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 JK^1,2, Rees-George J², Hill GN^1,2, Hahn M³, Kemen E⁴, Kucheryava N³, Templeton MD², Plummer KM^1,2. 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 (http://www.broad.mit.edu/annotation/fungi/fusarium). 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 1}The 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 fpr1^mut 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 MST7^{S212D 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 MST7^12-20 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 Denisov^1,2, 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 Frp1^L226S 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 S