Host Parasite Interactions Abstracts

304. Functional analysis of Pls1p, a tetraspanin involved in pathogenicity of Magnaporthe grisea on rice. Mathieu Gourgues¹, Christophe Fargeix¹, Fabien Cottier¹, Joaquim Cots¹, Marie Pascale Latorse² and *Marc-Henri Lebrun¹. ¹CNRS-Bayer CropScience, Lyon, France.²Fungicides Biology Dpt, Bayer CropScience, Lyon, France

PLS1 is required for pathogenicity on rice of the fungus Magnaporthe grisea. pls1- mutant produces melanised appressoria with normal turgor that fail to penetrate into host leaf. Pls1p is a 225 amino acids membrane protein related to animal tetraspanins. These proteins participate to membrane signaling complexes involved the control of cell adhesion, differentiation or motility. This type of signaling pathways could be involved in appressorial mediated penetration. We showed that Pls1p was only expressed in appressoria differentiated on leaves or artificial surfaces. Pls1p differential expression is regulated at the post-transcriptional level, since PLS1 mRNA was detected in all fungal cells. Deletion analysis of PLS1 UTR's showed that the controlling sequences are located in the 5'UTR. We constructed a Gfp-Pls1p fusion protein that is functional since it complements the pls1- deletion mutant. This fusion protein was localised in appressorial plasma membrane and vacuoles. The Gfp-pls1p fusion protein was also expressed under the control of the constitutive MPG1 promoter in mycelium without visible phenotypes. Transformants over-expressing Pls1p are currently used for immuno-precipitation experiments. Domains of Pls1p were modified by site directed mutagenesis and assessed by complementation of the pls1- mutant. The small domain between TM2 and TM3 is essential for Pls1p function. Since this domain is likely to be cytoplasmic, it may be required for the interaction between Pls1p and cytoplasmic proteins. A model ofPLS1 function during fungal penetration of rice leaves will be presented.

305. cAMP signaling pathway positively regulates germination and infectious growth inColletotrichum lagenarium. Junko Yamauchi, Kenichi Komeda, Naoyuki Takayanagi, Yoshitaka Takano, and Tetsuro Okuno. Agriculture, Kyoto University, Kyoto, Japan.

Colletotrichum lagenarium, the casual agent of cucumber anthracnose, develops a specialized infection structure called an appressorium to invade host plants. Functional analysis of the regulatory subunit gene of a cAMP-dependent protein kinase (PKA) has shown that hyper-activation of PKA impaired growth, conidiation and appressorium function in this fungus. To investigate the effect of inactivation of cAMP-PKA pathway, the adenylate cyclase gene (CAC1) and the PKA catalytic subunit gene (CPK1) of C.lagenariumwere functionally characterized. The CAC1 and CPK1 genes were isolated using a PCR-based strategy with degenerate primers. The targeted disrupted mutants of each gene were generated. Both the cac1 and cpk1 mutants lacked pathogenicity to the host plant cucumber, suggesting these genes are essential for fungal pathogenicity. Conidia of these mutants hardly germinated on both the glass and host plant surfaces. Germination of the cac1mutants was restored by the addition of cAMP, whereas that of the cpk1 mutants was not. These results indicate that the cAMP-Cpk1 pathway plays important roles for germination in C. lagenarium. Furthermore, in contrast to the wild-type strain, when mycelia of thecac1 and cpk1 mutants were inoculated on wounded sites of cucumber leaves, they failed to form lesions, suggesting CAC1 and CPK1 are necessary for infectious growth inside the host plant. These results indicate that cAMP signaling positively regulates germination and infectious growth. The Cmk1 MAP kinase has also been shown to regulate germination and infectious growth in C. lagenarium, suggesting that the Cmk1 MAP kinase and cAMP signaling pathways coordinately regulate germination and infectious growth.

306. Thermotolerance conferred to a broad plant host range by an endophytic fungus isolated from a thermotolerant plant. Joan Henson, Rusty Rodriguez, and Regina Redman. Department of Microbiology, Montana State University, Bozeman, MT and USGS, Seattle, WA.

Adaptation of plants to selective pressure is considered to be regulated by the plant genome. However, recent studies indicate that fitness benefits conferred by mutualistic fungi also contribute to plant adaptation. The survival of both a plant host (Dichanthelium lanuginosum) and a fungal endophyte (Curvularia sp.) in geothermal soils is dependent on symbiotically conferred thermotolerance. This fungus also asymptomatically colonizes and confers thermal and/or drought tolerance to nonthermal-adapted eudicots and monocots. The symbiotic communication responsible for thermal and drought tolerance is different, since at least one host becomes drought, but not heat tolerant, when colonized by the fungus. Possible mechanisms of conferred drought and thermotolerance will be discussed.

307. High affinity phosphate uptake in ectomycorrhizal Tricholoma species. Erika Kothe, Katrin Krause, Doreen Müller. Microbiology, FSU Jena, Germany.

Phosphate uptake and delivery is one essential function of the fungus in ectomycorrhizal symbiosis since woodland soils generally are limited in phosphate. The phosphate is mobilized by short range substrate acidification and then transported as polyphosphate from the substrate hyphae to the plant and into the vascular tissues. For phosphate uptake into the fungal hyphae, high affinity phosphate transporters are responsible. In T. vaccinum a gene hybridizing to the N. crassa proton symporter of the high affinity phosphate transporter family was shown. The use of proton as symported ion is limited to acidic environments which allow easy access to protons. This is in accordance with medium acidification by T. vaccinum under neutral or alkaline conditions. For T. terreum found on neutral to alkaline soils a different gene hybridizing to the N. crassa sodium symporter could be shown allowing this fungus access to phosphate independent of initial medium pH. For both transporters expression was shown to be higher under phosphorus starvation with a basal expression level of the sodium symporter in T. terreum at high phosphate concentrations.

308. APH1, a gene encoding a putative methyltransferase, is involved in appressorial penetration into the host plant by Colletotrichum lagenarium. Naoyuki Takayanagi, Yoshitaka Takano, Akiko Kimura, and Tetsuro Okuno. Department of Agriculture, Kyoto University, Kyoto, Japan.

Colletotrichum lagenarium, the casual agent of cucumber anthracnose, invades into the host plant using specialized infection structures called appressoria. A pathogenicity-deficient mutant KE51 was isolated by restriction enzyme-mediated DNA integration (REMI) mutagenesis. Molecular analysis of the mutant KE51 identified the APH1 gene as a gene disrupted by the plasmid insertion. The aph1 knockout mutants were generated by target gene replacement. The aph1 mutants showed significant reduction in pathogenicity the same as the original REMI mutant KE51. This indicates that the APH1 gene is required for fungal pathogenicity in C. lagenarium and that the plasmid insertion intoAPH1 is responsible for the reduced pathogenicity of KE51. Deduced amino acid sequence of Aph1 has a high homology with the methyltransferase-related proteins of other organisms. Aph1 conserved motifs commonly found in AdoMet-dependent methyltransferases. The aph1 mutants exhibited the phenotype similar to that of the wild type in colony growth, conidiation, conidial germination, and appressorium formation. By inoculation through wounded sites, the aph1 mutants formed lesions like the wild-type strain, suggesting that APH1 is not essential for infectious growth inside the host plant. However, the aph1 mutants showed a severe reduction in penetration into the host plant, indicating thatAPH1 is involved in a penetration step. In contrast, the aph1 mutants efficiently penetrated into cellulose membranes like the wild type. These data strongly suggest that a putative methyltransferase encoded by APH1 is specifically involved in appressorial penetration into the host plant.

309. The SNF1 gene is required for appressorium maturation and fungal pathogenicity inColletotrichum lagenarium. Makoto Asakura, Naoyuki Takayanagi, Yoshitaka Takano, and Tetsuro Okuno. Department of Agriculture, Kyoto University, Kyoto, Japan.

Colletotrichum lagenarium is the causal agent of cucumber anthracnose disease. C. lagenarium forms specialized infection structures called appressoria under poor nutrient conditions such as on host plant and artificial glass surfaces. Functional analysis of a peroxisomal biogenesis gene PEX6 (ClaPEX6) demonstrated that peroxisomal metabolic function is required for maturation of appressoria in this fungus. It has been shown that Snf1 protein kinases play a role as a metabolic sensor in several organisms. To assess the relation between appressorium formation and metabolic regulation, we investigated roles ofSNF1 (ClaSNF1) in C. lagenarium. ClaSNF1 was isolated using a PCR-based screen with degenerate oligonucleotide primers and the nucleotide sequence of this gene was determined. ClaSNF1 encodes a protein of 738 amino acids and exhibited significant homology to Snf1 proteins in other organisms. The clasnf1 knockout mutants were generated by target gene disruption. The clasnf1 mutants exhibited reduction in vegetative growth on nutrient media. The mutants lacked pathogenicity to the host plant, indicating essential roles of ClaSNF1 for fungal pathogenicity. The mutants germinated effectively on the glass surface, and germ tubes differentiated into swollen appressoria. However, appressoria formed by the clasnf1 mutants were relatively small and less melanized compared with those formed by the wild type, indicating that ClaSNF1 is required for appressorium maturation. Our findings that ClaSNF1 and ClaPEX6 are commonly required for appressorium maturation suggest a possibility of involvement ofClaSNF1 in regulation of peroxisomal metabolism.

310. Novel developmental processes associated with infection of roots by the rice blast fungus. Ane Sesma and Anne E. Osbourn. The Sainsbury Laboratory, John Innes Center, Colney Lane, Norwich NR4 7UH, U.K

Although the rice blast fungus Magnaporthe grisea is traditionally regarded as a foliar pathogen, this fungus can also cause disease symptoms on cereal roots. M. grisea is closely related to other pathogenic (M. poae, M. rhizophila andGaeumannomyces graminis) and non-pathogenic (Phialophora spp.) root-infecting fungi. Many of the genes that are required for pathogenesis-related development during infection of leaves are dispensable for root infection. Remarkably, M. grisea is capable of undergoing a range of developmental processes that are typical of root pathogens and forms microsclerotia, runner hyphae and hyphopodia. Bulbous swollen hyphae can be observed within the root cortex, and the fungus progresses through the cortex to invade the stele. Infection studies with GFP-expressing transformants indicate that M. grisea can spread from the roots to the leaves and produce lesions, suggesting that the soil may be a source of inoculum for the establishment of rice blast disease in the field. Mutants that are defective in infection of rice roots have been isolated following Agrobacterium -mediated random insertional mutagenesis and new genes required for root colonisation are being characterised. This research is financially supported by a Marie Curie Fellowship of the European Community. The Sainsbury Laboratory is supported by The Gastby Charitable Foundation.

311. Identification and characterisation of five hydrophobin genes in Fusarium verticillioides. Uta U Fuchs ¹ and James A Sweigard². ¹Department of Plant and Soil Sciences, University of Delaware, Newark, DE, USA ²DuPont, Crop Genetics, Newark, DE, USA

We have identified five hydrophobin genes in Fusarium verticillioides, a major corn pathogen. Hydrophobins are small, secreted fungal proteins with a characteristic spacing of eight cysteines. The hydrophobin genes were identified from cDNA libraries and from genomic sequencing efforts. FvHYD1 and FvHYD2, encoding class I hydrophobins, were both highly expressed in liquid mycelial cultures. Their potential gene products, FvHyd1p and FvHyd2p, are 80% similar. Class II hydrophobins FvHYD4 and FvHYD5 were obtained from transcripts in microconidia and from genomic sequence, respectively. FvHYD3 messages were found with low abundance in a range of culture conditions. Compared to other hydrophobins, FvHyd3p has only four amino acids between the third and fourth cysteine whereas a 17-39 amino acid loop is common in class I hydrophobins and an 11 amino acid loop in class II hydrophobins. Null mutants were created by gene replacement for each of the genes. None of the mutants showed phenotypic differences from the wild-type in the rate of radial growth and in the number of conidia produced on solid medium, as well as in the amount of disease caused in a corn seedling infection assay. Since FvHYD1 and FvHYD2 are highly expressed and since FvHyd1p and FvHyd2p are very similar, we speculate that either gene can compensate for the absence of the other and hypothesize that a double mutant missing both genes will show a measurable phenotype in growth and infection assays.

312. A unique binuclear zinc transcription factor regulates the Fusarium solani cytochrome P450 virulence gene responsible for detoxification of the host's phytoalexin. David Straney¹, Reynold Tan¹, Lydia Rivera², and Ever Ponciano¹. ¹ Dept. Cell Biol. & Mol. Genetics, University of Maryland, USA, ² University of Puerto Rico, USA.

Fusarium solani (teliomorph:Nectria haematococca MPVI) induces several virulence traits upon exposure to pisatin, the isoflavonoid phytoalexin produced by its host plant. One response is the induced transcription of a cytochrome P450 (PDA1) that detoxifies pisatin. Although regulators of xenobiotic detoxification-associated P450s belong to conserved families of nuclear receptors in metazoans, these families are absent in fungi. Do fungi regulate their detoxification-associated P450 genes in a parallel manner? We have identified a pisatin-responsive element in the promoter for the Fusarium cytochrome P450 gene and cloned a gene encoding a binuclear zinc transcription factor that binds this element. Use of RNAi to suppress expression of the native transcription factor abolishes pisatin induction of the native cytochrome P450 gene. Further, transfer of the Fusarium transcription factor and pisatin-responsive elements into a pisatin-naive heterologous fungal system confers strong pisatin-responsive expression of a reporter gene linked to the binding elements. High specificity in this heterologous pisatin-response suggests that the cloned transcription factor is acting as a receptor for pisatin. Such mode of action would provide a model for studying fungal perception of host plants through chemical cues.

313. Analysis of differential gene expression during the symbiosis betweenNeotyphodium lolii and perennial ryegrass. Richard D. Johnson, Shalome A. Campbell and Gregory T. Bryan. AgResearch Ltd., Palmerston North, New Zealand.

Neotyphodium lolii is a fungal endophyte that lives entirely within the intercellular spaces of its grass host, perennial ryegrass. The association is mutually beneficial since the endophyte confers a number of biotic and abiotic advantages to the host, including enhanced plant growth, protection from certain mammalian and insect herbivores, enhanced resistance to nematodes and some fungal pathogens and in some associations enhanced drought tolerance. We are interested in studying the molecular basis of this important symbiosis and hope to identify genes which are important in both its establishment and maintenance, some of which could be fundamental to understanding how plant-fungal interactions, particularly evasion of host defences, are regulated. Using Suppressive Subtractive Hybridisation (SSH), we have generated subtractive cDNA libraries from N. lolii infected and un-infected perennial ryegrass. Genes up-regulated or down-regulated during these symbioses are currently being identified. In a parallel approach, proteins which appear to be up or down regulated between the infected and un-infected state have been isolated by 2D-gel electrophoresis and subjected to MALDI-TOF mass spectrometry for identification. Crucial to this, is the generation of a fungal sequence database comprising random genomic sequences, SSH sequence data, and fungal expressed sequence tags derived from liquid culture. Funding by FRST (NZ) is acknowledged.

314. Cloning of a gene encoding an Alt a 1 isoallergen differentially expressed by the necrotrophic fungus Alternaria brassicicola during Arabidopsis infection. Robert A. Cramer, Juan Wang, and Christopher B. Lawrence. Colorado State University, Department of Bioag. Sciences Fort Collins, Colorado 80523-1170

Species of Alternaria are considered some of the most important fungi responsible for allergenic morbidity in humans. The Alternaria protein that elicits the most intense allergic reaction in humans is Alt a 1, yet, no known biological function has been identified for this protein. In this study, suppression subtractive hybridization and virtual northern blots were used to identify and characterize an Alt a 1 homolog in the phytopathogenic fungus Alternaria brassicicola. RNA was extracted from A. brassicicola spores germinated in water and on the leaf surface of the Arabidopsis ecotype Landsberg for 24 hours, and used to create cDNA using PCR. Double stranded cDNA was then used in suppression subtractive hybridization to identify differentially expressed genes. Messenger RNA transcript levels were assessed by virtual northern blots. A sequence with significant homology (90% amino acid, 92% cDNA) to the Alt a 1 subunit from Alternaria alternata was identified. Virtual northern blots demonstrated that this homolog, designated Alt b 1 precursor, was highly up-regulated during the infection process of A. brassicicola on Arabidopsis. The full length cDNA sequence of Alt b 1 was 815 bp, with an open reading frame of 477 bp. To functionally analyze the role of this allergenic protein, a 500 bp internal cDNA fragment was sub-cloned into the fungal transformation vector pCB1636. PEG-mediated protoplast transformation was performed to create Alt b 1 knockout mutants. We are currently analyzing phenotypic changes in the mutants with disrupted Alt b 1 ORFs.

315. Identification of Alternaria brassicicola genes differentially expressed during pathogenesis on Arabidopsis thaliana using Suppression Subtractive Hybridization. Robert A. Cramer and Christopher B. Lawrence. Colorado State University, Department of Bioagricultural Sciences Fort Collins, Colorado 80523-1170

Necrotrophic fungal pathogens are responsible for some of the world's most devastating plant diseases. Alternaria brassicicola (Schwein.) Wiltshire is a necrotrophic fungus that causes black spot disease on a wide range of cruciferous hosts including the model plant Arabidopsis. The objective of this study was to identify genes up-regulated during the early stages of A. brassicicola infection on Arabidopsis. Suppression subtractive hybridization (SSH) was employed to create a cDNA library enriched for such genes. Fungal spores were germinated either in sterile water or on leaves of the susceptible Arabidopsis ecotype Landsberg erecta (Ler). After a 24 hr incubation period at 24°C, RNA was extracted from these two fungal spore samples and used to create cDNA populations for use in SSH. Subtraction was performed between these cDNA populations to create a library enriched for genes unique to the spores germinated on the plant leaf surface. Up-regulation of clones corresponding to individual genes was confirmed using a dot blot technique coupled with virtual northern analysis. Fifty up-regulated clones were selected and sequenced. Database homology searches using blastn and blastx revealed sequences with homology to a putative arsenite ATPase translocase (ABC transporter), translation initiation factor, various glycoproteins, Alternaria allergen precursor, cyanide hydratase, and formate dehydrogenase that may be involved in pathogenesis. Based on the results obtained, SSH is an effective technique to identify fungal genes that may be important in the early stage of infection. Future research will involve functional characterization of these putatively important pathogenesis-related genes.

316. Molecular Dissection of the Stagonospora nodorum - wheat interaction. Peter S. Solomon, Kar-Chun Tan, T.J. Greer Wilson, Robert C. Lee, Simon Ip Cho, Kerrie Parker & Richard P. Oliver. Australian Centre for Necrotrophic Fungal Pathogens, W.A. State Agricultural Biotechnology Centre, Division of Science and Engineering, Murdoch University, Perth 6150, Western Australia, Australia.

The Australian Centre for Necrotrophic Fungal Pathogens (ACNFP) has been recently established on the west coast of Australia. The ACNFP was created to develop an understanding of necrotrophic fungal pathogen/host interactions at the molecular level, particularly those affecting Australian crops. One such project within the centre is focused on the interaction between the fungus Stagonospora nodorum and wheat. Stagonospora nodorum is the causal agent of leaf and glume blotch on wheat and is responsible for $$60M (AUD) of crop loss in Australia each year. Whilst also appearing to be an economically important pathogen throughout the world, very little is known at a molecular level about how the fungus infects wheat. We have begun dissecting this interaction using a variety of molecular techniques including the generation of EST libraries, gene expression analysis, bioinformatics and high throughput gene knockouts. Several genes, including those involved in transport, signal transduction and novel metabolic processes, have been characterised by gene disruption and expression analysis. This poster will review the phenotypic effects of these gene disruptions as well as examining their requirement for pathogenicity.

317. Random Insertional Mutagenesis of the fungus Leptosphaeria maculans, identifies two pathogenicity genes and leads to discovery of Repeat Induced Point (RIP). Alexander Idnurm *, Leanne M Wilson and Barbara J Howlett. School of Botany, The University of Melbourne, Victoria 3010, Australia. * Current address: Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC 27110 USA

Insertional mutagenesis generated pathogenicity mutants of Leptosphaeria maculans, the dothideomycete that causes blackleg disease of Brassica napus. Two mutants had single copy insertions of the plasmid pUCATPH that encodes hygromycin resistance. In one, the isocitrate lyase gene was mutated. As expected, this mutant did not grow on fatty acids including monolaurate and when the wildtype gene was reintroduced, growth on monolaurate was restored and pathogenicity was partially restored. When 2.5 % glucose was added to this mutant, pathogenicity was restored. These findings suggest that the glyoxylate pathway is essential for disease development by L.maculans. The second pathogenicity mutant had pUCATPH inserted upstream of an open reading frame of 529 amino acids with a weak database match tohet-s of Podospora anserina. Reintroduction of a wildtype copy of the gene restored this mutant's ability to form lesions on B. napus cotyledons. The role of this gene in plant disease is unknown. When seven pathogenicity mutants with multiple insertions of pUCATPH were crossed to an isolate that attacks B. napus, progeny with the hygromycin resistance gene were hygromycin-sensitive. Sequence analysis of an amplified fragment of pUCATPH in six clones derived from one ‘silenced' progeny showed mutation of GC to AT on one DNA strand, reminiscent of repeat-induced point mutation (RIP) in Neurospora crassa.

318. A putative secondary metabolite cluster of the blackleg fungus, Leptosphaeria maculans. Donald M. Gardiner¹ and Barbara J. Howlett¹.¹School of Botany, Melbourne University, Victoria, 3010 Australia.

The blackleg fungus, Leptosphaeria maculans causes major yield losses to canola (Brassica napus) worldwide. Random sequencing of clones in a library of Expressed Sequence Tags prepared from mycelia of L. maculans grown in complete medium identified a gene with homology to prenyl transferases in the ergot alkaloid biosynthesis cluster of Claviceps purpurea, and paxilline biosynthesis cluster of Penicillium paxilli. Sequencing of a L. maculans cosmid clone containing the prenyl transferase and an overlapping cosmid revealed the presence of a putative cluster of genes with predicted roles in various aspects of secondary metabolism. These genes include a zinc finger transcriptional protein, a peptide synthetase gene, two oxidoreductases, a methyl transferase, two cytochrome P450s, an ATP-binding cassette-type transporter protein and a glutathione-S-transferase. The function of genes in this cluster is being characterised by reverse genetics (gene knockout), transcriptional profiling and by complementation of isolates of a closely related Leptosphaeria species lacking this cluster.

319. Regulation of maize-induced genes in Ustilago maydis. Jan W. Farfsing, Regine Kahmann and Christoph W. Basse. Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Straße, 35043 Marburg, Germany

The facultative biotrophic fungus Ustilago maydis causes smut disease in its host plant maize. We have identified the fungal mig1 and mig2 genes as being specifically and strongly up-regulated during biotrophic growth. The mig2 genes are comprised in a gene cluster of five highly homologous and similarly regulated genes designated mig2-1 to mig2-5. All mig genes lack significant homologies in the database. The mig 1 as well as the mig2-5 promoters were subject to repression, which was partially relieved in U. maydis mutants of hda1, encoding the chromatin modifying enzyme histone deacetylase 1. However highest transcript levels were always encountered during biotrophic growth, indicating the presence of additional regulators. Reconstitution experiments comprising mig2-5 promoter fragments led to the identification of short, positive cis-acting elements required for high transcript levels during biotrophic growth. Negative as well as positive cis-acting regions of the mig2-5 promoter were mapped in close proximity to each other within the region from position –240 to –119 upstream of the translational start ATG. All mig2 promoters shared significant homologies in this region. For this reason we used this element to screen for the respective regulator based on a yeast one-hybrid assay comprising cDNA libraries from different stages of the fungal life cycle.

320. Prf1 integrates pheromone and cAMP signaling in the phytopathogen Ustilago maydis. Florian Kaffarnik, Marc Leibundgut, Philip Müller, Regine Kahmann and Michael Feldbrügge, Max-Planck Institute for Terrestrial Microbiology, Department of Organismic Interactions, Marburg, Germany

The basidiomycete Ustilago maydis causes smut disease on corn. The fungus switches from a haploid form that proliferates by budding to a filamentously growing dikaryon, the infectious form. A complex signalling network in which an evolutionarily conserved cAMP signalling pathway communicates with a MAP kinase branch regulates this developmental programme. To investigate the involved signalling processes we focus on initial events: the fusion of two haploid cells. Like in comparable model organisms such as Saccharomyces cerevisiae or Schizosaccharomyces pombe mating of two compatible cells is controlled by a pheromone/receptor system that regulates conjugation tube formation and gene expression via a MAP kinase module. Here, we present evidence that the activity of pheromone response factor 1 (Prf1), a transcription factor that activates expression of mating type genes, is regulated on the posttranscriptional level by cAMP as well as MAP kinase signalling. This regulation is most likely achieved by the action of the cAMP-regulated protein kinase A and / or the pheromone-activated MAP kinase. Thus, according to our current model Prf1 functions as an integrator between cAMP and pheromone signalling.

321. An ELAV-like RNA-binding protein regulates filament development and pathogenicity in Ustilago maydis. Philip Becht and Michael Feldbrügge, Max-Planck Institute for Terrestrial Microbiology, Department of Organismic Interactions, Marburg, Germany

The basidiomycete Ustilago maydis causes smut disease on corn. Prerequisite for pathogenic development is the fusion of two haploid cells resulting in formation of a filamentously growing dikaryon, the infectious form. The fusion process is regulated by a tetrapolar mating system that consists of the biallelic a locus and the multiallelicb locus. The genes at the a locus encode lipopeptide pheromone precursors and cognate receptors involved in intercellular recognition. At the blocus a pair of homeodomain proteins is encoded that is only functional as a heterodimeric transcription factor with monomers from different allelic origin. Since RNA-binding proteins are key regulators of differentiation processes in other model systems, such as D. melanogaster or C. elegans, we hypothesise that RNA-binding proteins are among the effectors regulating pathogenic development in U. maydis. Here we introduce Rrm4 identified by searching the genomic sequence for RRM domains. Rrm4 contains a novel domain structure containing 3 RRM domains as well as a C terminal polyA domain. Thereby it resembles ELAV proteins as well as polyA-binding proteins. In mating experiments with compatible rrm4delta strains a reduced filament formation was observed, indicating that formation of the infectious form is impaired. In plant infection experiments rrm4delta strains exhibit a drastically reduced virulence if compared to wild type. Hence, Rrm4 appears to be important for filament development and pathogenicity.

322. An extracellular protease inhibitor from Phytophthora infestans targets tomato serine proteases: a counter-defense mechanism? Miaoying Tian, Sophien Kamoun. Department of Plant Pathology, The Ohio State University, Wooster, OH44691, USA

The plant intercellular space is the battlefront where complex interactions between extracellular proteins from the oomycete pathogen Phytophthora infestans and its host plants occur. We used data mining of Phytophthora sequence databases to identify eleven genes encoding putative extracellular protease inhibitors (EPIs) with one to three predicted domains of Kazal-type serine protease inhibitors that are commonly found in animals. The EPI proteins did not show similarity to protease inhibitors from plant pathogens or plants suggesting that they might reveal novel molecular mechanisms in plant-microbe interactions. In vitro protease inhibition assays of three purified EPI fusion proteins against several commercial serine proteases revealed that EPI1 highly inhibits bacterial subtilisin, thus confirming the protease inhibition function predicted by bioinformatics. EPI1 was further demonstrated to inhibit and interact with tomato P69 subtilisin-like proteases, among which P69B and P69C are known to be pathogenesis-related proteins involved in plant defenses. We also found that EPI1, but not EPI2 and EPI3, is resistant to degradation by tomato intercellular fluids. Interestingly, EPI1 was able to protect EPI2 from degradation suggesting that inhibition of host proteases might help secreted proteins of Phytophthora resist proteolytic cleavage in the plant apoplast. Overall our results suggest that inhibition of plant proteases by P. infestans EPI1 could form a novel type of defense-counterdefense cross talk between plants and microbial pathogens.

323. cAMP signal transduction regulates virulence of the human-pathogenic fungus Aspergillus fumigatus. Burghard Liebmann, Stephanie Gattung, Bernhard Jahn and Axel Brakhage. Microbiology Institute, University of Hannover, Germany and University Hospital, Weisbaden.

Aspergillus fumigatus is an important pathogen of the immunocompromised host, causing pneumonia and invasive disseminated disease with high mortality rates. We have cloned three conserved components of the cAMP signal transduction cascade, the adenylate cyclase (acyA), a Ga subunit (gpaB) and a catalytic subunit of protein kinase A (pkaC). Deletion of the genes revealed, that all components influence growth, development and sporulation of A. fumigatus. Conidia of acyA and gpaB deletion mutants were killed more efficiently in a macrophage assay. Furthermore, a possible cAMP-dependent regulation of the A. fumigatus virulence determinant pksP, which encodes a polyketide synthase, was investigated by constructing a pksP-promoter lacZ-fusion. pksP-expression was significantly reduced in gpaB deletion backround compared with the expression of the gene fusion in a wild-type strain. We also started to investigate the cAMP-dependent pksP-regulation by promoter mutation analysis, in order to fully understand the role of PKSP during infection.

324. The analysis of the phylogenetic distribution of the pea pathogenicity (PEP) gene cluster of Nectria haematococca MPVI supports the hypothesis of its acquisition by horizontal transfer and uncovers a new fungal pathogen of garden pea: Neocosmospora boniensis. Esteban D. Temporini and Hans D. VanEtten. Department of Plant Pathology, University of Arizona, Tucson AZ.

The filamentous fungus Nectria haematococca Mating Population VI (MPVI) contains a cluster of genes required to cause disease on pea. The pea pathogenicity orPEP cluster is located on a supernumerary chromosome that is dispensable for normal growth in culture. A comparison of the G+C content and codon usage of the genes in thePEP cluster indicates that they differ from genes located on non-dispensable chromosomes in this fungus. These features, and the presence of several sequences with homology to transposable elements in this region, suggest the possibility that the PEPcluster was acquired through a horizontal gene transfer event. In this work, we show that homologs of the PEP genes have a scattered distribution among fungi belonging to theFusarium solani species complex, which are phylogenetically closely related to N. haematococca MPVI. However, homologs to most of the PEP genes were detected in Fusarium oxysporum f. sp. pisi, a pea pathogen distantly related to N. haematococca MPVI. This phylogenetic incongruence supports the hypothesis of a horizontal transfer origin of the PEP cluster. Our analysis has also determined that homologs for all the PEP genes are present inNeocosmospora boniensis, another member of the F. solani complex. A molecular characterization of the PEP homologs in this fungus has shown that they are organized as a cluster, which has a different physical organization compared to the N. haematococca PEP cluster. In addition, pathogenicity tests have revealed that N. boniensis is virulent on pea although no reports have been found to show this species as a naturally occurring pea pathogen.

325. Functions of Mg-NCS1 and LPL1, genes expressed in germ tube of Magnaporthe grisea. Takashi Kamakura¹, Ken-ichiro Saitoh^{1, 2}, Fumi Ishii², Masaki Kanamori², Minoru Yoshida¹ and Tohru Teraoka². ¹RIKEN Institute, Wako, Japan.²Tokyo University of Agriculture and Technology, Fuchu, Japan

 The conidial germ tube of the rice blast fungus, Magnaporthe grisea, differentiates an infection specialized cell, an appressorium, required for penetration into the host plant. We have constructed a cDNA subtractive differential library from the appressorium forming germ tube. From the library, some genes were pulled out and analyzed. A gene, Mg-NCS1, homologous to the Neuronal Calcium Sensor 1 (NCS1) family, was isolated and null mutants were generated in order to see its function. Different from other organisms' NCS1 family,Mg-NCS1 seemed to play an important role in tolerance against pH stress. Another gene,LPL1, was homologous with lysophospholipase gene. The disruptant of LPL1reduced ability to differentiate appressorium on hydrophobic surface and also showed delay of formation of infection-peg on onion epidermis strip and rice leaf sheath compared with the wild-type strain. Since phospholipids metabolic pathways are related to glycerol and diacylglycerol biosynthesis, LPL1 may be involved in the penetration of infection peg. However, these genes did not seem to have essential functions for pathogenic cycle because both of null mutants (Mg-ncs1- and Lpl1-) kept potential ability to make lesions in rice leaves.

326. The Cross-Pathway Control of the Opportunistic Pathogen Aspergillus fumigatus Cloning and Characterisation of its Transcriptional Activator CPCA. Sven Krappmann¹, Utz Reichard², Verena Grosse¹, and Gerhard H. Braus¹¹Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics. ²University Hospital, Center for Hygiene and Human Genetics, Department of Bacteriology. Georg-August University, Goettingen, Germany

The filamentous saprophyte Aspergillus fumigatus accounts for the majority of aspergillosis incidents, which represent a severe threat mainly to immunocompromised individuals. Detailed knowledge on factors contributing to pathogenicity of this opportunistic pathogen is scarce with only few determinants identified. Nutritional requirements and metabolic features rendering the fungus infective have not been investigated in great detail. We have focused on the Cross-Pathway Control (cpc) system of A. fumigatus, a global regulatory network acting on amino acid biosynthesis to counteract conditions of starvation or imbalance. The cpcA locus encoding the transcriptional activator protein of the system was identified and characterised. Its gene product represents a highly conserved protein that is the functional orthologue of Gcn4p, the yeast master regulator of gene expression upon starvtion and stress. Generation of cpcA deletion mutants revealed its central role for the cpc response of this fungus upon amino acid starvation. Next steps aim at the impact of the cpc system on pathogenicity of A. fumigatus, the current status of the project will be presented.

327. Molecular characterization of a calcineurin A gene, MgCNA, in Magnaporthe grisea. Jinhee Choi, Yangseon Kim, Soonok Kim and Yong-Hwan Lee, School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea

Magnaporthe grisea, the causal agent of the rice blast, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration. Pharmacological data suggests that calcium/calmodulin-dependent signaling is involved in appressorium formation in this fungus. Calcineurin is a calcium/calmodulin-activated protein phosphatase composed of a heterodimer of a catalytic (CnA) and a regulatory (CnB) subunit. To understand the role of calcineurin on appressorium formation at molecular level, a gene (MgCNA) encoding calcineurin catalytic subunit was cloned and characterized from M. grisea 70-15. MgCNA contains an ORF of 1,920 bp, encoding 537 amino acids. The overall order and amino acids of protein domains of MgCNA are conserved with those of other filamentous fungi. Transformants expressing antisense of MgCNA exhibited significant reductions on mycelial growth rate, conidiation and appressorium formation, and different colony morphology and hyphal branching patterns. These pleiotropic effects suggest that calcineurin A plays important roles on signaling transduction pathways involved in fungal developments of M. grisea.

328. Ruderal and combatative strategies in insect pathogenic fungi examined by EST analysis. Gang Hu, Florian M. Freimoser and Raymond J. St. Leger. Department of Entomology, University of Maryland, 4112 Plant Sciences Building, College Park, MD, 20742, USA

Metarhizium anisopliae (Ascomycota) and Conidiobolus coronatus (Zygomycota) are facultative saprophytes that are pathogenic to many insect species. About 2000 EST cDNA clones from each species were sequenced to analyze gene expression during growth on host insect cuticle and/or nutrient rich media. Proteases were the commonest transcripts with both species producing multiple subtilisins, as well as trypsin, metalloprotease and aspartyl protease activities capable of degrading host tissues and disabling anti-microbial peptides. However, compared to M. anisopliae, C. coronatus produced fewer extracellular hydrolases (e.g., no phospholipases), antimicrobial agents, toxic secondary metabolites and no ESTs with putative roles in the generation of antibiotics. Instead, C. coronatus produced a much higher proportion of ESTs encoding ribosomal proteins and enzymes of intermediate metabolism consistent with the rapid growth characteristics of C. coronatus. These results are consistent with M. anisopliae using a combatative strategy to defend captured resources, while C. coronatus has modified the saprophytic ruderal-selected strategy using rapid growth to overwhelm the host and exploit the cadaver before competitors overrun it. Both strategies are consistent with specialization to pathogenicity. Thus, induction of proteases by host cuticles (mucoralean fungi do not produce proteases under these conditions) demonstrates that C. coronatus is adapted to entomopathogenicity.

329. EST and microarray analysis of pathogenicity factors of the insect pathogen Metarhizium anisopliae. Gang Hu, Florian M. Freimoser, Steven Screen, Savita Bagga and Raymond St. Leger Department of Entomology, University of Maryland, 4112 Plant Sciences Building, College Park, MD, 20742, USA

ESTs and microarrays offer unprecedented opportunities for analysis of pathogenicity in fungi. ESTs of the insect pathogen Metarhizium anisopliae were obtained under growth conditions that optimize the secretion of many known pathogenicity factors. The EST sequences revealed new unsuspected stratagems of entomopathogenicity and previously unreported categories of biologically active molecules, as well as many enzymes involved in the degradation of host barriers. For example, M. anisopliae produces more proteinolytic enzymes than any other fungus studied to date. These comprise at least 11 different subtilisins as well as multiple trypsins, metalloproteases, chymotrypsins, etc. To analyze gene expression during the infection process cDNA fragments were arrayed onto glass slides. Genes encoding components of membrane biogenesis, synthesis of cell wall components, storage or mobilization of nutrient reserves and protein folding (particularly calnexin-always associated with protein secretion in 2575) are also highly expressed during appressorial differentiation indicative of manufacture and "remodeling" of cell structure. Other features of M. anisopliae physiology highlighted by this work include the production of antimicrobial molecules (presumably to defend limited resources within the insect cadaver) and the very early cuticle-induced production of variety of transporters and permeases that allow the fungus to "sample" the cuticle by absorbing peptides and amino acids and then respond with secretion of a plethora of proteins. Our results also suggest that many differences in the phenotypes of different strains result from genes with similar sequences but dissimilar expression patterns.

330. Patchy distribution of trypsin genes in fungi. Gang Hu, Florian M. Freimoser steven Screen, Savita Bagga, and Raymond J. St. Leger Department of Entomology, University of Maryland, 4112 Plant Sciences Building, College Park, MD, 20742, USA

The trypsin family of proteases have homologs in streptomycetes, five pathogenic ascomycetes and animals. This patchy distribution is consistent with: 1) components of the genetic apparatus of ascomycetes being derived from an ancestor of the streptomyces via horizontal gene transfer, or 2) rampant gene duplication, divergence and gene loss in different fungal lineages. In order to reconstruct the evolution of trypsin diversity we related the presence or absence of trypsins to the phylogenetic relationship of 46 representative fungi. Our results are consistent with niche-specific traits, that are traits shared by fungi that occupy the same niche irrespective of phylogenetic position. We found that trypsins are produced by: 1) many plant and insect pathogenic pyrenomycetous ascomycetes; 2) the basidiomycete Septobasidium canescens that has a symbiotic relationship with aphids that occasionally includes digesting them, and 3) some entomopathogenic entomophoralean zygomycetes (Conidiobolus coronatus, Zoophthora spp.). Trypsins are lacking in saprophytic ascomycetes including yeast, N. crassa and A. nidulans as well as saprophytic zygomycetes and basidiomycetes. The observed phylogenetic distribution of the trypsin orthologs is largely in agreement with an unconstrained rDNA tree that is itself consistent with the current consensus on fungal phylogeny. This indicates that these proteins have diverged in parallel with the organisms in which they are expressed. Several species such as Paecilomyces, which have no trypsins, are nested between species that do, indicating that the trypsin has been lost in the lineage leading to that species. Overall, comparative studies suggest that individual genes, such as the trypsins have been lost many times independently in different lineages, and that the flux of genes is an ongoing process. This is clearly seen in the specialized M. anisopliae strain 324 that unlike generalist strains of the same species contains a silent trypsin pseudogene.

331. In Ustilago maydis the MAP kinase Kpp6 is required for successful penetration of the plant surface. Andreas Brachmann, Jan Schirawski, Philip Müller and Regine Kahmann. Max Planck Institute for terrestrial Microbiology, Marburg, Germany.

To complete its life cycle, the plant pathogenic fungus Ustilago maydis has to invade the host plant tissue. During this process dikaryotic filaments form appressorium-like infection structures from where growing hyphae extend into the plant tissue. In Ustilago maydis filamentous growth and pathogenic development depend on the expression of the regulatory bE/bW complex. We have identified by RNA fingerprinting a b-regulated gene, kpp6, encoding a MAP kinase with similarity to other fungal MAP kinases involved in mating and pathogenicity. Kpp6 is unusual in that it contains an N-terminal domain unrelated to other proteins. kpp6 deletion mutants are unaffected in mating and dikaryon formation but show attenuated pathogenic development. kpp6^T355A,Y357F mutants carrying a non-activatable allele of kpp6 are severely compromised in pathogenicity. These strains can still form appressoria, but are defective in the subsequent plant surface penetration step. A Kpp6-GFP fusion is expressed during all stages of the sexual life cycle. Expression of the kpp6 gene yields two transcripts that are differentially expressed. We have analyzed these transcripts and present evidence that the smaller one is regulated through Prf1 while the longer one is regulated indirectly through the bE/bW heterodimer.

332. Signalling in the plant pathogen Ustilago maydis: A comparative genomics approach. Heiko Eichhorn, Joerg Kaemper, Philip Mueller and Regine Kahmann. Max Planck Institute for terrestrial Microbiology, 35043 Marburg, Germany.

Ustilago maydis is the causative agent of corn smut disease. The pathogenic dikaryotic form is generated after mating of two compatible cells. Results from several laboratories have shown that the signalling pathways required for transmission of the pheromone signal during mating are also needed during pathogenic development. In particular, the components of a MAP kinase module as well as tightly regulated cAMP signalling are needed for disease progression. Recent results suggest that these pathways have partially overlapping as well as distinct functions. To analyse these pathways in more detail we have performed a comparative transcriptome analysis. To this end, strains were generated which allow to activate the different signalling pathways. Wild type alleles or non-phosphorylatable alleles of the two MAP kinases kpp2 and kpp6, respectively, were introduced into strains that harbour a constitutively active allele of the MAPKK fuz7. The expression of the fuz7 allele is made inducible by the crg1 promoter which is ON in arabinose and OFF in glucose. In addition we have generated a strain that expresses the catalytic subunit of the PKA (adr1) under control of the crg1 promoter. The transcription profile of pheromone stimulated wild type cells was included for comparison. RNA was isolated at various time points and analysed by whole genome microarrays (Affymetrix). We will present these data and discuss their impact for the identification of genes that are functionally significant for pathogenic development.

333. Establishment of the sexual cycle of Cryptococcus neoformans varietygrubii and virulence of congenic a and alpha isolates. Kirsten Nielsen and Joseph Heitman. Dept. Molecular Genetics and Microbiology, Duke University Medical Center and The Howard Hughes Medical Institute, Durham, NC

Cryptococcus neoformans is an opportunistic human pathogen that infects the central nervous system of immunocompromised individuals. This basidiomycete has evolved into three distinct varieties. A heterothallic sexual cycle with haploid a and alpha cells has been defined for one variety (neoformans, serotype D), but the most common pathogenic variety (grubii, serotype A) was thought to be clonal and restricted to the alpha mating type. We have recently identified multiple serotype A a-mating type strains from a worldwide screen of greater than 500 strains. We characterized one of these unusual isolates (strain 125.91) and show it is an authentic haploid serotype A a-mating type strain. The aA strain 125.91 is capable of mating with a subset of pathogenic serotype A alpha strains to produce filamentous dikaryons with fused clamp connections, basidia, and viable recombinant basidiospores. Congenic serotype A a and alpha strains were generated and analyzed for virulence potential in animal models. These strains provide the platform to conduct a broad scale genetic analysis of the molecular determinants of virulence.

334. Ustilago maydis uac mutants elicit a host response in maize leaves. Aarthi Gopinathan, Karen Snetselaar, Michael McCann. Biology Department, Saint Joseph's University, Philadelphia PA

The basidiomycete Ustilago maydis causes corn smut disease. This dimorphic fungus is normally non-pathogenic when growing vegetatively by budding. Infection occurs when compatible cells form mating filaments that fuse, and the resulting filamentous dikaryon enters host tissues. Adenylate cyclase (uac) mutants of the fungus are constitutively filamentous (Gold S, Duncan G, Barrett K, Kronstad J; 1994; Genes and Dev 8:2805-16), yet they are non-pathogenic. Filaments formed by uac mutants differ from mating and infection filaments in features of polarity and cell division. Exogenous cAMP restores the ability of the uac mutants to produce mating and infection filaments in vitro and on host surfaces. However, although uac mutants provided with cAMP make infection filaments that grow in plant tissue and may initiate tumor formation, the tumors fail to complete development. To determine whether host defense responses accompanied the impaired pathogenicity, we used diaminobenzidine to localize H₂O₂ in the presence of peroxidase, indicating a hypersensitive response, (Vanacker, H, Carver, T, Foyer, C; 2000; Plant Phys 123:1289-1300). Leaves inoculated with uac mutants supplemented with exogenous cAMP produced a much stronger host response than leaves inoculated with wild-type cells where no cAMP was added. However, control inoculations involving wild-type cells and added cAMP also resulted in a host response. These and additional experiments indicate that regulation of cAMP is required for normal completion of the U. maydis pathogenic program. The work was partially funded by NSF MCB 9807807 to K. Snetselaar and M. McCann.

335. The HMG-box protein Rop1 is essential for pheromone-responsive gene expression in Ustilago maydis. Thomas Brefort, Philip Muller and Regine Kahman. Max-Planck-Institute for terrestrial Microbiology, Marburg, Germany.

In the phytopathogenic fungus Ustilago maydis fusion of compatible haploid cells is a prerequisite for infection. This process is genetically controlled by the biallelic a locus encoding pheromone precursors and receptors. Binding of pheromone to its cognate receptor triggers the so-called pheromone response leading to an activation of the HMG-domain transcription factor Prf1. Prf1 binds to the PRE-boxes located in the promoters of the a- and b-genes. As a result, stimulated wildtype cells show elevated transcription of these genes as well as conjugation tube formation, while prf1 mutants do not. Here, we present the identification of rop1 encoding a second sequence-specific HMG-domain protein. While the HMG-domain of Rop1 is 17,7% identical to the HMG-domain of Prf1, it shows highest identity (42,6%) to the HMG-domain of Pcc1 of Coprinus cinereus. rop1 deletion strains display a severe mating defect and do not form conjugation hyphae upon pheromone stimulation. Northern analyses revealed that rop1 is essential for prf1, mfa1, pra1 as well as b gene expression. Since constitutive expression of prf1 fully complements the observed mating defect ofrop1 deletion strains it is likely that Rop1 regulates prf1 gene transcription. We are now investigating whether Rop1 binds directly to the prf1 promoter or whether its effect on prf1 gene transcription is indirect.

336. Functional analysis of a thiamine biosynthetic gene in the interaction of Epichloë typhina with perennial ryegrass. Xiuwen Zhang¹, Michael Christensen², Barry Scott¹. ¹Institute of Molecular BioScience, Massey University, Private Bag 11 222, Palmerston North, New Zealand. ²AgResearch Limited, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand.

Epichloë/Neotyphodium endophytes are a group of clavicipitaceous fungi that form symbiotic associations with temperate grasses. The asexual N. lolii form asymptomatic mutualistic associations with ryegrass whereas the sexual E. typhina behaves as a mutualist during the vegetative phase of plant growth but switches to epiphytic growth and formation of an external stroma upon development of the floral inflorescence. We are interested in the metabolic interaction between these endophytes and their perennial ryegrass host. We have chosen to examine the role of endophyte thiamine biosynthesis in this interaction, because of its key role as a coenzyme in primary cellular metabolism. The orthologue (thi1) ofSaccharomyces cerevisiae THI4 was isolated from N. lolii and E. typhina by PCR using degenerate primers designed to conserved regions of known thiazole biosynthetic genes. This gene is strongly expressed in culture and in planta and shows alternative splicing, with distinct patterns of the isoforms expressed under different nutritional conditions. A knockout of the E. typhina thi1 has been constructed and shown to have reduced hyphal density and branching compared to the wild-type on defined media lacking thiamine. Both thiamine and thiazole complemented this defect. No differences in infectivity were observed between wild-type and mutant in their ability to establish stable artificial associations with perennial ryegrass. However, some differences were observed in host colonisation, with the mutant strain behaving more like the asexual N. lolii. Both wild-type and mutant formed stromata on reproductive tillers, and, unexpectedly, both formed some stromata on vegetative tissue.

337. Smu1, a Ste20p homologue from Ustilago maydis with roles in mating and pathogenicity. David Smith¹, Zhanyang Yu¹, Scott Gold², and Michael H. Perlin¹. ¹ University of Louisville, Louisville, Kentucky, USA and ² University of Georgia, Athens, Georgia, USA

Ste20p is a member of the PAK family of protein kinases responsible for regulation of a series of mitogen-activated protein kinase (MAPK) signal transduction cascades conserved from yeast to humans. We isolated genes, smu1 and smtE, encoding homologues of the PAK-like Ste20p from, respectively, the corn smut, U. maydis, and from the anther smut, Microbotryum violaceum. Though both proteins were similar to the PAK-like kinases, they were distinct from the germinal-center kinases, including Don3, also from U. maydis. When both U. maydis partners in a mating pair were disrupted for smu1 there was significant impairment of mating. Over-expression of the catalytic region of the M. violaceum SmtE failed to complement U. maydis with smu1 knock-outs. Interestingly, such expression interfered with wildtype mating and made the mating defect of the smu1 knock-out even more pronounced. Smu1 knock-out strains also showed significantly reduced ability to cause disease when used to infect maize. In such infections, the majority of symptoms were limited to those observed early in infection; gall formation and plant death were almost never observed. Thus, unlike the Don3 kinase involved in cell separation, smu1 is part of the mating pathway, and its disruption greatly impairs the ability of U. maydis to cause disease.

338. Genome Rearrangement in Magnaporthe grisea: Translocation of an Avirulence Gene. M.W. Harding, M.A. Mandel and M.J. Orbach. University of Arizona, Department of Plant Pathology, Tucson, AZ.

We are working to characterize the fungal effector (avirulence) gene AVR1-MARA from M. grisea. An incompatible (avirulent) phenotype is observed on the rice cultivar Maratelli challenged with strain 4224-7-8. Incompatibility is controlled by a single gene (AVR1-MARA) that does not readily or spontaneously mutate to virulence.AVR1-MARA originated in G-22, an M. grisea strain that is pathogenic on finger millet. G-22 is highly fertile and was backcrossed to a rice pathogen (0-17) to increase fertility in a rice pathogen field isolate (Dobinson & Hamer, 1992). Incompatibility of 4224-7-8 progenitor strains on Maratelli is also defined by a single genetic locus. However, we discovered that incompatibility in progenitors segregated independently with respect to AVR1-MARAwhen crossed with 4224-7-8. Our working hypothesis is that these two unlinked incompatibility loci contain the same effector gene. Data that support this hypothesis will be presented. We propose that during a backcross with G-22 (cross 4134) an insertional translocation was responsible for moving the effector gene from the ancestral location in G-22 to theAVR1-MARA locus described in 4224-7-8 (Mandel et al., 1997). DNA sequence from G-22 and 4224-7-8 indicate that a region of AT-rich sequence larger than 40kb moved during the translocation. Mandel et al. 1997. MPMI 10:1102-1105. Dobinson & Hamer. 1992. Magnaporthe grisea. In: Molecular Biology of Filamentous Fungi. U. Stahl & P. Tudzynski, eds. Weinheim, NY.

339. The role of reactive oxygen species in plant-pathogen interaction. P. Tudzynski, K. B. Tenberge, S. Joshi, S. Moore, Y. Rollke, E. Nathues; Institut fuer Botanik, Westf. Wilhelms-Universitaet, Schlossgarten 3, D-48149 Muenster, Germany

One of the earliest reactions of plants against pathogens is the transient formation of reactive oxygen species (ROS), termed in analogy to mammalian systems "oxidative burst". Its impact on plant defense has been studied in detail; the active and passive role of the pathogen facing this oxidative stress during the early stages of interaction is less clear. Two different strategies seem to exist: in the necrotroph Botrytis cinerea the formation of ROS in planta is directly correlated with aggressiveness of fungal isolates ; there is evidence for production of ROS by the fungus itself, triggering enhanced production by the plant. This leads to rapid death of plant tissue, facilitating fungal growth. In more balanced systems like in the biotrophic cereal pathogen Claviceps purpurea, the fungus obviously tries to overcome the oxidative stress by building up a protective shield of secreted AOS-scavenging enzymes. We are studying both types of interactions using cytological, biochemical and molecular genetic techniques including functional analysis by targeted gene inactivation. We will focus here on the C. purpurea/rye system and present evidence that the oxidative stress response system has impact on the interaction. We have identified a bZIP transcription factor (cptf1) involved in oxidative stress response, which controls catalase activity and is essential for full virulence of the fungus on rye.

340. Isolation and characterization of genes preferentially expressed during asexual sporulation in the oomycete plant pathogen Phytophthora cinnamomi. Reena Narayan, Weixing Shan, and Adrienne R. Hardham. Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, Canberra, Australia.

The genus Phytophthora contains at least 60 species, many of which are destructive pathogens causing diseases in hundreds of commercially important plants. P. cinnamomi is capable of infecting over a thousand plant species and causes severe economic and ecological losses to agriculture and forest industries in Australia and worldwide. Under nutrient-deprived conditions, vegetative hyphae of P. cinnamomi sporulate to produce multinucleate, asexual sporangia that cleave to form uninucleate, motile zoospores, which are the primary means of infection of new hosts. Our current understanding of the molecular basis of sporulation is extremely limited. Identification and characterization of Phytophthora genes that serve key roles in sporulation and spore function would make a significant contribution towards increasing our understanding of these processes. In this study, differential hybridization techniques were used to screen over 5000 cDNA clones from a P. cinnamomi cDNA library made from an early stage of sporulation, and 328 putative sporulation-specific genes were isolated and partially sequenced. Candidate genes were identified through Genbank and Phytophthora Genome Consortium database comparisons. Of the 328 sequenced clones, 195 were found to represent unique genes of which 27% were homologous to metabolic and structural proteins, 31% were homologous to ribosomal proteins, and 42% were unknown genes. Three candidate genes were selected for further molecular characterization. A macroarray of the unique genes was screened with probes made from mRNA isolated at nine different stages in the sporulation process and cohorts of genes expressed at different stages of sporulation were identified. The macroarray results were complemented by RNA blot analysis.

341. Identification of a cysteine-rich protein secreted by Fusarium oxysporum during growth in tomato xylem vessels. Martijn Rep¹, Charlotte van der Does¹, Michiel Meijer¹, Henk L. Dekker², Petra M. Houterman¹, Chris G. de Koster² and Ben J.C. Cornelissen¹. ¹Plant pathology and ²Mass spectrometry, Swammerdam Institute for Life Sciences, University of Amsterdam

Fusarium oxysporum f. sp. lycopersici colonizes tomato plants through invasion of xylem vessels, resulting in wilt disease. Proteins secreted by the fungus in xylem sap are likely to play a crucial role during colonization. On the one hand, they can serve to promote colonization, for instance through suppression of plant defense mechanisms. On the other hand, they may elicit plant defense responses. In order to clarify the molecular basis of Fusarium pathogenicity, we set out to identify proteins secreted by the fungus in tomato xylem vessels. Besides several tomato PR proteins, a 12 kD Fusarium protein (SIX1, for Secreted in Xylem 1) has now been identified. The corresponding gene was isolated and potentially encodes a 30 kD protein, from which SIX1 is derived through proteolytic processing. The protein is cysteine-rich and does not resemble any other known protein. In a mutant with an altered virulence phenotype (less virulent on some tomato cultivars, more virulent on another), the SIX1 gene is lost along with neighbouring sequences. Characterization of the genomic locus of the SIX1 gene and that of a truncated homolog on the same chromosome revealed the presence of several repetitive elements. The potential role of SIX1 in pathogenicity and/or avirulence is being investigated through complementation and knock-out studies.

342. Signalling and pathogenicity in the grey mould Botrytis cinerea. Christian Schulze Gronover, Philipp Hantsch and Bettina Tudzynski. Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schlossgarten 3, 48149 Münster, Germany

Heterotrimeric G proteins were shown to play an important role in pathogenicity of many fungi. We cloned and deleted two different genes (bcg1 and bcg2). bcg1 mutants differ in colony morphology from the wild-type, show a reduced growth rate and extracellular protease activity. Tomato and bean leaves inoculated with conidia from bcg1 mutants caused only primary necrosis, but never spreading lesions. bcg-2 mutants developed secondary lesions but much slower than the wild-type. Biochemical analysis showed that bcg1, but not bcg2 mutants lost the ability to produce the phytotoxin botrydial. In a molecular approach (SSH) we identified in planta expressed genes which are not longer expressed in bcg1 mutants. Furthermore, we deleted the B. cinerea adenylate cyclase gene (bac) resulting in reduced vegetative growth and a colony morphology reminding that of bcg1 mutants. Conidia of bac mutants were still able to germinate, to penetrate bean leaves, and, in contrast to bcg1 mutants, to form soft rot. However, the development of secondary spreading lesions is much slower, and in contrast to the wild-type, no conidiation was obtained on bean leaves. In contrast to bcg1 mutants, bac mutants still produce botrydial and extracellular proteases. The much stronger effect of bcg1 mutation on pathogenicity in comparison to the bac mutation suggests that BCG1 controls at least one more signalling pathway in addition to the cAMP pathway. In addition, several protein kinase-encoding genes, e.g. a homologue of the S. cerevisiae sch-9 gene, and genes encoding small GTP-binding proteins of the ras family were cloned and their role in pathogenicity was analyzed.

343. Signalling cascades regulating growth and virulence in Fusarium oxysporum. Jesús Delgado, Ana Lilia Martínez-Rocha, Raquel Roldán, Carmen Velasco, M. Isabel G. Roncero, Michael W. Rey and Antonio Di Pietro. Universidad de Cordoba, Department of Genetics, Cordoba, Spain.

Fusarium oxysporum is a soilborne fungus that causes vascular wilt disease in a wide variety of crops and has also been reported as an emerging opportunistic pathogen of humans. A signalling cascade controlled by the extracellular-regulated mitogen-activated protein kinase (MAPK) Fmk1 was shown previously to be required for infection. Here we report the presence of a second, independent pathway that regulates development and virulence in F. oxysporum by identifying one of its components, the G protein beta subunit Fgb1. Strains carrying either a fgb1 loss-of-function mutation (delta fgb1) or a dominant activating allele (fgb1^W115G) show strongly reduced virulence on tomato. Similar to delta fmk1 strains, fgb1 mutants produce altered levels of extracellular virulence factors such as polygalacturonases and hyphal hydrophobicity determinants. Levels of Fmk1 phosphorylation in fgb1 mutants are comparable to those in the wild type strain as shown by immunoblot analysis with anti-phospho-p44/p42 MAPK antibodies, suggesting that Fgb1 does not signal upstream of Fmk1. Delta fgb1 mutants display a strongly elongated and unbranched hyphal growth pattern which is reversed by the protein kinase A (PKA) inhibitor H-89. Conversely, strains overexpressing the dominant activating fgb1^W115G allele show increased branching and premature submerged conidiation, which can be reversed by the phosphodiesterase inhibitor IBMX. We propose that the G-protein beta subunit Fgb1 controls proliferative growth, differentiation and virulence in F. oxysporum by negatively regulating a cAMP-dependent PKA cascade.

344. ACE1: a complex evolution from avirulence to virulence in populations of the rice blast fungus Magnaporthe grisea. Isabelle Fudal¹, Heidi U. Böhnert¹, Didier Tharreau², Jean-Loup Notteghem³ and Marc-Henri Lebrun¹. ¹CNRS/Bayer, France. ²CIRAD-CA, France.³ENSA-M, France.

Rice resistance to the blast fungus Magnaporthe grisea depends on specific interactions controlled by fungal avirulence genes and their corresponding plant resistance genes. The avirulence gene ACE1 that interacts with the rice resistance gene Pi33 was isolated by positional cloning and encodes a combined polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) (4035 aa). The basis of virulence towards Pi33 in nature was investigated through population studies. Most rice-pathogenic M. grisea isolates collected at diverse locations such as China, Philippines or Columbia turned out to be avirulent towardsPi33. The rare virulent isolates were detected in different geographic populations but are genetically related. We analysed ACE1 polymorphism in a subset of avirulent and virulent isolates representative of the world-wide diversity. This revealed that all avirulent isolates have an ACE1 allele similar to the avirulent isolate Guy11, while virulent isolates display significant polymorphism. Three distinct virulent alleles can be distinguished. The first type of virulent isolate has a RFLP pattern identical to Guy11-ACE1 suggesting that it may result from point mutations in ACE1 open reading frame (ORF) or promoter. CM28-ACE1 allele differs from the Guy11-ACE1 allele by several restriction site polymorphisms. Sequencing revealed 12% nucleotide diversity when compared to Guy11-ACE1 suggesting that the CM28-ACE1 allele is not derived from the Guy11-ACE1 allele by recent accumulation of point mutations. The third virulentACE1 allele (PH14-ACE1) has a combination of patterns corresponding to Guy11-ACE1 and CM28-ACE1 patterns. The corresponding alleles are localised on two different chromosomes, indicating that these normally haploid isolates are partially diploid for ACE1. A possible scenario of ACE1 evolution in M. grisea populations will be proposed.

345. A Data Mining Strategy to Identify in planta Induced Genes from the Oomycete Pathogen Phytophthora infestans. Luis da Cunha, Edgar Huitema, Miaoying Tian and Sophien Kamoun. Department of Plant Pathology, The Ohio State University - OARDC, Wooster, OH

The oomycete Phytophthora infestans causes late blight, a devastating disease of tomato and potato. A key step in understanding pathogenicity of P. infestans is to define the transcriptional changes that take place during colonization of host tissue. For example, in planta-induced (ipi) genes are more likely to encode virulence or avirulence factors and form attractive candidates for detailed functional analyses. Here, we describe a strategy to identify ipi genes from expressed sequence tags (ESTs) obtained from infected tomato tissue. To select a subset of ipi candidate genes, computational analyses based on GC counting and differential BLAST searches against tomato and Phytophthora databases were performed on 2808 "interaction" ESTs. A total of 523 ESTs (19%) were predicted to originate from P. infestans. Additional BLAST searches against 72.000 in vitro P. infestans ESTs identified a set of 55 sequences that are over-represented in the interaction. To validate this data mining strategy, we performed PCR on P. infestans and tomato genomic DNA, semi-quantitative RT-PCR, and northern blot analyses. All genes examined were confirmed to be from P. infestans. Of these, 52 were expressed during colonization of tomato and 10 were found to be up-regulated during infection using semi-quantitative RT-PCR. Current work focuses on functional analyses of the novel ipigenes to determine whether they play a role in pathogenesis.

346. Secondary metabolism and avirulence in Magnaporthe grisea: is ACE1 part of an avirulence gene cluster? Heidi U. Böhnert, Isabelle Fudal, Anne-Elodie Houlle and Marc-Henri Lebrun. UMR 1932 CNRS / Bayer CropScience, Lyon, FRANCE.

Resistance of rice to the blast fungus Magnaporthe grisea depends on specific interactions controlled by fungal avirulence genes and their corresponding plant resistance genes. The avirulence gene ACE1 that interacts with the rice resistance gene Pi33 encodes a combined polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS), a novel type of eukaryotic enzyme involved in the biosynthesis of an as yet unidentified secondary metabolite. Comparison of ACE1 to known fungal PKS shows that it is related to LNKS from Aspergillus terreus, involved in the biosynthesis of lovastatin. ACE1 is expressed exclusively in penetrating appressoria. High turgor of the appressorial cell appears to be required but not sufficient for the induction of ACE1 expression. Since Ace1p is localized in the cytoplasm of the appressorium and the enzymatic function of Ace1p required for avirulence, we conclude that the signal recognized by resistant rice is not Ace1p itself, but the secondary metabolite produced by Ace1p. Sequence analysis of the genomic region adjacent to ACE1 revealed a cluster of genes potentially involved in secondary metabolism. Several of these appear to be co-regulated withACE1. Our current research focuses on two aspects: identification of the common regulator of the ACE1 gene cluster and whether the entire gene cluster is involved in avirulence.

347. Characterisation of Trehalose Biosynthetic and Metabolic Mutants of Magnaporthe grisea. Joanna M. Jenkinson and Nicholas J. Talbot. School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, EX4 4QG, United Kingdom.

The heterothallic ascomycete Magnaporthe grisea causes rice blast disease of cultivated rice. The potentially devastating effects of this disease are determined by the ability of the fungus to mechanically penetrate the plants cuticle and epidermis. This force is due to the enormous hydrostatic turgor that accumulates within the appressorium as a result of the high concentrations of glycerol present. Glycogen, lipid and trehalose are thought to be potential precursors for glycerol biosynthesis in M. grisea. Trehalose is also one of the major carbohydrates in dormant conidia and can act as a stress metabolite.

The aim of this project is to understand the role of trehalose in the infection cycle of Magnaporthe grisea. Using a trehalose-6-phosphate synthase mutant we have shown that trehalose synthesis is required for plant infection and affects the ability of appressoria to generate turgor. We have also identified two trehalases, but only one of these activities is important for pathogenesis, affecting post-penetration development.

348. Structure-function relationship studies on the CBEL glycoprotein of Phytophthora parasitica var. nicotianae, by PVX expression in N. benthamiana. E. Gaulin^1,2, T. Torto², Y. Martinez¹ M. Khatib¹, A. Bottin¹, M.T. Esquerré-Tugayé¹, S. Kamoun², and M. Rickauer¹. ¹Pôle de Biotechnologie Végétale UMR5546, B.P.17 Auzeville, 31326 Castanet-Tolosan, FRANCE² Dept of Plant Pathology, OARDC, 1681 Madison Av., Wooster, Ohio, USA

CBEL is a cell wall glycoprotein produced by Phytophthora parasitica var. nicotianae, an oomycete pathogen of tobacco. It binds to cellulose and plant cell walls in vitro and induces defense reactions in the host plant (1). The protein structure of CBEL consists of two repeated domains separated by a linker region ; each domain contains a motif similar to the cellulose-binding domain (CBD) found in fungal glycanases. In order to determine the roles of the different domains in elicitor activity of CBEL, we adopted the PVX expression system for our studies. The coding sequence of CBEL, including its proper signal peptide sequence, as well as various deletions and point mutations, were introduced into the pGR106 expression vector (2). Production of CBEL in planta induces necrosis in N. benthamiana leaves, leading to death of the whole plant. The necrosis-inducing activity, together with western blot analysis and immunocytolocalisation of CBEL, show that the oomycete secretion signal peptide directs CBEL towards the cell wall in N. benthamiana. Results obtained with mutant forms of CBEL indicate that the CBDs are involved in its necrosis-inducing activity, and hence in its perception by the plant cell.

1. Villalba-Mateos et al., 1997. Mol. Plant-Microbe Interact. 10, 1045-1053 2. We thank D. Baulcombe, Norwich, for the gift of pGR106

349. The corn pathogen Ustilago maydis responds to triglycerides by switching from budding to filamentous growth. J. Klose, M. Moniz de Sa and J. Kronstad. Biotechnology laboratory, Department of Microbiology and Immunology, and Faculty of Agricultural Science, The University of British Columbia, Vancouver, B.C V6T 1Z3, Canada

Yeast-like cells of the corn smut pathogen Ustilago maydis mate to form the filamentous dikaryon that is capable of infecting corn plants. We found that the dimorphic switch from budding to filamentous growth was triggered by the presence of triglycerides supplied as corn oil (or other oils) or fatty acids (supplied as tweens) in the culture medium. The ability of the fungus to respond required components of the ras/MAPK and the cAMP/PKA signal transduction pathways that are known to mediate morphological changes in U. maydis. For example, a mutant with a defect in the regulatory subunit of protein kinase A (encoded by the ubc1 gene) failed to form filaments in response to the lipid signals. Similarly, a mutant defective in a MAPK gene (ubc3) also failed to repond. An extracellular lipase activity was evident in culture supernatants during the morphological transition as determined by a turbidimetric enzyme assay. Glucose repressed the switch to filamentous growth in response to triglycerides and fatty acids, and inhibited the extracellular lipase activity in culture supernatants. It is possible that one or more of secreted lipases may contribute to the response of the fungus to triglycerides, and may ultimately contribute to virulence. Overall, these results provide the framework for a model for triglyceride/fatty acid signaling in U. maydis and establish a foundation for subsequent molecular genetic experimentation.

350. Insertional mutagenesis of Fusarium graminearum from rice in Korea. You-Kyoung Han¹, In Young Jang¹, Hun Kim¹, Sung-Hwan Yun², and Yin-Won Lee¹. ¹School of Agricultural Biotechnology, Seoul National University, Suwon, 441-744, Korea,²Division of Life Sciences, Soonchunhyang University, Asan, Choongnam, 336-745, Korea

Fusarium graminearum is an important pathogen of cereal crops in many areas of the world causing head blight and ear rot of small grains. In addition to serious economic losses, this fungus produces mycotoxins, such as trichothecenes and zearalenone on diseased crops and has been a potential threat to human and animal health. Recently, it has been confirmed that F. graminearum was associated with epidemic of rice head blight occurred in Korea. More than 200 filed isolates of F. graminearum obtained from disease symptoms of rice in Korea were investigated for their pathogenicity against rice as well as other mycological characteristics. Meanwhile, genetic diversity of the F. graminearum population was evaluated by using amplified fragment length polymorphisms (AFLP). To massively identify pathogenesis-related genes from F. graminearum, two representative strains (SCKO4 from rice and Z03643 from wheat) were mutagenized using restriction enzyme-mediated integration (REMI). In total, 20,000 REMI transformants have been collected from the two strains. So far 60 mutants for several traits involved in disease development such as virulence, mycotoxin production, and sporulation have been selected from 2,000 transformants. Now, selected mutants of interest are genetically analyzed using a newly developed outcross method (See Jungkwan Lee et al poster). In addition, cloning and characterization of genomic DNA fragments flanking the insertional site in the genome of selected mutants are in progress.

351. Functional analysis of CLPT1, a RAB/GTPase gene from the bean pathogen Colletotrichum lindemuthianum. Piyawane Siriputthaiwan¹, Corentin Herbert¹, Alain Jauneau², Marie-Thérèse Esquerré-Tugayé¹ and Bernard Dumas¹. 1: UMR5546 CNRS-UPS and 2: IFR 40 Pôle de Biotechnologie Végétale Castanet-Tolosan, France

During colonization of their host, phytopathogenic fungi secrete enzymes that degrade plant cell wall polymers. Since the secretory pathway could be a major control step for the production of these extracellular proteins, we have undertaken the cloning and the functional characterization of a Rab-GTPase from C. lindemuthianum, a filamentous fungi causing anthracnose disease on bean. Rab/GTPases are included in the Ras superfamily of GTPases and play a major role in the regulation of vesicle trafficking. Recently we have isolated a Rab gene from C. lindemuthianum, named CLPT1 (C. lindemuthianum Protein Transport 1). CLPT1 encodes a functional homologue of the yeast RAB/GTPase, Sec4p. To study more precisely the role of this protein, a CLPT1 gene carrying a dominant-negative mutation (N123I) was expressed in C. lindemuthianum. Transgenic strains expressing this mutated gene accumulated large amount of vesicles randomly distributed in the fungal cells, were unable to produce extracellular enzymes and were non-pathogenic. However, they grew normally on synthetic media and differentiated appressoria on glass surface. Altogether, these results showed that CLPT1 plays a key role in pathogenesis being essential for the post-golgi vesicular transport allowing the secretion of extracellular enzymes.

352. Identification of FDB1, FDB2, and other Fusarium verticillioidesgenes expressed in response to BOA, a maize antimicrobial compound. Anthony E. Glenn and Charles W. Bacon. USDA, ARS, Russell Research Center, Toxicology & Mycotoxin Research Unit, Athens, GA

Maize produces antimicrobial compounds (DIMBOA, DIBOA, MBOA, and BOA) implicated in disease resistance and insect feeding deterrence. Fusarium verticillioides, the most common fungal pathogen associated with maize, has the physiological capacity to biotransform these compounds into non-toxic metabolites. While data suggest such biotransformation is not a major virulence factor, the metabolic capacity may enhance the ecological fitness of F. verticillioides in a cornfield environment. Genetic analyses showed at least two loci, FDB1 and FDB2, are necessary for biotransformation. The biotransformation pathway for BOA is suggested to involve hydrolysis (Fdb1p) to produce 2-aminophenol, which is subsequently modified by addition of a malonyl group (Fdb2p) to produce N-(2-hydroxyphenyl) malonamic acid. If either gene is mutated, detoxification does not occur and the fungus cannot grow on BOA-amended medium. In an effort to molecularly characterize FDB1 and FDB2 as well as other genes involved in biotransformation, suppression subtractive hybridization (SSH) was used to target genes up-regulated in response to BOA. Among the clones identified, those with similarities to amidase and arylamine N-acetyltransferase were of particular interest, since these enzymes catalyze chemical modifications similar to those postulated for Fdb1p and Fdb2p. Genomic cosmid clones were identified for each using the respective cDNAs as probes. The putative amidase cosmid genetically complemented an fdb1 mutation, while the putativeN-malonyltransferase cosmid complimented an fdb2 mutation. Thus, the proposed chemical modifications and the putative proteins involved are mutually supported. Also, these results demonstrate the utility of SSH for cloning genes previously identified by forward genetics.

353. Functional analysis of Ustilago maydis Ubc2, a putative novel adapter protein. Steven J. Klosterman, Alfredo D. Martinez-Espinoza, and Scott E. Gold. Department of Plant Pathology, University of Georgia, Athens, GA 30602-7274.

The plant pathogenic fungus Ustilago maydis alternates between a haploid budding form and a dikaryotic filamentous form. Genes encoding proteins involved in the MAP kinase pathway that controls mating and morphogenesis have been identified previously by complementation of mutants that suppress the constitutively filamentous phenotype of auac1 (Ustilago adenylate cyclase) mutant. Thus these proteins were named Ubc (Ustilago bypass of cyclase) proteins. Three of the Ubc proteins share homology with MAP kinase cascade members that control mating and morphogenesis in other fungi. Another of these genes, ubc2, is a critical virulence factor and enocdes a protein which appears basidiomycete-specific in its overall structure. Ubc2 possesses four distinct protein interaction domains, indicating that Ubc2 is likely a novel adapter protein functioning in the MAP kinase pathway. To ascertain the role of the various protein interaction domains and to determine the functionally important amino acids within these domains of Ubc2, site-directed mutagenesis and complementation studies are being conducted. To gain further insight into the function of Ubc2, the yeast two-hybrid assay is being employed to identify interactions between Ubc2 and other proteins in the MAP kinase and potentially related pathways. The results of these studies will be discussed.

354. Molecular evolution of the AVR-Pita avirulence gene family in Magnaporthe grisea. Chang Hyun Khang¹, Seogchan Kang¹, and Barbara Valent². ¹ Dept. Plant Pathology, The Pennsylvania State University, University Park, PA 16802. ² Dept. Plant Pathology, Kansas State University, Manhattan, KS 66506.

Magnaporthe grisea, the causal agent of the devastating rice blast disease, can infect many gramineous species. The host specificity of this fungus follows the gene-for-gene model. Deploying disease resistance (R) genes for disease control has had limited success, mainly due to the evolution of new races with the ability to overcome resistance. Considering its mainly asexual population structure, frequent variation of pathogen avirulence (AVR) genes likely underpins such evolutionary events. Therefore, understanding the mechanisms of variation and biological role of AVR genes is of importance for designing durable resistance. AVR-Pita, one of the M. grisea AVR genes, prevents the fungus from infecting the rice cultivars that express the Pi-ta R gene. AVR-Pita is located near a telomere and predicted to encode a 223 amino acid protein with similarity to metalloproteases. Southern blot analyses revealed that AVR-Pita is a member of a widespread gene family in the M. grisea strains from various hosts including rice. Phylogenetic analyses and comparative study of their genome organization suggest that the AVR-Pita gene family consists of at least three members that have been duplicated and rearranged via recombination events, probably mediated by repetitive DNA elements flanking the gene family. The conservation of AVR-Pita protein structures within M. grisea populations and the presence of strains lacking any characterized family members suggest that members of this family may function as a host-specific fitness factor.

355. Characterization of a gene cluster for host-specific AAL-toxin biosynthesis in the tomato pathotype of Alternaria alternata. Hajime Akamatsu, Hiroshi Otani and Motoichiro Kodama. Laboratory of Plant Pathology, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan

AAL-toxins are host-specific toxins produced by Alternaria alternata tomato pathotype, the causal agent of Alternaria stem canker of tomato. AAL-toxins and fumonisins of the maize pathogen Gibberella moniliformis are structurally related to sphinganine and are termed sphinganine-analogue mycotoxins. Since AAL-toxins are polyketide-derived compounds similar to fumonisins, we have cloned several polyketide synthase (PKS) gene fragments from the tomato pathotype of A. alternata by PCR. Gene targeting with a vector harbouring a PKS sequence specific to the tomato pathotype created AAL-toxin-minus mutants that lost virulence to the susceptible tomato. Analysis of flanking sequences of the PKS fragment from a genomic library of the tomato pathotype revealed a Type I PKS gene of 7.8 kb in length, designated ALT1. Flanking ALT1, several genes were identified which constitute an AAL-toxin biosynthetic (ALT) gene cluster, and some of these genes resembled those found in fumonisin biosynthetic (FUM) gene cluster. The predicted products of the genes in the cluster were similar to fungal Type I PKSs, cytochrome P450 fusion proteins, alcohol dehydrogenases, aminotransferases, ABC transporters and longevity assurance factors. However, the order of the genes in the ALT gene cluster was different from that of the FUM gene cluster. The ALT gene cluster reside on a 1.0 Mb conditionally dispensable chromosome found only in the AAL-toxin-producing strains of A. alternata and homologues of the genes were not detected in nonpathogenic strains of A. alternata. This suggests that the ALT gene cluster may have been acquired by horizontal gene transfer and provides a possible mechanism whereby new pathotypes could arise in nature.

356. The phosphoinositide-specific phospholipase C gene, MPLC1, of Magnaporthe grisea is required for fungal development and plant colonization. Hee-Sool Rho, and Yong-Hwan Lee, School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea

Magnaporthe grisea, the casual agent of rice blast, forms an appressorium to penetrate its host. Much has been learned about environmental cues and signal transduction pathways, especially those involving cAMP and MAP kinases, on appressorium formation during the last decade. More recently, pharmacological data suggest that calcium/calmodulin-dependent signaling system is involved in its appressorium formation. To determine the role of phosphoinositide-specific phospholipase C (PI-PLC) on appressorium formation, a gene (MPLC1) encoding PI-PLC was cloned and characterized from M. grisea strain 70-15. Sequence analysis showed that MPLC1 has all five conserved domains present in other phospholipase C genes from several filamentous fungi and mammals. Null mutants (mplc1) generated by targeted gene disruption exhibited pleiotropic effects on conidial morphology, appressorium formation, fertility and pathogenicity. mplc1 mutants developed nonfunctional appressoria and are also defective in infectious growth in host tissues. Defects in appressorium formation and pathogenicity in mplc1 mutants were complemented by a mouse PLCdelta-1 cDNA under the control of the MPLC1 promoter. These results suggest that cellular signaling mediated by MPLC1 plays crucial and diverse roles in development and pathogenicity of M. grisea, and functional conservation between fungal and mammalian PI-PLCs.

357. Identification of race-specific avirulence genes in Phytophthora infestans by transciptional profiling. Rays H.Y. Jiang¹, Guo Jun^1,2 and Francine Govers¹. ¹Laboratory of Phytopathology, Wageningen University, and Graduate School Experimental Plant Sciences, Wageningen, The Netherlands.²Institute of Vegetable Crops and Flowers, Chinese Academy of Agricultural Sciences., Beijing, China.

Phytophthora infestans is a destructive oomycete pathogen causing potato late blight worldwide. Genetic analyses of potato and P. infestans have demonstrated that in this pathosystem, monogenic resistance mediated by resistance (R) genes, is based on a gene-for-gene interaction. Our aim is to clone and characterise avirulence (Avr) genes in P. infestans. Previously, we constructed high density linkage maps of two regions carrying Avr genes (van der Lee et al. 2001 Genetics 157: 949-956). Currently, cDNA-AFLP analysis is performed on F1 progeny of the mapping population. Twenty-one isolates with different avirulence phenotypes have been selected and mRNA has been isolated from the stage that Avr genes are most likely to be expressed, i.e., the germinating cyst stage. Based on the transcriptional profiles we can identify genes that are differentially expressed in different races and this may lead to identification of Avr genes in P. infestans.

358. Effects on virulence of the tomato pathogen Cladosporium fulvum by RNAi-mediated silencing of avirulence genes. Bas Brandwagt^1,2, Maarten de Kock², Matthieu Joosten¹ and Pierre de Wit¹. Laboratories of Phytopathology¹ and Plant Breeding², Wageningen University, The Netherland

The interaction between the biotrophic fungus Cladosporium fulvum and tomato (Lycopersicon esculentum) complies with the gene-for-gene model. Fungal growth is restricted to the extracellular spaces of tomato leaves, where C. fulvum secretes avirulence (AVR) proteins. AVR proteins are strain-specific and elicit a hypersensitive response in tomato genotypes with the corresponding resistance (Cf) genes. AVR proteins secreted by all known strains of C. fulvum are called extracellular proteins (ECPs) and are presumed to be virulence factors. Our goal is to study the function of the AVRs and ECPs in establishing or aborting infection of tomato by C. fulvum. We aimed to silence the (a)virulence genes of C. fulvum by transformation with constructs inducing RNA interference (RNAi). First, we proved that RNAi-mediated silencing in C. fulvum was possible for the UidA (GUS), EGFP and hydrophobin marker genes. Subsequently, two highly virulent C. fulvum strains were independently transformed with Ecp1, Ecp2, Ecp4, Ecp5 or Avr4E RNAi constructs. TheEcp2 and Avr4E RNAi transformants could colonise Cf-ECP2 and Cf-4E tomato plants through the absence of the ECP2 and AVR4E proteins, respectively. These observations prove that the reduction of avirulence gene expression by RNAi is sufficient to prevent resistance responses in tomato plants with the corresponding Cf gene. The Ecp1, Ecp4 and Ecp5 RNAi transformants, however, were still avirulent on plants with the corresponding Cf genes. We are now further analysing this phenomenon. In conclusion, RNAi in C. fulvum can be used as a versatile tool to determine virulence functions of avirulence proteins in the interaction between C. fulvum and tomato.

359. The role of the mitochondrial Mrb1 protein in pathogenic development of Ustilago maydis. Miriam Bortfeld, Kathrin Auffarth and Christoph W. Basse. Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg/Lahn, Germany

The smut fungus Ustilago maydis causes tumor development in infected maize plants. The constitutively expressed Ustilago maydis mrb1 gene encodes a protein with significant similarity to mitochondrial, acidic proteins of the p32 family known from different eukaryotic organisms. Mitochondrial localization of Mrb1 was demonstrated and the mitochondrial targeting sequence was delineated. Compatible U. maydis mrb1-null mutants derived from strains FB1 and FB2 were able to mate, however, the resulting dikaryotic hyphae were severely attenuated in pathogenicity. Despite their abilities to develop appressoria-like structures on maize leaf surfaces and to penetrate throughout epidermal layers, ramification and proliferation within infected tissue as well as the capacity to induce tumor development were drastically reduced compared to wild-type hyphae. Intriguingly, pathogenicity of haploid, solopathogenic U. maydis strains derived from FB1 was not affected by the deletion of mrb1, whereas mrb1 was required for pathogenicity of haploid, solopathogenic FB2 strains, suggesting that FB2 specific factors compromise pathogenicity in the absence of Mrb1.

360. Sensing, signalling and stress in the barley powdery mildew fungus. Ziguo Zhang, Catherine Henderson, Gemma Priddey, Emma Perfect and Sarah Gurr: Department of Plant Sciences, University of Oxford, OX1 3RB, UK.

Blumeria graminis is the causal agent of barley powdery mildew disease. Infection is spread by asexual conidia, which, on contact with the leaf surface, undergo a complex and highly regulated programme of development. Conidia germinate and produce a short primary germ tube followed by a second formed germ tube which elongates, swells and produces a specialised, hooked infection structure, the appressorium. B. graminis is an obligate biotroph; it cannot be grown axenically and consequently, tissue for experiments is limiting. We have described a range of techniques to assess how B. graminis perceives, integrates and relays signals for morphogenesis up to the point of penetration. Previously, our work demonstrated that both physical properties of the leaf surface, such as hydrophobicity, and cuticle-derived chemicals promote B. graminis differentiation. But how does B. graminis transduce signals to drive differentiation and development? Applications of exogenous agonists and antagonists have allowed us to demonstrate a role for cAMP signalling and PKA in germling differentiation, but this work also highlights that cAMP alone is not sufficient to trigger the complete programme of differentiation. Hitherto, we have identified other genes involved in signal transduction and cell integrity pathways in B. graminis, notably PKC, MAPKinases and cell wall genes. We will discuss this work, considering also the pathogen's management of host-derived oxidative stress during development and penetration.

361. Characterisation of the MPS1 MAP Kinase signalling pathway in Magnaporthe grisea. Zac Cartwright and Nicholas J. Talbot. School of Biological Sciences, University of Exeter, EX4 4QJ, United Kingdom.

Fungal plant pathogens have evolved diverse methods of gaining entry to their particular host plant tissue. One fungal pathogen that makes use of mechanical entry is the ascomycete Magnaporthe grisea. Once present on a leaf surface the spore quickly attaches to the hydrophobic leaf surface by secreting an adhesive from within the spore apex. The spore produces a germ tube, and after four hours, differentiates into an appressorium. Penetration into the leaf epidermis occurs when the pressure within the appressorium reaches an estimated 8.0 MPa, causing a penetration peg to rupture the cuticle. The mitogen-activated protein kinase (MAPK) MPS1 is involved in an uncharacterised pathway in the pathogenicity of M. grisea. It is known that mutants lacking the MPS1 gene are able to produce appressoria, but are unable to penetrate the plant cuticle and are therefore non-pathogenic. MPS1 has homologues in a variety of other fungal species; SLT2/MPK1 in Saccharomyces cerevisiae, MKC1 in Candida albicans and CPMK2 in Claviceps purpurea. SLT2 in yeast is a component in the pathway that regulates maintenance of cell wall integrity. The aim of this project is to investigate the biological function of the MPS1 MAP kinase gene in M. grisea. Studies to identify genes regulateded by the MPS1 pathway have been undertaken and preliminary analysis indicates differential regulation of a glycogen synthase kinase-encoding gene in mps1 mutants. Results of diff