barriers made up by the host to block infection. The mode of penetration and invasion of F. graminearum is still not fully elucidated. However, the formation of appressoria has been excluded, as such structures were never found. Instead, the fungus probably enters the host through natural openings, such as the glume stomata, or penetrates the epidermal cell walls directly with short infection hyphae. F. graminearum secretes cell wall degrading enzymes during colonization of its host. Jenczmionka and Schaefer (Curr. Genet., 2004) could show that the regulation of various cell wall degrading enzymes is mediated by the MAP kinase pathway. Gpmk1 MAP kinase disruption mutants of F. graminearum show in vitro a reduced lipolytic activity in comparison to the wild type strain. Here we show the regulation of the secreted lipase FGL1 in dependence of MAP kinase Gpmk1.

336. Visualizing the impact of trichothecenes on the development of Fusarium Head Blight. Frank J. Maier, Hadeler, B., and W. Schaefer. Department of Molecular Phytopathology and Genetics, Biocenter Klein Flottbek, University of Hamburg, Germany.

Head blight (FHB) caused by the ascomycetic fungus Fusarium graminearum (Gibberella zeae) is one of the most destructive diseases of cereals. It causes yield reductions and contaminates grain with mycotoxins, which constitutes a potential risk for human and animal nutrition. One important class of mycotoxins produced by several Fusarium species are the trichothecene derivatives (e.g. nivalenol, deoxynivalenol). Trichothecenes accumulate in Fusarium-infested food and non-specifically affect most eukaryotes.

To depict the importance of the trichothecenes on spreading and development of FHB we expressed constitutively the gene for the green fluorescent protein (gfp) in F. graminearum wild type and tri5 disruption mutants, the latter carry the gfp cassette in the disrupted tri5 gene. To evaluate the induction of the trichothecenes in the host plants, the gfp gene was fused to the tri5 promoter and integrated into the genome by homologous recombination. The resulting mutants exhibit a gfp gene driven by the endogenous tri5 promoter and a fully functional tri5 gene. Monitoring gfp expression, we evalute the infection of wheat spikes by wild type and trichothecene deficient mutants as well as the induction of the trichothecene pathway.

337. Surprising variation to the general scheme: The mating type genes of Sporisorium reilianum. Jan Schirawski, Bernadette Heinze, Martin Wagenknecht and Regine Kahmann. Max-Planck-Institute for terrestrial Microbiology, Marburg, Germany.

Sporisorium reilianum causes head smut of maize and sorghum. Infection takes place at seedling stage with disease symptoms appearing only in the inflorescence. With respect to the infection process S. reilianum is very similar to the related species Ustilago hordei and U. avenae. However, genetically, S. reilianum is more closely related to U. maydis that can induce disease symptoms on all green parts of the maize host. In U. maydis, mating and pathogenesis are regulated by a pheromone/receptor system and a pair of homeodomain proteins, which are encoded on two unlinked mating type loci, a and b. Mating and sexual development require that sporidia differ at both mating type loci.

To elucidate the differences in infection mode of the closely related smut fungi S. reilianum and U. maydis, we have investigated the mating type loci of S. reilianum. We isolated haploid sporidia from spore samples collected on different continents. In contrast to published reports, which state that S. reilianum has four mating types, we found five different b alleles and three different a alleles. We have sequenced all eight loci. Both the a and b alleles show high synteny to the respective loci of U. maydis. However, the a alleles contain one additional pheromone precursor gene each. With respect to specificity only three different functional mating pheromones exist, with two of the three being present on any one locus. This is the first report of a smut fungus having three different a alleles, i.e. three different pheromone/receptor systems.

338. The role of ‘Necrosis and Ethylene inducing Proteins' (NEPs) of Botrytis cinerea in plant cell death and pathogenicity. Alexander Schouten, Yaite Cuesta Arenas, Peter van Baarlen & Jan van Kan. Wageningen University, Laboratory of Phytopatholgy, Wageningen, The Netherlands

The plant pathogenic fungus Botrytis cinerea is considered a necrotroph, since it kills the host tissue prior to colonization. Little information about the mechanism of cell killing is available but it was proposed to involve (possibly concerted) action of diffusible toxins, oxalic acid, cell wall degrading enzymes and the production of active oxygen species (AOS).

We characterized two genes in B. cinereaI, Bcnep1 and Bcnep2, which are both homologs of the ‘necrosis and ethylene inducing protein' gene (nep1) initially described in Fusarium oxysporum, f.sp. erythroxyli. Currently, approximately 25 NEP homologs have been characterized in various bacteria, oomycetes and fungi. Generally, NEPs are phytotoxic for dicotyledons. In hemibiotrophic plant pathogens, the nep genes are expressed during the onset of the necrotrophic phase. However, it is currently still not clear whether NEPs do play a key role in pathogenicity.

The Bcnep1 and Bcnep2 genes are differentially expressed during the infection of tomato, suggesting specific roles for each protein during pathogenesis. When synthesized in heterologous expression systems, both BcNEP1 and BcNEP2 have phytotoxic activity. Prior to cell death, tobacco leaves respond by an increase in ethylene production and the accumulation of AOS. Currently, Bcnep disruption mutants of B. cinerea are generated. The mechanism of plant cell death and the role of these proteins in pathogenesis is discussed.

339. Physiological and molecular responses of Alternaria brassicicola to exposure to host plant antimicrobial compounds. Adnane Sellam, Pascal Poupard, Philippe Simoneau. UMR PaVé N̊77, Faculté des Sciences, 2 Bd Lavoisier, 49045 Angers cedex, France

Alternaria brassicicola is a necrotrophic seed-borne pathogen responsible for the black spot disease of crucifers. During the colonization of the host tissues, this fungus is exposed to several antimicrobial plant compounds such as phytoanticipins and phytoalexins. In order to better understand how the fungus deals with such toxic environment, we first analyzed in vitro the effects of two glucosinolate-derived isothiocyanates (ITC) and of camalexine (CAM) on different growth parameters of Alternaria brassicicola . Briefly, allyl-ITC and to a lesser extent benzyl-ITC strongly inhibited Alternaria brassicicola developement. Concerning CAM, inhibitory effects on spore germination and germ-tube elongation were observed at concentrations significantly lower than those measured in planta. At higher concentrations, effects on radial growth and sporulation were also recorded. In a second step, we analyzed the expression of fungal target genes after exposure to ITC or CAM at sub-inhibitory concentrations. The two types of ITC induced the expression of cyanide hydratase and of an ABC-transporter gene similar to BcAtrB fromBotrytis cinerea ; benzyl-ITC also significantly affected the expression of a glutathione S-transferase homologue. Up-regulation of the expression of the ABC transporter gene by CAM was also observed. We now plan to study the expression patterns of these Alternaria brassicicola genes in planta using experimentally inoculated Arabidopsis thaliana plants.

340. Physiological and molecular responses of Alternaria brassicicola to exposure to host plant antimicrobial compounds. Adnane Sellam, Pascal Poupard, Philippe Simoneau UMR PaVé N̊77, Faculté des Sciences, 2 Bd Lavoisier, 49045 Angers cedex, France

Alternaria brassicicola is a necrotrophic seed-borne pathogen responsible for the black spot disease of crucifers. During the colonization of the host tissues, this fungus is exposed to several antimicrobial plant compounds such as phytoanticipins and phytoalexins. In order to better understand how the fungus deals with such toxic environment, we first analyzed in vitro the effects of two glucosinolate-derived isothiocyanates (ITC) and of camalexine (CAM) on different growth parameters of Alternaria brassicicola . Briefly, allyl-ITC and to a lesser extent benzyl-ITC strongly inhibited Alternaria brassicicola developement. Concerning CAM, inhibitory effects on spore germination and germ-tube elongation were observed at concentrations significantly lower than those measured in planta. At higher concentrations, effects on radial growth and sporulation were also recorded. In a second step, we analyzed the expression of fungal target genes after exposure to ITC or CAM at sub-inhibitory concentrations. The two types of ITC induced the expression of cyanide hydratase and of an ABC-transporter gene similar to BcAtrB fromBotrytis cinerea ; benzyl-ITC also significantly affected the expression of a glutathione S-transferase homologue. Up-regulation of the expression of the ABC transporter gene by CAM was also observed. We now plan to study the expression patterns of these Alternaria brassicicola genes in planta using experimentally inoculated Arabidopsis thaliana plants.

341. Withdrawn

342. Withdrawn

343. Physiological and molecular responses of Alternaria brassicicola to exposure to host plant antimicrobial compounds. Adnane Sellam, Pascal Poupard, Philippe Simoneau. UMR PaVé N̊77, Faculté des Sciences, 2 Bd Lavoisier, 49045 Angers cedex, France

Alternaria brassicicola is a necrotrophic seed-borne pathogen responsible for the black spot disease of crucifers. During the colonization of the host tissues, this fungus is exposed to several antimicrobial plant compounds such as phytoanticipins and phytoalexins. In order to better understand how the fungus deals with such toxic environment, we first analyzed in vitro the effects of two glucosinolate-derived isothiocyanates (ITC) and of camalexine (CAM) on different growth parameters of Alternaria brassicicola . Briefly, allyl-ITC and to a lesser extent benzyl-ITC strongly inhibited Alternaria brassicicola developement. Concerning CAM, inhibitory effects on spore germination and germ-tube elongation were observed at concentrations significantly lower than those measured in planta. At higher concentrations, effects on radial growth and sporulation were also recorded. In a second step, we analyzed the expression of fungal target genes after exposure to ITC or CAM at sub-inhibitory concentrations. The two types of ITC induced the expression of cyanide hydratase and of an ABC-transporter gene similar to BcAtrB fromBotrytis cinerea ; benzyl-ITC also significantly affected the expression of a glutathione S-transferase homologue. Up-regulation of the expression of the ABC transporter gene by CAM was also observed. We now plan to study the expression patterns of these Alternaria brassicicola genes in planta using experimentally inoculated Arabidopsis thaliana plants.

344. Dissection of Distinct Pathogenesis-related Processes in the Blast Fungus Magnaporthe grisea. Ane Sesma and Anne E. Osbourn. The Sainsbury Laboratory, John Innes Center, Colney Lane, Norwich NR4 7UH, U.K.

The blast fungus Magnaporthe grisea , which causes disease in a wide variety of grasses including rice, wheat and barley, has emerged as a paradigm system for investigation of foliar pathogenicity. This fungus undergoes a series of well-defined developmental steps during leaf infection, including the formation of elaborate penetration structures (appressoria). Remarkably, M. grisea can also infect roots. We have shown that it can undergo a different (and previously uncharacterised) set of programmed developmental events that are typical of root-infecting pathogens that can lead to systemic invasion and the development of classical disease symptoms on the aerial parts of the plant. However, the ability to infect roots does not depend on appressoria. In contrast, hyphal swellings resembling the simple penetration structure (hyphopodia) of root-infecting fungi are evident at infection sites. In addition, experiments with defined mutants defective in leaf infection confirm that there are key differences between the molecular determinants and signal transduction requirements for penetration of leaves and roots. Using cytological approaches (scanning electron microscopy and confocal microscopy) we are extending these studies to identify the step in which these mutants are unimpaired during root colonisation. We are also testing a collection of M. grisea wild-type and mutant strains on a range of grooved topographies with different chemical, hydrophobic and thickness properties to find in vitro conditions for hyphopodium formation.

345. FSR1, a putative WD40 repeat homolog in Fusarium verticillioides, plays a role in maize stalk rot virulence. W. B. Shim, H. W. Wilkinson, U. S. Sagaram, and Y. E. Choi. Department of Plant Pathology and Microbiology, Program for the Biology of Filamentous Fungi, Texas A&M University, College Station, TX77843-2132

F. verticillioides (teleomorph Gibberella moniliformis) causes stalk rot of maize, the most prevalent disease in all maize-growing regions. Using a forward genetics approach, we have identified a locus, FSR1, implicated in Fusarium stalk rot pathogensis; the fsr1 mutant exhibits a loss of virulence and fails to cause rot in maize stalks. Sequencing of the disrupted locus revealed a 2718-bp ORF with two introns. Conceptual translation resulted in a 824-amino acid polypeptide with 60% identity to Sordaria macrospora Pro11, which is a multimodular protein with two distinct functional domains known to be involved in protein-protein interactions: coiled-coil motif and seven-WD40 repeats. Pro11 plays an important role in cell differentiation, particularly sexual development in S. macrospora. Significantly, Pro11 homologs are highly conserved and found in a number of mammalian systems, filamentous fungi and Drosophila, but not in yeasts and plants. While the role of Pro11 homologs in pathogenic fungi has not been previously characterized, our data indicate that F. verticillioides FSR1 is involved in fungal virulence. Additional studies are in progress to characterize the relationships among genes in this gene family and to assess whether FSR1 is a key molecular component in a putative signaling pathway regulating stalk rot virulence.

346. Dissecting the role of signal transduction in Stagonospora nodorum during infection on wheat. Peter S. Solomon1, Kar-Chun Tan1, Ormonde D.C. Waters1, A. Harvey Millar2, Richard P. Oliver1. 1Australian Centre for Necrotrophic Fungal Pathogens, DHS, SABC, Murdoch University, Perth 6150, WA, Australia. 2 University of Western Australia, School Biomedical and Chemical Sciences, 35 Stirling Highway, Perth 6009, WA Australia.

Stagonospora nodorum is a fungal pathogen of wheat that has the potential to severely affect the economic viability of Australia's wheat crop. Whilst having been identified as serious wheat disease for many years, the molecular understanding of how the fungus and its host interact is poorly understood. A reverse genetics approach has been adopted to find genes involved in pathogenicity focusing predominantly on signalling and primary metabolism pathways. To better understand the role of signal transduction in pathogenicity, genes encoding a Gα subunit (Gna1) from the cAMP pathway and a MAP kinase (Mak2) from the MAP kinase pathway were characterised by gene disruption. Fungal strains developed with mutations in either gene were severely reduced pathogenicity and both were unable to sporulate. Infection assays revealed the mak2 strain was virtually non-pathogenic whilst the gna1 strain appeared weakly pathogenic and was able to penetrate the leaf through natural openings. Both mutations also revealed severe phenotypic alterations specific for each mutation including melanin synthesis deficiencies, sensitivity to osmotic stress, reduced protease production/secretion. These results suggest that these key signalling genes control processes downstream which are required for pathogenicity. To determine what these processes are, a proteomics approach has been adopted. A comparison of the wild-type, gna1 and mak2 proteomes has identified proteins that are controlled by either gna1, mak2 or both. A detailed analysis of these key signalling mutants and a synopsis of the proteomics will be presented.

347. Three novel retrotransposons from Magnaporthe grisea mini-chromosome. Teruo Sone, *Akio Oguchi, *Hisashi Kikuchi, **Akihiro Senoh, **Satoshi Nakagawa and Fusao Tomita. (Grad. Sch. Agr., Hokkaido Univ., Sapporo JAPAN * NITE, Tokyo, JAPAN **XANAGEN, Co. Tokyo Japan)

Most of Magnaporthe grisea rice-pathogenic field isolates have mini-chromosomes. These mini-chromosomes are less than 2 Mb in size and known to be related to fertility. Strain 70-15, used in the genome sequence project of M. grisea lacks mini-chromosomes. Thus we attempted to sequence the mini-chromosome of this fungus. A shotgun library of 1.6 Mb mini-chromosome from the strain 9439009, Japanese field isolate was constructed. Sequencing of both ends of inserts were completed for 28800 clones of the library, but assemble was not successful even by the masking of known transposable elements. One of the estimated reasons for this failure was the occurrence of many unknown repetitive elements. Therefore we manually extracted 71 repetitive sequences with consed program. Three novel retrotransposon-like sequences were identified from these repetitive sequences.

Further characterization of these sequences was attempted. Two of these elements, named Inago 1 and Inago 2, were 58.8 % homologus in amino-acid sequence of pol gene product, showing the structural similarity to Gypsy/Ty3 family of retrotransposons. Interestingly, LTR (Long terminal repeat) of these elements were known repetitive elements of M. grisea, MGR608 and REP1, respectively. Other one element, named Swarm, indicated structural similarity with Copia/Ty1 family of retrotransposons. LTR of Swarm exhibited homology with IGS (intergenic spacer sequence) of rDNA.

348. Genetic Evidence for a Role of Phytophthora infestans protease inhibitors in disease. Jing Song, Nicolas Champouret, Joe Win, Miaoying Tian, Sophien Kamoun. Department of Plant Pathology, The Ohio State University-OARDC, Wooster, OH 44691

The oomycete Phytophthora infestans causes late blight, a reemerging and ravaging disease of potato and tomato. Phytophthora infestans has evolved 18 extracellular protease inhibitor genes belonging to two major structural classes: (i) Kazal-like serine protease inhibitors (EPI1-14) and (ii) cystatin-like cysteine protease inhibitors (EPIC1-4). Biochemical evidence suggests that some of the protease inhibitors target apoplastic proteases from the host plant tomato. EPI1 and EPI10 inhibit and interact with the subtilisin-like protease p69B, whereas EPIC2 interacts with the papain-like cysteine protease PIP1. To complement the biochemical studies, we first carried out stable DNA transformation of protoplasts of Phytophthora infestans using lipofectin, polyethylene glycol and CaCl2. We used the Gateway technology to generate transformation constructs in which the gene of interest is bordered by the oomycete ham34 promoter and terminator, followed by the bacterial resistance gene G418 (Geneticin) fused to hsp70 promoter and terminator. Both sense and antisense constructs of epi genes were used. We also used RNA interference (RNAi) triggered by dsRNA to silence epigenes. Preliminary evidence suggests that alteration of epi1 expression resulted in altered virulence on tomato. These studies will lead to a better understanding of the role of protease inhibitors in disease progression.

349. Coordinate regulation of metabolic genes and identification of a virulence gene expression cluster in Blumeria graminis. M Both, M Czukai, PD Spanu. Imperial College London and Syngenta Ltd

We used cDNA microarrays to measure the expression of 2027 unigenes of Blumeria graminis f. sp. hordei, an obligate biotrophic pathogen of barley. The relative expression of these genes was followed throughout the normal course of B. graminis development from the ungerminated conidium, its early germination stages (4, 8 and 15hpi) to the proliferation on the host surface and in barley epidermis containing haustoria (3 and 5 dpi). Global correlation analysis showed that there was a marked shift in gene expression between the pre- and post-penetrative stages. This was due primarily to large scale changes in the metabolism of the fungus as it progressed though its development. For example, genes devoted to translation were significantly up-regulated when the fungus proliferates in the post-penetrative stages. Some of the genes encoding enzymes on common primary metabolic pathways showed striking coordinate expression. Thus, "glycolytic transcripts" increased significantly immediately after germination, then decreased and increased again in the samples containing haustoria. Glycogen degradation transcripts were abundant at early stages (4 and 8 hpi) and later decreased; this was matched by a significant peak in glycogen branching transcript when conidia are formed. Many lipid degradation transcripts levels were high at the early stages and decrease to low levels thereafter. The AdoMet cycle transcripts were most abundant in the proliferating and sporulating epiphytic mycelium.

There are a number of B. graminis cDNAs on our microarrays that are homologous to genes, such as cap20, known to affect pathogenicity in other fungi. Expression cluster analysis identified genes whose expression correlated with a cap20 homolog. One such expression cluster included a striking proportion of genes that are associated to pathogenicity. Applying the "guilt-by-association" principle identified a number of cDNAs that are potential candidates for pathogenicity genes in B. graminis.

350. A gene involved in modification of transfer RNA is required for fungal pathogenicity and stress tolerance of Colletotrichum lagenarium. Yoshitaka Takano1, Naoyuki Takayanagi1, Hiroyuki Hori2, Yoshiho Ikeuchi3, Tsutomu Suzuki3, Akiko Kimura1, and Tetsuro Okuno1. 1Kyoto University, Japan. 2Ehime University, Japan. 3University of Tokyo, Japan.

7-methylguanosine (m7G) modification of tRNA occurs widely in eukaryotes and prokaryotes, although there is little information about its biological roles. We report here that a gene involved in m7G modification of tRNA is required for pathogenicity in Colletotrichum lagenarium that causes cucumber anthracnose. The pathogenicity mutant KE51 of C. lagenarium was identified by insertional mutagenesis. Analysis of KE51 identified a plasmid-tagged gene APH1. The aph1 deletion mutants failed to infect host plants. Aph1 showed strong similarity to Saccharomyces cerevisiae Trm8 involved in m7G modification of tRNA. RNA from the aph1 mutant has much reduced m7G compared with the wild type, indicating that APH1 is required for m7G methyltransferase activity. Appressoria formed by the mutants failed to develop penetration hyphae into the host plant, suggesting requirement of APH1 for appressorium function for plant infection. Interestingly, the aph1 mutants increased sensitivity to several stresses such as H2O2 treatment, implying that penetration defects of the aph1 mutant depend on a failure to protect itself against plant defense systems. Consistent with this, heat shock treatment on the host plants enabled the aph1 mutant to penetrate into them. These data suggest a possibility that the fungal pathogen require Aph1-mediated tRNA modification to overcome plant defense.

351. Reactive oxygen species generated by a fungal NADPH oxidase regulate hyphal differentiation and growth in Epichloe/N festucae, a mutualistic symbiont of temperate grasses. Aiko Tanaka1, Michael Christensen2 and Barry Scott1. 1Institute of Molecular BioSciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand. 2AgResearch Limited, Grasslands Research Centre, Private Bag 11 008, Palmerston North, New Zealand.

Epichloe /Neotyphodium endophytes are a group of clavicipitaceous fungi (Clavicipitaceae, Ascomycota) that form symbiotic associations (symbiota) with temperate grasses of the sub-family Pooideae. The hyphae of these endophytes grow systemically through the intercellular spaces of the leaf as seldom branched linear strands and the host remains symptomless. Plasmid insertional mutagenesis was used to identify genes of E. festucae responsible for maintaining the mutualistic association. From a screen of 220 transformants, one mutant FR2 was identified that causes severe stunting of the grass host characterized by an increase in tiller number, and in most cases eventual death of the host. Associations containing FR2 show hyphal hyper-branching and a dramatic increase in fungal biomass, although the growth of FR2 was normal in culture. The genomic DNA flanking the single-copy plasmid insertion in FR2 was isolated by plasmid rescue. Sequence analysis of the recovered fragment showed that the plasmid was inserted into the coding region of a NADPH oxidase gene (designated EfnoxA). Nox genes are homologous to gp91phox, the glycosylated transmembrane subunit of the mammalian NADPH oxidase that catalyses production of reactive oxygen species (ROS). Complementation and deletion analysis confirmed that the phenotype of FR2 is caused by disruption of EfnoxA. Using the cerium chloride method ROS accumulation was detected cytochemically near cell walls of fungal hyphae in wild-type associations. These results demonstrate that ROS production by the endophyte NADPH oxidase is crucial in maintaining a mutualistic association between the endophyte and its grass host.

352. Molecular characterization of the pea pathogenicity (PEP) gene cluster of Neocosmospora boniensis and other Neocosmospora species. Esteban D. Temporini, Dai Tsuchiya, and Hans D. VanEtten. Division of Plant Pathology & Microbiology, University of Arizona, Tucson AZ.

In 1989, Udagawa and colleagues (SYDOWIA 41: 349-359) described a new species belonging to the genus Neocosmospora: N. boniensis. Although Neocosmospora species have been described as pathogens of several plant species, there are no reports on the literature showing this group of fungi as naturally occurring pea-pathogens. We have recently shown that N. boniensis contains homologs of the genes present in the pea pathogenicity (PEP) cluster of Nectria haematococca MPVI, which are required by N. haematococca to cause disease on pea. Moreover, we have shown that the PEP homologs are also clustered in N. boniensis and that this species is capable of causing disease on pea. In this work, DNA fiber-FISH experiments were used to analyze the physical organization of the homologs in the N. boniensis PEP cluster. The results indicate that several rearrangements have occurred in the N. boniensis PEP cluster, compared to the N. haematococca PEP cluster. These rearrangements have been confirmed by determining the nucleotide sequence of this region, which shows that transposable elements may have played a role in shaping the N. boniensis PEP cluster. This analysis has also revealed that the DNA homology between the PEP genes of N. boniensis and N. haematococca is very high (around 95% identity). We also show here that PEP homologs are present in other species of Neocosmospora and that these species are capable of causing disease on pea.

353. Dissection of the transcriptome of Phytophthora sojae under oxidative stress: Source of pathogenicity factors? Trudy Torto-Alalibo, Dianjing Guo, Regina Hanlon, Hua Li and Brett Tyler. Virginia Bioinformatics Institute, Virginia Tech, Blacksburg VA, 24061

One of the hallmarks of plant defense responses against pathogens is the generation of reactive oxygen species (ROS) in a process referred to as oxidative burst. Extensive studies have been conducted to investigate the role of ROS in plant defense; however, little has been done to determine the role of the pathogen under oxidative stress. Recent studies have shown that the production of ROS in Botrytis cinerea, a necrotroph, leads to increased levels of ROS production in the plant, which causes death of plant tissue. Subsequently, increase in fungal growth is observed. Alternatively, the biotrophic fungus, Claviceps purpurea, produces scavenging enzymes to protect itself from oxidative stress. Studies have shown that some key genes such as catalase and the bZIP transcription factor identified when pathogens are under oxidative stress contribute to pathogenicity. Phytophthora sojae is a hemibiotroph, which causes root rot to soybean resulting in billions of dollars in losses to farmers annually. Responses of either the pathogen or the plant at the point of interaction (interactome) are not well studied. Genes differentially expressed under oxidative stress by P. sojae may have a role in the modulation of host defense reactions. We used the technology of microarray, which helps to look at a global expression of genes under a particular condition to study the transcriptome of P. sojae under oxidative stress. P. sojae mycelium was grown in vitro and subjected to 0.3mM cumene hydroperoxide. Samples were taken at six time points viz. 0, 5 mins , 15 mins, 30 mins, 1hr and 2hrs. Results from the data generated from the microarray will be presented.

354. Investigating the interaction between Magnaporthe grisea and rice through a comprehensive timecourse experiment using microarray analysis. Sara L. Tucker1, Nicole Donofrio2, Ralph Dean2, David A. Henderson1 and Marc J. Orbach1. 1University of Arizona, Tucson, AZ 85721. 2North Carolina State University, Raleigh, NC 27695, USA.

Magnaporthe grisea is responsible for blast disease of rice. We are using this system to identify fungal and plant genes that are differentially expressed during infection and growth of the fungus in planta. To study global changes in gene expression, an interactions microarray was developed in collaboration with Agilent Technologies, which contains identifiers for 13666 M. grisea genes and 7137 Oryza sativa genes. We are characterising pathogen and host gene expression in a compatible interaction over a six-day period following infection. Hybridisations were carried out using a loop design to best compare gene expression at several time points during the infection cycle. This also allows the comparison of expression at additional time points after analysis of the initial data. The initial loop compares gene expression of uninoculated plants with infected plants at nine time points over a six-day period following infection. Additional infected tissue was collected up to 10 days post infection for potential future analysis. The data generated from this experiment has allowed us to cluster genes with similar expression patterns over the infection cycle and provisionally assign them to certain developmental stages of the rice/M. grisea interaction. The data is currently being validated using Real Time-PCR and these results will be presented.

355. The role of ROS generating and scavenging systems in host-pathogen interaction. N. Segmüller, S. Giesbert, E. Nathues, P. Tudzynski. Institut für Botanik, Westf. Wilhelms-Universität, Schlossgarten 3, 48149 Münster, Germany

Reactive oxygen species (ROS) are normal cellular components and recently have been shown to be involved in differentiation processes in several fungi. Since the generation of ROS is a major defense reaction of plants (“oxidative burst”), in fungi/plant interactions ROS are produced by both partners. We study the role of ROS scavenging systems (catalases, etc.) and ROS generating systems (toxins, NADPH oxidases) in pathogenic processes in two interaction systems with different strategy: the biotrophic rye pathogen, Claviceps purpurea, and the necrotrophic grey mould, Botrytis cinerea. We can show that the latter efficiently reduces ROS formation in planta 1, wherease B. cinerea, uses ROS as “weapon”. In both systems components of the stress-activated MAPK cascade are investigated.

1 Nathues E, Joshi S, Tenberge KB, von den Driesch M, Oeser B, Bäumer N, Mihlan M, Tudzynski P (2004) CPTF1, a CREB-like transciption factor is involved in the oxidative stress response in the phytopathogen Claviceps purpurea, and modulates ROS level in its host Secale cereale. Mol Plant Microbe-Interact 17: 383-393.

356. The G alpha subunit BCG1 of Botrytis cinerea controls at least two signalling cascades in the interaction with plants. Julia Schumacher, Christian Schulze Gronover, and Bettina Tudzynski, Institut für Botanik, Westfälische-Wilhelms-Universität Münster, Schlossgarten 3, D-48149 Münster, Germany

The G-alpha subunit BCG1 plays an important role during the infection of plants by B. cinerea. bcg1mutants are able to penetrate host tissue and to produce small primary lesions. However, the mutants completely stop invasion of plant tissue at this stage never producing spreading lesions. SSH was used to identify fungal genes which are up-regulated in planta in the wild-type, but not expressed anymore in bcg1 mutants. Among the differentially expressed genes we found those encoding proteases, enzymes involved in secondary metabolism, hexose transporters, and cell wall-degrading enzymes. To examine if the expression of the BCG1 target genes is also affected in the adenylate cyclase (BAC) mutant, we performed Northern blot analysis with RNA from bean leaves infected with the wild-type, bcg1 or bac mutants. Unexpectedly, most of the BCG1-controlled genes are still expressed in the bac mutant in planta, suggesting that BCG1 is involved at least in one additional signalling cascade beside cAMP-depending pathway. Only four of the genes are indeed regulated by BCG1 and BAC. To find out whether the G-beta/gamma dimer controls the unknown signalling pathway, we cloned and characterized the G-beta subunit-encoding gene, bcgb1. Furthermore, GUS-reporter gene assays with different promoter fragments of a xylanase-encoding gene (cAMP-pathway) and a metalloprotease-encoding gene (yet unknown pathway) were performed in order to identify binding motifs for the corresponding transcription factors for ‘yeast one-hybrid' assays.

357. Comparison of gene expression in trap cells and vegetative hyphae of the nematophagous fungus Monacrosporium haptotylum. Dag Ahrén1,2, Margareta Tholander1, Csaba Fekete1, Balaji Rajashekar1, Eva Friman1, Tomas Johansson1, and Anders Tunlid1. 1Department of Microbial Ecology, Lund University, Lund, Sweden. 2European Bioinformatic Institute, Hinxton, Cambridge, UK.

Nematode-trapping fungi enter the parasitic stage by developing specific morphological structures called traps. We have compared the global patterns of gene expression in traps and mycelium of the fungus Monacrosporium haptotylum. The trap of this fungus is a unicellular spherical structure called the knob, which develops on the apex of a hyphal branch. RNA was isolated from knobs and mycelium and hybridised to a cDNA array containing probes of 2,822 EST clones of M. haptotylum. Despite the fact that the knobs and mycelium were grown in the same medium, there were substantial differences in the patterns of genes expressed in the two cell types. In total, 23.3 % (657 of 2,822) of the putative genes were differentially expressed in knobs versus mycelium. Several of these genes displayed sequence similarities to genes known to be involved in regulating morphogenesis and cell polarity in fungi. Several homologues to genes involved in stress response, protein synthesis and protein degradation, transcription, and carbon metabolism were also differentially expressed. A number of the genes that were differentially expressed in trap cells are also known to be regulated during the development of infection structures in plant pathogenic fungi.

358. The transcription-associated proteins of Fusarium graminearum identified by sequence clustering and profile analyses. Richard Coulson1, Martin Urban2, John Antoniw2, and Kim Hammond-Kosack2. 1European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, CB10 1SD, UK and 2Rothamsted Research, Herts, AL5 2JQ, UK

The trichothecene mycotoxin producing Ascomycete fungus Fusarium graminearum causes ear blight disease of small grain cereals. Infections lower grain quality and safety, and are of increasing global concern. In 2003, its genome was sequenced to ~10 × coverage by the Broad Institute ( As part of the global initiative to complete the manual annotation of the genome, we have explored in depth F. graminearum sequences involved with the transcriptional process. Eukaryotic transcription is a highly regulated process involving interactions between large numbers of proteins, exhibiting a high degree of taxon-specificity. To identify transcription-associated proteins (TAPs), the genome was queried with a reference set of TAPs, extracted from the protein sequence databases via keyword searches (Coulson & Ouzounis (2003) Nucleic Acids Research 31, 653-660). The TRIBE-MCL algorithm was employed to detect TAP families in F. graminearum, in addition to those present in six model organism species: Schizosaccharomyces pombe, Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus. Four plant pathogens Ashbya gossypii, Magnaporthe grisea, Aspergillus oryzae, Ustilago maydis, two human pathogens, Candida albicans and Aspergillus fumigatus and two free living saprophytes, Aspergillus nidulans and Neurospora crassa were also included in the study. The findings from this TAP-TRIBE analysis, and a second complementary approach utilising profile-hidden Markov models of domains present in well-characterised transcriptional regulators, will be presented. Currently, we are exploring the physical distribution of each TAP gene amongst the four F. graminearum chromosomes.

359. The Fusarium oxysporum avirulence protein Six1 is required for full virulence, and is expressed early during infection of tomato. H. Charlotte van der Does, Michiel Meijer, Ben J.C. Cornelissen and Martijn Rep. University of Amsterdam, Swammerdam Institute for Life Sciences, Plant Pathology, Kruislaan 318, 1098 SM Amsterdam, The Netherlands

Fusarium oxysporum f.sp. lycopersici (Fol), is a soil inhabiting fungus that can infect tomato plants via the roots and colonize the xylem. In xylem vessels Fol secretes a 12 kD, cysteine rich protein (Six1) which appears to be derived from a 30 kD precursor through proteolytic processing by fungal proteases. The SIX1 gene is required for resistance of tomato plants carrying the I-3 resistance gene against Fol, but is also required for full pathogenicity of Fol, especially on older (4 week old) tomato plants. SIX1 expression is highly induced in planta, compared to in vitro conditions. Expression starts already before the 4th infection day, when no disease symptoms are visible yet, and deminishes at later stages of infection, when plants show severe disease symptoms. To monitor expression in more detail, we have constructed SIX1 promoter-GFP fusion genes, which have been placed at the native SIX1 locus. In addition, to search for proteins interacting with Six1, we have produced the Six1 protein with an internal His-tag in Pichia pastoris. P. pastoris appears to process the 30 kD Six1 precursor in the same way as Fol does.

360. Characterization and functional analysis of a Botrytis cinerea aspartic proteinase gene family. Arjen ten Have, Ester Dekkers, John Kay, Lowri H. Phylip and Jan A.L. van Kan. Wageningen University, Laboratory of Phytopathology, Wageningen, The Netherlands

Botrytis cinerea secretes aspartic proteinase (AP) activity in axenic cultures. No cysteine, serine or metalloproteinase activity was detected. Proteinase activity was higher in cultures containing BSA or wheat germ extract as compared to minimal medium. AP was also the only proteinase activity in fluid obtained from B. cinerea-infected tissue of apple, pepper, tomato and zucchini. Five B. cinerea genes encoding an AP were cloned and named Bcap1-5. Especially BcAP1 has novel characteristics. A phylogenetic analysis was performed comprising sequences originating from different kingdoms. BcAP1 and BcAP5 did not cluster in a bootstrap supported clade. BcAP2 clusters with vacuolar APs. BcAP3 and BcAP4 cluster with secreted APs in a clade that also contains GPI-anchored proteinases from S. cerevisiae and C. albicans. All five Bcap genes are expressed in liquid cultures. Transcript levels of all genes except Bcap5 are subject to glucose and peptone repression. All five Bcap genes are expressed in infected tissue, indicating that at least part of the AP activity in planta originated from the pathogen. Previous research showed that pepstatin could inhibit infection by B. cinerea, suggesting an important role for APs in the infection process. We analysed the role of APs by gene replacement. Mutants were generated in all five genes separately, as well as in combinations of two Bcap genes. None of the mutants displayed a reduction in virulence or in secreted AP activity.

361. Withdrawn

362. Growth and colonization of Colletotrichum graminicola inside corn stalk tissues. C. M. Venard and L. Vaillancourt. University of Kentucky, Plant Pathology Dpt.

Anthracnose disease, caused by the fungus C. graminicola, is one of the greatest threats to corn production in the U.S. today. The pathogen can attack any part of the plant at any time during the growing season. The most common symptoms are those affecting leaves (blight) and stalk (rot). Anthracnose stalk rot has the greatest economic impact on harvestable yield, but this phase of the disease is poorly understood. We developed an inoculation method that reproduces ASR field symptoms in the greenhouse and used a cytological approach to study pathogen development inside stalk tissues. Fungal strains expressing green fluorescent markers were used to inoculate a highly susceptible sweet corn hybrid. We observed the formation of a highly reproducible, highly organized hole-like lesion under the rind that we called an infection chamber. Necrotic tissues, on which the fungus produces falcate spores in acervuli, surround the hole. The pathogen also colonized the necrotic tissues at the margins of the infection chamber, where it was observed to produce oval conidia inside the parenchyma cells. The fungus was observed to move through the stalk primarily as mycelium inside fibers under the rind, and via the vascular bundles that were damaged by the expansion of the initial lesion. Secondary lesions were initiated from these colonized vascular tissues. When the fungus reached the node, its progression was stopped until the tissues in the plexus collapsed. The fungus then progressed across the node into the upper internode, where it initiated new lesions.

363. FGL1, a secreted lipase of Fusarium graminearum is a novel virulence factor during infection of cereals. Christian A. Voigt, Wilhelm Schaefer, and Siegfried Salomon. Department of Molecular Phytopathology and Genetics, University of Hamburg, Germany.

Fungal pathogens have evolved a number of different strategies to infect and colonize host plants. A lot of fungal pathogens secrete various extracellular enzymes like xylanases, pectinases, cutinases, and proteinases which are supposed to be involved in host infection. We could detect, clone, and characterize a secreted lipase (FGL1) of F. graminearum. The functional identity of the lipase was examined by heterologous gene expression in Pichia pastoris.

In planta, FGL1 transcripts were already detected one day after inoculation of wheat spikes. To evaluate the role of FGL1 during the infection process we created lipase deficient mutants by gene disruption and compared them to the wild type strain. Gene disruption of FGL1 resulted in a significantly reduced extracellular lipolytic activity of the mutants. After infection of wheat spikes, the FGL1 deficient strains showed a drastically reduced virulence. In contrast to F. graminearum wild type infected wheat spikes, FGL1 deficient strains were unable to colonize the rachis of the spike. Infections of spikes were therefore restricted to the point of inoculation. Additionally, maize ears inoculated with F. graminearum wild type conidia are fully infected. In contrast, the maize ears develop normally and showed minor disease symptoms when inoculated with FGL1 deficient strains. Our data are the first molecular proof that a secreted lipase is a major virulence factor of a fungal pathogen.

364. Genetics of avirulence in Mycosphaerella graminicola, the wheat septoria leaf blotch fungus. Sarah B. Ware1 and Gert H.J. Kema1. 1Plant Research International, Wageningen, The Netherlands.

To elucidate the genetics of avirulence in Mycosphaerella graminicola, 163 progeny of an in vivo cross between a Dutch bread wheat-derived isolate, IPO323, and an Algerian durum wheat-derived isolate, IPO95052, are currently undergoing pathogenicity testing on a total of seven cultivars of bread and durum wheat. Preliminary results show many phenotypic recombinants and very few parental phenotypes. Although IPO323 did not infect any of the durum wheat cultivars and IPO95052 did not infect any of the bread wheat cultivars, 25% of progeny were able to infect both. Neither IPO323 nor IPO95052 were able to infect the bread wheat cultivar Shafir, but 10% of the progeny were able to produce pycnidia on this cultivar. Ten percent of progeny infected only bread wheat cultivars (at least one of four) and 25% of progney infected only durum wheat cultivars (at least one of three). Thirty percent of progeny were not able to infect any of the seven cultivars tested. Certainly, there are many genes involved in avirulence in M. graminicola. The genetics of avirulence on bread wheat and durum wheat cultivars in M. graminicola is apparently under simple genetic control. Furthermore, because a virulent isolate and an avirulent isolate can cross and produce viable offspring, it can be concluded that for M. graminicola, a resistant host does not necessarily stop gene flow.

365. Cercosporin is a virulence factor in the infection of sugar beet by Cercospora beticola. John J.Weiland1, Kuang-Ren Chung2 and Jeffrey C. Suttle1. 1Sugarbeet and Potato Research, USDA-ARS-RRVARC, Fargo, ND, 58105 and 2University of Florida, Lake Alfred Research Station, Lake Alfred, FL, 33850

A portion of the CTB gene encoding an enzyme in the cercosporin biosynthetic pathway of Cercospora species was cloned from C. beticola. Using a transformation vector harboring these sequences, the endogenous CTB gene in C. beticola was disrupted by homologous recombination. Southern and northern blot analysis confirmed that transformants lacking secretion of the red pigmented cercosporin toxin possessed a disrupted CTB gene and lacked vector integration events in other regions of the genome. Five CTB mutants (ctb-delta2, -delta3, -delta21, -delta23, and -delta24) examined produced reduced cercosporin as compared to parent C. beticola isolate 303B. All isolates possessed radial growth rates indistinguishable from 303B and retained the ability to sporulate in culture. Inoculation of sugar beet plants with the five mutants induced a lower number of leaf spot lesions that expanded at a reduced rate as compared to that produced by isolate 303B. Cercosporin accumulation in leaves infected with the mutants as compared to that from leaves infected by the parent will be presented. The data indicate that cercosporin is a virulence factor in the infection of sugar beet by C. beticola.

366. Functional analysis of CLSTE12, an STE12-like gene isolated from the bean pathogen Colletotrichum lindemuthianum. Wong Sak Hoi J., Herbert C., Dumas B. UMR 5546 CNRS-Université Paul Sabatier, Pôle de Biotechnologie Végétale, 24 chemin de Borde-Rouge, 31326 Castanet-Tolosan, France

STE12 is a transcription factor that regulates yeast mating and invasive growth. This factor is activated by a MAPKinase cascade involving KSS1. In many phytopathogenic fungi, orthologue genes of KSS1 are essential for infection, suggesting a role for STE12-like factors in pathogenicity. We isolated an STE12-like gene (CLSTE12) from Colletotrichum lindemuthianum, the causal agent of bean anthracnose. This gene encodes a 705 amino acid protein containing a homeodomain at the N-terminal region and two Cys2His2 zinc fingers at the C-terminal region. The homeodomain is present in all the proteins from the STE12 family ever identified, whereas zinc fingers are specific to filamentous fungi. We produced truncated versions of CLSTE12 in Escherichia coli, including both, one or no zinc fingers. Gel shift experiments revealed that the protein was able to bind to the yeast STE12 recognition site despite the deletions, indicating that the zinc fingers are not essential for DNA-binding. To define the function of CLSTE12, clste12 mutants were obtained by targeted gene disruption. Saprophytic growth of the mutants was indistinguishable from that of the wild type strain. These mutants were able to form appressoria, but did not produce anthracnose lesions on bean leaves, indicating that CLSTE12 is essential for the pathogenicity of C. lindemuthianum.

367. G protein signaling mediates developmental processes and pathogenesis of Alternaria alternata. Daisuke Yamagishi, Hiroshi Otani and Motoichiro Kodama. Plant Pathology Lab, Dept Agriculture, Tottori University, Tottori 680-8553, Japan.

To investigate possible signal transduction pathways that might be related to basic compatibility of toxigenic and necrotrophic A. alternata pathogens, we have cloned and characterized a Ga gene from the apple pathotype of A. alternata. In addition, we observed whether the gene controls the production of secondary metabolites, particularly host-specific toxins responsible for host-specific pathogenicity or vilrulence of the pathogen. A PCR-based approach was employed to isolate a Ga gene (designated AGA1) from the apple pathotype. AGA1 shows a high degree of similarity to other fungal Ga genes. Targeted disruption of AGA1 gave rise to mutants that differed in colony morphology, compared to the wild type, and sporulation of the mutants dramatically decreased in vitro. In addition, the mutants showed decreased conidial length and width, and few longitudinal septa were formed. The development of infection structures on cellulose membranes was also compared between the wild type and the mutants. The conidia of wild type, an ectopic transformant and the mutants showed equal germination. However, wild type germ tube formed readily from different points around the conidia, grew randomly and were often branched, whereas those of the mutants formed only at one or both ends of the conidia and tended to grow in straight paths. In addition, targeted disruption of AGA1 resulted in reduction of pathogenicity on apple leaves, although the mutant produced host-specific AM-toxins at levels similar to the wild type strain. Measurement of the intracellular cAMP levels of the mutant indicated that it was consistently higher than that of the wild type, indicating that AGA1 negatively regulates cAMP levels similar to mammalian Gai systems. These results indicate that the signal transduction pathway represented by AGA1 appears to be involved in developmental pathways leading to sporulation and pathogenesis of A. alternata

368. Molecular characterization of yeast Hsp40 homologues MHF17 and MHF23 from Magnaporthe grisea. Mihwa Yi and Yong-Hwan Lee. School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea

Magnaporthe grisea is a causing agent of rice blast disease considered as the most serious concern in rice growing areas world wide. M. grisea is a model plant pathogenic fungus with molecular genetic tractability and released genome sequences. Relied on recently developing bioinformatics tools, we discovered 26 putative HSP40s including conserved J domain in M. grisea genome database. To elucidate the functions of HSP40s in M. grisea, we preferentially determined to characterize two unique HSP40s, named as MHF17 and MHF23. MHF23, type II HSP40, is yeast Sis1 homologue, which is essential and function on initial translation stage. MHF17 has no homologue in S. cerevisiae, and belongs to type III Hsp40 family. The expression of MHF17 and MHF23 under heat shock was down regulated and no changes were detected in cold shock condition. MHF17 and MHF23 deletion mutants were generated by Agrobacterium tumefaciens-mediated transformation, which has well established as high efficient manner adopting positive negative selection strategy. Mhf17 mutant showed sectoring morphology instead of radial growth on all solid media tested, most severe on oatmeal agar media, and reduction in asexual sporulation. Abilities on germination, appressorium formation, and pathogenicity on rice were not affected, and the defects on conidiation and radial growth on solid media were recovered with insertion of MHF17 ectopically. Mhf23 mutant undergoes germination and appressorium formation normally, and fully pathogenic on susceptible rice cultivar, and further characterization of phenotypes is in progress.

369. Functional genomics of Phytophthora infestans effectors of plant disease. Thirumala-Devi Kanneganti, Carolyn Young, Jorunn Bos, Joe Win and Sophien Kamoun. Department of Plant Pathology, The Ohio State University-OARDC, Wooster, OH 44691, USA

The oomycete Phytophthora infestans is a devastating pathogen of potato and tomato. During infection, P. infestans secretes a diverse array of effector proteins that manipulate host processes, leading to virulence. We hypothesize that P. infestans secretes two classes of effectors, one that is secreted into the plant apoplast and another that is translocated into the plant cell. Our goal is to understand the molecular mechanisms underlying P. infestans-plant interactions. For this purpose, computational data mining tools and robust high throughput functional assays were combined to identify candidate effector genes. We have selected ~200 full length cDNAs encoding extracellular proteins that fulfilled at least two of the following criteria: (1) up-regulated during infection; (2) conserved between Phytophthora and saprophytic/plant pathogenic fungi; (3) contain a nuclear localization signal; (4) possess R-dEER, a highly conserved novel amino acid motif present in virulence/avirulence proteins from three different oomycetes; (5) polymorphic between P. infestans strains. Single candidate genes were expressed in planta using virus vectors to identify genes that trigger cellular and molecular responses in plant cells. We aim to support our functional data with expression profiling and subcellular localization experiments. This research will provide the basis towards understanding Phytophthora effector gene function and will establish functional connections between P. Infestans effectors and plant processes.