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 Saitoh1, 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. kpp6T355A,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 (fgb1W115G) 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 fgb1W115G 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. Gaulin1,2, 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 Jun1,2 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 Brandwagt1,2, 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 differential expression studies will be presented.
362. Fungal H2O2 scavenger activity influences pathogenesis in the true obligate barley powdery mildew pathogen. Ziguo Zhang, Catherine Henderson & Sarah J. Gurr, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK
The barley powdery mildew, Blumeria graminis, is exposed to acute host-derived oxidative stress at critical times during germling morphogenesis, notably at the primary and appressorium germ tube (PGT, AGT) stages of differentiation. To evaluate the antioxidant capacity of this fungus, in particular to assess how Bgh protects itself against damage caused by the accumulation of H2O2 and if fungal catalase contributes towards pathogenicity, the Bgh catB gene was cloned and characterised. An anti-CATB antibody revealed an intense circle of immunofluorescence at the host-pathogen interface at the AGT tip and within the halo area surrounding the host papilla, whilst Diaminobenzidine (DAB) revealed H2O2 accumulation in the papilla. RT-PCR profiling of catB transcript activity, alongside other genes implicated in the management of oxidative stress (catalase-peroxidase, cpx; glutathione peroxidase, gpx and superoxide dismutase, sod1), revealed enhanced numbers of catB transcripts at mature PGT and AGT stages of differentiation in a susceptible host. Moreover, comparison of susceptible (S) barley cv Pallas with its resistant counterpart carrying the papilla-based resistance gene (R) mlo5, P22, revealed intense DAB staining in the papilla and halo beneath the PGT in both S and R lines, but rare staining of the papilla and halo beneath AGTs in the S interaction, provoking speculation that Bgh catalase scavenger activity influences pathogenicity in the barley powdery mildew fungus.
363. The mitogen-activated protein kinase Tvk1 from Trichoderma virens regulates mycoparasitism related genes and conidiation. Artemio Mendoza-Mendoza1, Darlene Grzegorski2, María J. Pozo2, Pedro Martínez1, Juan García2, Vianey Olmedo-Monfil1, Carlos Cortés1, Charles Kenerley2 and Alfredo Herrera-Estrella1.1Department of Plant Genetic Engineering, Centro de Investigación y Estudios Avanzados. Unidad Irapuato. Apartado Postal 629. 36500, Irapuato, Guanajuato., México.2Department of Plant Pathology & Microbiology, TEXAS A&M University. College Station, TX 77843, USA.
Trichoderma virens has a potent antagonic activity against a broad range of phytopathogenic fungi. In the parasitic interaction Trichoderma produces lytic enzymes that have been associated with penetration of the host. In phytopathogenic fungi, the production of lytic enzymes that degrade the cell wall of the host is regulated through a mitogen-activated protein kinase (MAPK) pathway. A MAP kinase-encoding gene from T.virens, named tvk1, was cloned and its physiological role was assessed. Cell growth, conidiation and the expression of mycoparasitic related genes (MRGs) were examined in tvk1 disruptants. Null mutants showed a reduction in aerial growth and production of spores when grown in solid medium. On the other hand a massive production of conidia was achieved in submerged cultures, suggesting that this kinase can act as a negative regulator of conidiation under this condition. During simulated mycoparasitism or direct confrontation with a host (Rhizoctonia solani), the mutants showed a clear augment in the level of expression of all MRGs tested when compared with the wild type strain. The increased transcription of lytic enzyme-encoding genes correlated with an increase in protein secretion as measured by in gel activities. Finally, biocontrol activity assays showed that tvk1 null mutants are more effective in disease control than the wild type strain.
364. Possible function for a F-box protein in pathogenicity of Fusariumoxysporum f. sp. lycopersici. Roselinde Duyvesteijn, Petra M. Houterman, Martijn Rep, and Ben J. C. Cornelissen. University of Amsterdam, Faculty of Science, Swammerdam Institute for Life Sciences, Plant Pathology, Amsterdam, The Netherlands
Fusarium oxysporum f. sp. lycopersici is a soil-borne pathogen that infects tomato. The fungus invades the plant via the roots and subsequently colonizes the xylem vessels of the stem. Ultimately, the sap stream is blocked and the plant wilts. We used insertional mutagenesis to identify, isolate and characterize genes involved in pathogenesis. From among 398 transformants, 19 mutants were identified that showed reduction in or complete loss of pathogenicity but normal growth on agar plates. Plasmid rescue was used to obtain flanking regions of the inserted plasmid from mutant N40 which has complete loss of pathogenicity. Approximately 3 kb Fusarium DNA was retrieved and used to screen a BAC-library in order to obtain the wild type locus. Sequence analyses revealed that the insertion point was in a gene for a 527 AA protein containing an F-box domain. F-box proteins are involved in the ubiquitination machinery of the cell. The protein has homology with a protein fromMagnaporthe grisea, a plant-pathogenic fungus on Rice, and one from Neurospora crassa, a non-pathogenic fungus. The genomic regions containing the genes for the F-box proteins showed synteny over at least 12 kb in all three fungi. A transcript for the F-box protein was found in a cDNA library made of tomato plants infected withFusarium, indicating that the gene is active during infection. Complementation and knockout studies will be done to determine the relation between the insertion and the loss of pathogenicity.
365. ABC transporters of Mycosphaerella graminicola involved in pathogenesis and multidrug resistance. Lute-Harm Zwiers, Ioannis Stergiopoulos and Maarten A. De Waard Laboratory of Phytopathology, Department of Plant Sciences, Wageningen University, Wageningen, the Netherlands
ATP-binding cassette (ABC) transporters are membrane-bound proteins that in plant pathogenic fungi are implicated to act as virulence factors by providing protection against plant defense compounds or by secreting pathogenicity factors. Moreover, ABC transporters are involved in the establishment of multi-drug resistance (MDR) and thus impose a serious threat to the successful control of fungi with antimycotics. We are interested in the role of ABC transporters in Mycosphaerella graminicola, the causal agent of septoria tritici leaf blotch of wheat. Five single copy ABC transporter genes (MgAtr1 to MgAtr5) have been cloned and characterised from this pathogen. The function of these genes was investigated with knockout mutants, generated by PEG- and Agrobacterium-mediated transformation. All transformants were tested for virulence on wheat seedlings. Furthermore, the role of these transporters in MDR was studied by complementation of Saccharomyces cerevisiae mutants with the M. graminicola ABC transporter genes and by testing the sensitivity of ABC transporter knockout mutants of M. graminicola to natural toxic compounds, xenobiotics, and antagonistic bacteria. Our results show that MgAtr4 plays a role in virulence, whereas other ABC transporters provide protection against fungicides, plant metabolites and antibiotics produced by antagonistic bacteria. Therefore, the data presented suggest that ABC transporters contribute to the competitive ability of M. graminicola.
366. Transcript profiling of the fungus Monacrosporium haptotylum during infection of the nematode Caenorhabditis elegans. Tunlid, A., Fekete, C., Tholander, M., Ahren, D, Friman, E. and Johansson, T. Department of Microbial Ecology, Lund university, Lund, Sweden
Monacrosporium haptotylum infects nematodes using adhesive knobs, which are developed on branches of vegetative mycelium. To identify genes and metabolic pathways that are expressed during development of traps and infection of nematodes, we have analyzed 7113 expressed sequence tags from four different cDNA libraries, vegetative mycelium, knobs, and knobs infecting the nematode Caenorhabditis elegans for 4h and 24 h, respectively. The ESTs were clustered into 2729 unigenes (contigs) representing 2729 putatively unique transcripts. Approximately 400 of these unigenes were of nematode origin. Approximately 23-31 % of all ESTs displayed significant similarities to sequences found in the GenBank database and this information was used for functional and metabolic annotations. Only 10.4 % of the contigs contained ESTs from more than one cDNA library, thus the patterns of genes expressed in the three libraries were significantly different. To obtain further information on the expression levels of the ESTs, the total set of unigenes were spotted on a cDNA array. Data on the patterns of expression of these genes will be presented.
367. Mating, conidiation and pathogenicity of Fusarium graminearum, the main causal agent of the head blight disease of wheat, are regulated by the MAP kinase Gpmk1. Nicole J. Jenczmionka, Frank J. Maier, Anke P. Loesch, and Wilhelm Schaefer. University of Hamburg, Institute of General Botany, Department of Molecular Phytopathology and Genetics (AMP III), Ohnhorststr. 18, D-22609 Hamburg, Germany.
Up to date only very little is known about the molecular infection mechanisms of the head blight pathogen of wheat, Fusarium graminearum (teleomorph Gibberella zeae). Here we report on the isolation and characterization of the Fus3 / Pmk1 MAP kinase homologue Gpmk1 from F. graminearum. Transformation-mediated gene disruption was used to create strains with a non functional Gpmk1 coding sequence. RT-PCR proved the absence of the corresponding mRNA. The deletion mutants exhibit a reduced conidia production and are sexually sterile. Inoculation into wheat spikeletts demonstrate that the DGpmk1 mutants are fully apathogenic. Therefor, this MAP kinase is involved in the regulation of several important steps of the fungal life cycle, including pathogenicity.
368. Functional characterization of the transcription factor Fopta1 in strains of Fusarium oxysporum f.sp. phaseoli. B. Ramos, A. García-Sánchez, A.P. Eslava and J.M. Díaz-Mínguez. Área de Genética, Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca, 37007 Salamanca, Spain.
Fusarium oxysporum f.sp. phaseoli J.B. Kendrick & W.C. Snyder is the fungus wich causes Fusarium wilt, a serious disease of common bean (Phaseolus vulgaris L.). There are over 120 formae speciales described capable of causing this disease. It shows a wide range of infection as species and host specificity as formae speciales, that makes it an attractive model for the study of the molecular interactions involved in pathogenicity and/or virulence. We have isolated the Fopta1 gene (F. oxysporum f.sp. phaseoli transcription activator), belonging to the GAL4 family of transcription factors. Copies of this gene are present both in pathogenic and nonpathogenic strains of F. oxysporum f.sp.phaseoli, but only the copies in highly virulent strains seem to be fully functional. We have analized the differences in the promoter region to study how some minor nucleotide changes in the TATA box can drastically reduce the expression of Fopta1 in weakly virulent and nonpathogenic strains. Previously we had detected the expression of Fopta1 by RT-PCR both in mycelium of highly virulent strains grown in vitro and in bean plants inoculated with these strains. Here we report the analysis of expression of Fopta1 in the course of the infection process in bean plants by means of real-time PCR, in comparison with a constitutive expressed gene coding for beta-tubulin. Also, we show the analysis of gene inactivated mutants obtained by using a transformation procedure based on Agrobacterium tumefaciens. This research was supported by grant AGL 2001-2052 (Ministerio de Ciencia y Tecnología of Spain). Brisa Ramos was the recipient of a fellowship from the INIA (Ministerio de Ciencia y Tecnología of Spain) and Asunción García-Sánchez was the recipient of a fellowship from Caja Rural de Salamanca (Castilla y León, Spain).
369. Disruption of the homoaconitase gene of the fungal barley pathogen Pyrenophora teres results in lysin auxotrophy, disturbed conidiation and strongly reduced virulence. Karen Sonnenberger, Frank J. Maier, and Wilhelm Schaefer. University of Hamburg, Institute of General Botany, Department of Molecular Phytopathology and Genetics (AMP III), Ohnhorststr. 18, D-22609 Hamburg, Germany.
The lysin biosynthesis pathways of fungi and plants differ greatly. Therefor, fungal enzymes involved in lysin biosynthesis may serve as targets for new fungicides. We cloned the homoaconitase gene lys4) from the fungal barley pathogen Pyrenophora teres. Transformation-mediated gene replacement was used to create strains which lack a major part of the lys4 coding sequence. RT-PCR proved the absence of the homoaconitase mRNA. Homoaconitase enzyme activity was measured in a crude extract of mycelia grown in complete medium. The D lys4 mutants exhibit a 65 % reduced activity in comparison to wild type. D lys4 mutants are unable to grow in-vitro on minimal medium, whereas wild type like growth can be restored by addition of lysin to the medium. Conidiation of the Dlys4 P. teres strains was reduced to less then 10%, even under optimal conditions. Pathogenicity studies of D lys4. teres strains show that they are greatly reduced in virulence and unable to colonize barley leaves beyond the point of inoculation. For the first time, we could show that lysin prototrophy is necessary for virulence of a phytopathogenic fungus.
370. Analysis of a non ribosomal peptide synthase gene from Alternaria brassicae and flanking genomic sequences. Thomas Guillemette, Babu Subramanian, Adnane Sellam, Philippe Simoneau UMR PaVé N̊77, Faculté des Sciences, 2 Bd Lavoisier, 49045 Angers cedex, France
Alternaria brassicae, a necrotrophic seed-borne pathogen of crucifers, produces host-specific toxins called destruxins. These metabolites are cyclic depsipeptides that may be synthesized by the fungus via a non ribosomal route using a multifunctional modular enzyme called non-ribosomal peptide synthase (NRPS). A 22 kbp NRPS gene was identified in A. brassicae by screening a cosmidic genomic library with an homologous PCR probe obtained with degenerate primers. This gene encodes a protein of ca. 790 kDa that contains four amino-acid activation, three epimerization, six condensation and six thiolation modules. The modular architecture of this peptide synthase suggests the presence of at least one additional NRPS gene in the A. brassicae genome. Approximatively 4.5 kbp upstream the NRPS start codon, a gene encoding an ABC-transporter (MRP type) was found. In addition, a dicarboxylic acid permease gene and an ORF similar to the Bys1 gene of the dimorphic fungus Blastomyces are present immediately downstream the NRPS stop codon. Structural analysis of these genes and corresponding proteins as well as results of expression studies will be presented.
371. Enzyme characteristics of Candida albicans secreted lipases LIP1, LIP4, and LIP10. Siegfried Salomon, Inga M. Melzer, Christian A. Haase, Frank Stehr, and Wilhelm Schaefer. University of Hamburg, Institute of General Botany, Department of Molecular Phytopathology and Genetics (AMP III), Ohnhorststr. 18, D-22609 Hamburg, Germany.
The human pathogen Candida albicans is able to cause several different types of infections such as oral, vaginal, or systemic candidosis. Various virulence factors have been suggested to be important during different types of C. albicans infections. These factors include the yeast-to-hyphal transition, adhesion factors, or surface hydrophobicity, phenotypic switching, molecular mimicry, and the secretion of hydrolytic enzymes. While secreted aspartic proteinases and phospholipases are well characterized, the role of other secreted hydrolytic enzymes such as esterases and lipases as virulence factors is unknown. Lipolytic activity enablesC. albicans to grow on lipids as the sole carbon source. Southern blot analysis and screening of the sequence data from the C. albicans genome project revealed a family of lipase genes with at least ten members. The ORFs of the ten lipase genes consist of between 1281 and 1416 bp and encode highly similar proteins with up to 80 % identical amino acid sequences. For detailed biochemical characterization of lipase 1 (LIP1), 4 (LIP4), and 10 (LIP10), the enzymes were expressed functionally in the methylotrophic yeast Pichia pastoris. After transformation and selection, the heterologous expressed enzymes were tested for pH and temperature optimum, substrate specificity, and sensitivity against drugs which inhibit lipases. Our data clearly demonstrated that LIP1, LIP4, and LIP10 shows distinguished activities in spite of their high sequence homology.
372. Pathogenesis of Mycosphaerella graminicola : two complementary approaches for the identification of molecular determinants. A. Cousin, M. Dufresne and T. Langin. Laboratoire de Phytopathologie Moléculaire, Institut de Biotechnologie des Plantes, Université Paris-Sud, France.
Septoria leaf blotch of wheat caused by Septoria tritici (teleomorph, Mycosphaerella graminicola) is a serious foliar disease that becomes increasingly important in all temperate regions of the world. This fungus penetrates the leaf through stomatal openings without the differentiation of any penetration structures. The infection cycle of M.graminicola is relatively well known at the cytological level, however the molecular basis of pathogenesis is poorly understood. In order to identify pathogenicity genes, different strategies have been developed in our laboratory. The first one consists in random insertional mutagenesis using a transforming plasmid. 195 stable hygromycin-resistant transformants of M.graminicola were obtained and screened for loss of pathogenicity using two infection assays, (i) on detached leaves and (ii) on seedlings. Among 11 mutants showing a reduced ability to induce blotch symptoms, two have been studied in more details : A18, a non pathogenic mutant and D22 showing a great reduction of pathogenicity. Molecular analysis and more detailed phenotypic characterization of these two strains will be presented. The second strategy consists in a candidat approach. Based on homology with the pmk1 gene of M.grisea encoding a MAP kinase essential for appressorium formation and invasive growth, the mgmk1 gene from M.graminicola encoding a putative PMK1-like MAP kinase was cloned. The gene is present in the genome as a single copy. In order to assess the role of MgMK1 in the life cycle of M.graminicola, targeted gene disruption is currently conducted. Studies of mutants carrying an interrupted copy of the gene will be performed. Is MgMK1 essential for plant infection? May MAP kinase pathway be widely conserved in pathogenic fungi for regulating infection?
373. Transcriptome-like approach to study global and fine appressorium development and functionality in Colletotrichum lindemuthianum. R. Lauge, C. Veneault and T. Langin. Laboratoire de Phytopathologie Moléculaire, Institut de Biotechnologie des Plantes, Université Paris-Sud, France.
Colletotrichum lindemuthianum is the causal agent of anthracnose on common bean. This ascomycete is an interesting plant pathogen model, as it displays a hemibiotrophic cycle and differentiates several well-defined infection structures (appressorium, infection vesicle, primary hyphum, secondary hyphum) on its host. We have used a cDNA library to initiate a transcriptome-like approach on the different development stages of its infection cycle. 5000 independent clones have been archived, of which half have been spotted on filter replicates for expression studies. The setup of a method for in vitro differentiation of appressoria has allowed us to start the study of specific genes expression during this critical step for penetration. Clones presenting a differential expression have been identified and analysed. Among others, cla1 the C. gloeosporioides cap5 orthologue (unknown function) and cla3 an ubiquitin encoding gene were found upregulated, and. cla2 the yeast DSK2 orthologue (mitosis control via requirement for spindle body duplication) was found downregulated during appressorium development, respectively. Using this method, as well as SSH, we have also identified genes displaying a differential expression between wild-type appressorium and appressorium obtained from the clk1 kinase mutant which is blocked at the penetration step (Dufresne et al., 1998). Such genes, named clak for C. lindemuthianum appressorium kinase, could correspond to targets of the signal transduction pathway that CLK1 is part of. Possible involvement of these genes in appressorium development and/or functionality will be presented, as well as new genes that are under characterization.
374. Signalling in the interaction Claviceps purpurea and rye. Jan Scheffer and Paul Tudzynski. Institut für Botanik, Westfälische Wilhelms-Universität Münster, Germany.
Claviceps purpurea is a common phytopathogenic ascomycete which colonizes only grass florets. This interaction represents an interesting fungal model for oriented growth comparable e.g. to the guidance of axons to neural synapses in animals. Our main interest is to identify signalling cascade components involved in directed growth during infection. Strong candidates are those known to be essential for the pathogenicity of Claviceps, such as the cpmk2 MAP kinase (Mey et al. 2002). Deletion mutants invade the host tissue but they are not able to tap the vascular tissue. These mutants are a valuable tool for the identification of other signal chain components mediating directed growth. Further interesting genes possibly involved in directed and/or oriented growth are homologues to COT1, a Ser/Thr protein kinase responsible for hyphal elongation in Neurospora crassa and to CDC42 and Ras, small G proteins involved in the mediation of cell polarity. The influence of these genes in the orientation of C. purpurea is to be investigated. Another approach for the isolation of genes involved in oriented growth is the creation of an insertional mutant library based on the Agrobacterium-mediated T-DNA-transfer. Mutants with impaired growth can be identified easily in an in vitro system for cultivation and infection of rye ovaries, which has been established recently.
375. Gene regulation by the histone deacetylase Hdc1, and the identification of genes important for pathogenesis in Cochliobolus carbonum. Jennifer A.Bieszke1, Johannes Galehr2, Paolo Amedeo3, Olen C. Yoder3 and Jonathan D. Walton1. 1MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA. 2Department of Molecular Biology, University of Innsbruck, Innsbruck Austria. 3Torrey Mesa Research Institute, 3115 Merryfield Row, San Diego, CA 92121, USA.
Hdc1 is a histone deacetylase important for pathogenesis of Cochliobolus carbonum on maize. A null hdc1 mutant of C. carbonum has reduced virulence and down-regulation of genes encoding cell wall degrading enzymes (CWDEs) implicated in pathogenesis (Baidyaroy et al., 2001, Plant Cell 13:1609). Two approaches are being used to determine if Hdc1 has a direct role in transcriptional regulation of CWDEs during pathogenesis. The first is to characterize the global acetylation state in the hdc1mutant as compared to wild type using quantitative protein blot analysis with antibodies specific to different acetylated forms of histones. The second approach is to analyze differences in the acetylation states among CWDE promoters using chromatin immunoprecipitation (ChIP). In addition to characterizing the mechanism of gene regulation by Hdc1, a second objective is to identify additional genes whose expression is affected by HDC1 or byccSNF1 (which also causes reduced transcription of CWDE genes and reduced virulence; Tonukari et al., 2000, Plant Cell 12:237). To do this an Affymetrix GeneChip array with ~20,000 DNA sequences of C. heterostrophus, which is also a foliar pathogen of maize, was probed with RNA from wild type or from hdc1 or ccsnf1 mutants. All strains were grown under conditions that either repress or induce CWDE gene expression. Under inducing conditions, ~600 genes were regulated differentially in wild type. Further analysis has differentiated the regulation of some of these genes in the two mutants as well as identified a subset of these genes that appears to be co-regulated by HDC1 and ccSNF1, whereas regulation of other genes differs between the two mutants. Genes identified by GeneChip analysis will be confirmed by RNA blotting, then tested for a role in growth and pathogenesis by targeted gene disruption.
376. Pathogenicity factors and host responses in the Heterobasidion annosum conifer pathosystem. Jan Stenlid, Åke Olson, Magnus Karlsson, Mårten Lind and Fred Asiegbu. Department of Forest Mycology and Pathology, University of Agricultural Sciences, SWEDEN
The presentation will provide information on factors that are important to determine the success of Heterobasidion annosum as a pathogen of conifers. Important basic traits include ample spore production, colonisation and growth in host tissue including ability to attach to cell surfaces, to degrade macromolecules, to produce toxins, to handle low oxygen tension, and to detoxify host defence. Recent EST projects have indicated induction of a range of proteins including hydrophobins, mitochondrial proteins and proteins involved in signal transduction pathways. Infection experiments with defined strains of H. annosum have shown the importance of nucleus-mitochondria interaction for the expression of virulence. A genetic map is constructed for positioning of the pathogenicity factors. A search for host genes involved in the interaction with H. annosum. Following challenge with the pathogen, expressed genes in Pinus sylvestris has been hybridised to microarrays of P. taedagenes. A range of induced responses have been identified. Future work will focus on characterising early responses and to compare with the reactions of the host to saprotrophs and mycorrhizal fungi.
377. Abscisic acid biosynthesis in Botrytis cinerea. Verena Siewers and Paul Tudzynski. Institut fuer Botanik, Westfaelische Wilhelms-Universitaet Muenster, Germany
Botrytis cinerea causes the grey mould disease in more than 200 plant species. Like several other phytopathogenic fungi, B. cinerea has been shown to produce different kinds of phytohormones in axenic culture. Although the impact of this biosynthetic capacity on host-parasite interaction is still unclear, it has been suggested that production of the plant hormone abscisic acid (ABA) is supporting the infection process. Our aim is to investigate the role of fungal ABA by cloning genes encoding enzymes of the biosynthetic pathway, studying their expression and analysing the pathogenicity of deletion mutants. In plants, ABA is derived from cleavage products of carotenoids. We could exclude the presence of a similar pathway in B. cinerea, as inhibition of carotenoid biosynthesis with diphenylamine (DPA) and inactivation of a putative gene of this pathway did not affect the ability of the fungus to produce ABA. A group of enzymes probably involved in fungal ABA biosynthesis are P450 monooxygenases. Therefore, about 20 different P450 monooxygenase encoding genes of B. cinerea were cloned and characterized. Two of them (P450-12, P450-16) were found to be differentially expressed during ABA production. Gene deletion of P450-12 had no influence on the synthesis of the plant hormone. In another approach to investigate the possible role of P450 monooxygenases the cytochrome P450 oxidoreductase encoding gene (cpr) was isolated and deleted.
378. Isolation and characterization of Fusarium graminearum mutants compromised in mycotoxin production and virulence. Frances Trail 1, Martin Urban 2, Iffa Gaffoor, 1, Ellie Mott 3, Corrie Andries 1 and Kim Hammond-Kosack 2. 1Departments of Plant Biology and Plant Pathology, Michigan State University, East Lansing MI 48824, USA. 2 Plant-Pathogen Interactions Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK. 3 Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK.
A population of random plasmid insertion F. graminearum mutants, derived from the strain PH-1, (n=1170) was screened for their ability to cause head blight disease of wheat. Nine disease attenuated Fusarium, daf mutants were recovered. Each daf mutant differed in its ability to (a) cause disease symptoms on the head and stem bases of wheat plants, (b) affect grain fill and (c) synthesize trichothecene mycotoxins. Additional characteristics of each daf mutant will be presented.
379. Expression Analysis of the Candida albicans Lipase Gene Family During Experimental Infections and in Patient Samples. Frank Stehr1, Marianne Kretschmar2, Birgit Maehnss3, Karsten Neuber3, Bernhard Hube1,4, and Wilhelm Schaefer1. 1 University of Hamburg, Institute of General Botany, Department of Molecular Phytopathology and Genetics (AMP III), Ohnhorststr. 18, D-22609 Hamburg, Germany, 2Institute of Medical Microbiology and Hygiene, Faculty of Clinical Medicine, Mannheim, 3University Hospital Hamburg-Eppendorf, Department of Dermatology and Allergology, 4Robert Koch-Institute, Berlin.
Secreted lipases of Candida albicans are encoded by a gene family with at least ten members (LIP1-LIP10). The role of these putative virulence factors in pathogenicity still needs to be elucidated. Therefore, the expression pattern of this multigene family was investigated using RT-PCR in experimental infections and in samples of patients suffering from oral candidosis. The findings illustrate that individual LIPs were differentially regulated in a mouse model of systemic candidosis with some members showing sustained expression, while others were transiently expressed or even silent. It is demonstrated, that the LIP expression profile depended on the stage of infection rather than on the organ localization. This temporal regulation of lipase gene expression was also detected in an experimental model of oral candidosis. Furthermore, the expression of candidal lipase genes in human specimens is shown for the first time. The present study indicates, that LIP1-10 constitute a new family of virulence factors.
380. Mating Types of Pyrenophora teres and P. graminea, the causal agents of net blotch and leaf stripe of barley.
Frank J. Maier, Gerrit Mohrmann, Maram Girgi, Bettina Wagner, and Wilhelm Schaefer. University of Hamburg, Institute of General Botany, Department of Molecular Phytopathology and Genetics (AMP III), Ohnhorststr. 18, D-22609 Hamburg, Germany.
Net bloch, caused by the leaf pathogen Pyrenophora teres is, one of the most devastating diseases of cultivated barley (Hordeum vulgare). Only moderate resistance to this disease is available in the field and nothing is known about the fungal genes that condition pathogenicity or that cause the cultural variability observed. We initiated molecular genetic studies of this pathogen to address these issues and understand its life cycle. Mating in fungi is controlled by mating type genes. The fungal class of ascomycetes is characterized by, as regards their mating behaviour, three types. 1. sterility, 2. self-incompatibility (heterothallism) and 3. self-compatibility (homothallism). In most ascomycetes, mating is controlled by a single locus, the MAT genes. The two alternate forms are not classical alleles, but rather idiomorphs, because the highly dissimilar MAT genes (MAT 1 and MAT 2) are located between highly conserved flanking sequences at the same locus. The barley pathogen P. teres is a heterothallic ascomycte. We cloned the responsible genes for sexual reproduction (MAT 1 and MAT 2) of different P. teres isolates and of the closely related P. graminea, causal agent of stripe disease of barley. Sequence comparisons of the different mating type genes will be presented.
381. Analysis of AVR-Pita Gene Function in Pathogenicity and Host Specificity. Barbara Valent1, Melinda Dalby1, Prasanna Kankanala1, Darcey Klaahsen1, Yulin Jia2 and Gregory T. Bryan3. 1Department of Plant Pathology, Kansas State University, Manhattan, KS 66506-5502. 2USDA-ARS, Dale Bumpers National Rice Research Center, Stuttgart, AR, 72160-0287. 3AgResearch Grasslands, Palmerston North, New Zealand
We continue our characterization of the rice blast AVR-Pita gene and its corresponding resistance gene Pi-ta. Sequence analysis suggests that AVR-Pita encodes a secreted zinc metalloprotease of the Deuterolysin metalloprotease (M35) family. Our current data suggest that the Pi-ta protein is an intracellular receptor that binds directly to the mature AVR-Pita protease inside the plant cell, initiating defense responses. Expression ofAVR-Pita is infection-specific, and the gene is highly expressed during the colonization phase of the susceptible interaction. Current research objectives include demonstrating protease activity for AVR-Pita in vitro, defining its natural substrates within the infected plant tissue, and ultimately determining the relationship between protease activity and the molecular recognition event that triggers disease resistance. AVR-Pita has properties characteristic of bacterial "effector" proteins that are delivered into the cytosol of living plant cells through a specialized type III secretion system. We have initiated studies to define when and how AVR-Pita is delivered into the plant cytosol to interact with Pi-ta. A thorough understanding of AVR-Pita biology will lead to strategies for identification of additional fungal effector molecules and for understanding the mechanism for delivery of critical pathogenicity factors into living plant cells.
382. Xylanolytic transcriptional activators in Cochliobolus carbonum. John Scott Craig, Scott Baker, Olen Yoder and Jonathan Walton. MSU-DOE Plant Research Lab, Michigan State University, East Lansing, MI. and TMRI, San Diego, CA.
Indirect evidence suggests that Cochliobolus carbonum penetrates maize leaves by enzymatic and not mechanical means. C. carbonum produces a variety of extracellular cell wall degrading enzymes (CWDEs), including xylanases, pectinases, and glucanases, which may be involved in enzymatic penetration. Disruption of ccSNF1, the C. carbonum ortholog of yeast SNF1, results in down-regulation of CWDE expression, reduced growth on alternate carbon sources, and attenuated leaf penetration efficiency (Tonukariet al., 2000, Plant Cell 12:237). To understand further the role of CWDEs in fungal pathogenesis, genes related to XlnR, which encodes a transcriptional activator involved in the control of expression of xylanolytic and other CWDE genes in Aspergillus niger(van Peij et al., 1998, Mol Microbiol. 27:131) were studied. Two putative C. heterostrophus homologues of this gene were identified in the genomic sequence database at the Torrey Mesa Research Institute. The corresponding genes were isolated from C. carbonum and used to create strains with targeted deletions. Experiments designed to determine the effect, if any, of XlnR deletion on xylanolytic gene expression and pathogenicity of C. carbonum are underway; results will be reported in the relevant poster session.
383. The role of the trichothecens in different isolates of Fusarium graminearum causing Fusarium head blight (FHB) in cereals. Frank J. Maier1, Simon Henning1, Thomas Miedaner2, and Wilhelm Schäfer1. 1University of Hamburg, Institute of General Botany, Department of Molecular Phytopathology and Genetics (AMP III), Ohnhorststr. 18, D-22609 Hamburg, Germany; 2University of Hohenheim, State Plant Breeding Institute (720), 70593 Stuttgart, Germany.
Head blight caused by the ascomycetic fungus Gibberella zeae (Fusarium graminearum) is one of the most destructive diseases of cereals. It causes yield reductions and contaminates grain with mycotoxins, which constitutes a potential risk for human and animal nutrition. One important class of mycotoxins produced by several Fusarium species are the trichothecene derivates (e.g. nivalenol, deoxynivalenol). Trichothecenes accumulate in Fusarium-infested food and non-specifically affect most eukaryotes.We want to investigate whether virulence is only determined by the presence of the trichothecenes or is a quantitative character that is heterogeneously determined by several factors that differ from one isolate to the other. Three isolates of F. graminearum, well characterized in field experiments, were selected: one medium aggressive isolate that produces nivalenol, another medium aggressive isolate which forms deoxynivalenol. The third one is highly virulent and produces high levels of deoxynivalenol. The Tri5 genes in these three isolates were disrupted. A general biochemical and molecular biological analysis as well as the phytopathogenic properties of the mutants will be presented.
384. Plant recognition and appressoria formation in arbuscular mycorrhizal symbiosis. Natalia Requena and Esther Serrano Plant Ecophysiology Dept., University of Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen.
Symbiosis formation in AM fungi is induced upon a partner recognition event that switches the developmental programme of the fungus and triggers appressorium formation. In the absence of host root recognition the fungus retracts back its protoplasm and suffers a temporary growth arrest. This developmental switch is the subject of our study. We have identified a fungal novel gene, GmGIN1, exclusively expressed during the out planta phase. The full-length GmGIN1 cDNA encodes a protein of 429 amino acids with two-domain structure and a putative self-splicing activity. The N-terminal domain shares sequence similarity to a novel family of GTP binding proteins. The C-terminus has a striking homology to the C-terminal part of hedgehog proteins from metazoa. Hedgehog proteins suffer autoproteolysis catalyzed by the carboxy terminus. The splicing reaction renders an active N-terminal domain covalently modified by a cholesterol moiety at its carboxy end. Our hypothesis is that GmGIN1 also suffers autoproteolysis to generate the functional GTPase protein. In order to show the splicing activity of GmGIN1, 207 aa from the carboxy terminus were expressed in E. coli as an N-terminal His6 tag fusion protein. A splicing reaction involving the conserved GCF motif should render two peptides with molecular masses of 21 and 7 kDa. A small amount of spliced protein could be observed after induction of splicing activity with DTT. Further work is in progress to improve the yield of active protein and the splicing efficiency.
385. Hipervirulent strains of Botrytis cinerea show altered respiration. Arranz, M., Eslava, A.P., Díaz-Mínguez, J.M. and Benito, E.P. Área de Genética. Centro Hispano-Luso de Investigaciones Agrarias (CIALE). Universidad de Salamanca. 37007. Salamanca. Spain.
Botrytis cinerea is an important plant pathogenic fungus with a wide host range. In plant-fungal pathogen interactions the analysis of differential gene expression has been utilized to isolate genes specifically induced during pathogenesis. This strategy allowed us to isolate gene Bde47. Sequence analysis predicted mitochondrial inner membrane localization for the protein encoded by Bde47 . Mutants altered in gene Bde47, obtained by gene replacement, show increassed aggressiveness on several hosts and produce higher amounts of active oxygen species, in addition to several other alterations during saprophytic growth. We propose that these phenotypes are consequence of altered mitochondrial activity. First, to confirm the mitochondrial localization of the Bde47 gene product we have obtained transformants expressing the GFP fused to the B. cinerea Bde47 gene. Confocal microscopy analysis demonstrated that the Bde47 gene product is targeted to the mitochondria. Second, to investigate whether or not the hipervirulent mutant strains are altered in mitochondrial activity, we measured and compared O2 consumption in the B. cinerea wild type strain and in two representative gene replacement mutant strains. The data obtained demonstrated a higher level of oxygen consumption in both mutant strains. Studies using inhibitors either of complex IV or of the alternative oxidase (Antimycin A and Salicylhydroxamic acid, respectively) indicated that the activity of complex IV is severely reduced in the mutant strains, while oxygen consumption through the alternative oxidase pathway is highly increased.
This research was supported by grants SA069/01 (Junta de Castilla y León, Spain) and AGL 2001-2052 (Ministerio de Ciencia y Tecnología, Spain).
386. Characterisation of aminophospholipid translocases during infection by the rice blast fungus Magnaporthe grisea. Martin J. Gilbert and Nicholas J. Talbot. School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter EX4 4QG, United Kingdom.
Plant infection by the rice blast fungus Magnaporthe grisea is brought about by the action of specialised infection cells called appressoria. These cells generate enormous turgor pressure, which is translated into the necessary force that allows the fungus to breach the plant cuticle. Targeted gene disruption of the M.grisea PDE1 gene showed its importance in penetration hypha development and pathogenicity. The predicted PDE1 gene product showed highest similarity to members of the aminophospholipid translocase group of P-type ATPase and was shown to be a functional homologue of the yeast ATPase gene ATC8. Expression studies showed that PDE1 is expressed at low levels in all stages of the M. grisea development. The sub-cellular localisation of PDE1(p) is being examined using a C-terminal GFP-tagging approach and also by using purified antibodies raised against the large central loop of PDE1(p). Biochemical studies are also currently underway to ascertain the substrate specificity of the PDE1(p). To further analyse the possible roles of aminophospholipid translocases during M. grisea infection, another member of this group MgDRS2 has been identified and its relationship to PDE1 is being investigated.
387. PEP2 serves as a gratuitous pathogenicity gene in Nectria haematococca. Li-Feng Chen1, Yinong Han2, Haruhisa Suga1,3 and H. Corby Kistler1,4. 1University of Minnesota, St. Paul, MN 55108, USA; 2National Research Council, Halifax, Canada; 3Molecular Genetics Research Center, Gifu University, Gifu, Japan; 4USDA ARS Cereal Disease Laboratory, St. Paul, USA
A gene cluster consisting of several genes that contribute to the pathogenicity of Nectria haematococca to pea plants is located on a fungal supernumerary chromosome. Among the cluster's pea pathogenicity (PEP) genes, PEP1, PEP2 and PEP5 as well as PDA1 (the gene for pisatin demethylase) can independently increase pathogenicity to pea when added individually to an isolate that lacks the 1.6 Mb chromosome containing the PEP cluster. PEP2 is a gene encoding a 233 amino acid protein with sequence similarity to polyadenylate binding proteins. To further examine the contribution of the PEP2 gene to fungal pathogenicity a plasmid was constructed for PEP2 gene replacement. A 13 kb BamHI / SalI fragment including PEP2 and flanking sequences was cloned in E. coli, and an internal 3.5 kb of Bgl II fragment including the entire PEP2 gene was replaced by a fungal hygromycin resistance cassette to produce the new plasmid pUCPEP2. This plasmid was used to transform wild type strain 77-13-5. Of the several transformants obtained, two (called P4 and P5) were confirmed by Southern hybridization to be gene replacements, while four transformants (P1, P6, P7 and P8) were due to ectopic integration of pUCPEP2. Another transformant (P40) was found to have lost the entire 1.6 Mb supernumerary chromosome. Tests on pea epicotyls and roots showed there was no significant difference in pathogenicity between gene replacement and ectopic transformants but that P40 was significantly lower in pathogenicity than 77-13-5. We conclude that while PEP2 can increase pathogenicity of a strain lacking the chromosome containing the PEP gene cluster, it is not essential for maximum pathogenicity in strains containing an otherwise intact 1.6 Mb chromosome. PEP2 thus may represent a gratuitous or functionally redundant pathogenicity factor in some genetic backgrounds.
388. Analysis of the MAP kinase-signalling pathway in the mycoparasitic response of Trichoderma atroviride. Velázquez-Robledo Rocío, Artemio Mendoza-Mendoza, and Alfredo Herrera-Estrella. Department of Plant Genetic Engineering, Centro de Investigación y Estudios Avanzados. Unidad Irapuato. Apartado Postal 629. 36500, Irapuato Guanajuato., México.
Fungi belonging to the genus Trichoderma are ubiquitous microorganisms in soil. Several species of the genus have been used as biocontrol agents against a broad spectrum of phytopathogenic fungi. Parasitic response in Trichoderma has been well documented and is now referred as mycoparasitism. Mycoparasitic response involves the production of volatile antibiotics, rhizosphere competence and production of hydrolytic enzymes such as beta-1,3 glucanases, chitinases and proteases. In the presence of a potential host, T. atroviride tends to coil around the host hyphae followed by penetration due to the degradation of the outer surface of the host. Expression of a basic protease (prb1), and an endochitinase (ech42) are linked to this later event. These two hydrolytic enzymes seem to be regulated by different environmental factors. The use of specific inhibitors revealed the relevance of a MAPK signalling pathway in the induction of prb1 and ech42 mediated by nitrogen and carbon limitation respectively. A gene encoding a MAPKK kinase homologous to the yeast kinase Ste11 was cloned from the mycoparasite T. atroviride and named tmk11. This gene has a single copy in Trichoderma genome and showed high similarity to ncr1 from Neurospora crassa. Tmk11 null mutants were obtained by antisense strategy. The relevance of this kinase during the interfungal relationship between T. atroviride and Rhizoctonia solani will be discussed.
389. Isolation and characterization of GCN5 histone acetyltransferase gene from Ustilago maydis. Juan González-Prieto, Cristina Reynaga-Peña, Angel Domínguez, and José Ruiz-Herrera. Depto. de Ingeniería Genética, CINVESTAV Unidad Irapuato, Irapuato, Mexico.
Isolation of a histone acetyltransferase gene from Ustilago maydis was carried out by specific hybridization to a gene fragment obtained by polymerase chain reaction. The gene (UmGCN5) contains an open reading frame of 1421 bp encoding a putative protein of 473 aminoacids. This protein exhibits a high degree of homology with histone acetyltransferases from different organisms. Null mutants were constructed by substitution of most of the coding sequence with the hygromycin B resistance cassette. Mutants displayed a slight reduction in growth rate under different conditions, grew as mycelial cells, and formed fuzz-like colonies under all conditions where wild-type strains grow yeast-like. cAMP addition failed to revert this phenotype. The most important phenotypic characteristic of the null mutants was their loss of virulence, and their incapacity to form teliospores.
390. An inducible defense mechanism against nitric oxide in Candida albicans. Wiriya Chiranand1, Breanna Ullmann1, Hadley Myers1, Qiang Zhao1, Paul R. Gardner2, and Michael C. Gustin1. 1Biochem & Cell Biol, Rice Univ, Houston, TX. 2Children's Hosp Med Ctr, Cincinnati, OH
The opportunistic pathogen Candida albicans poses a serious threat to patients with compromised immune systems. Immune cells respond to C. albicans with a complex arsenal that includes production of damaging reactive oxygen and nitrogen radicals. WhetherCandida possesses a means to defend against free radicals such as nitric oxide (NO) is yet undetermined. In the yeast Saccharomyces cerevisiae and various bacteria, defense against nitric oxide involves an NO-consuming flavohemoglobin, and we have identified three homologous genes in the C. albicans genome. Using deletion mutant strains constructed for each gene, we demonstrated that just one homologue, CaYHB1, is responsible for significant nitric oxide consumption and detoxification. In contrast to nonpathogenic S. cerevisiae, NO consumption by C. albicans is greatly enhanced by exposure to nitric oxide. Correspondingly, CaYHB1 mRNA levels are rapidly and dramatically increased by various NO-generating agents or low concentrations of NO sources but not by other oxidants. Loss of CaYHB1 results in a higher sensitivity of C. albicans to NO-mediated growth inhibition. Thus C. albicans utilizes a rapid, specific and highly inducible NO defense mechanism involving one of three putative flavohemoglobin genes. Funding: NSF and NIH
391. Genetic characterization of Fusarium wilt of Arabidopsis. Andrew C. Diener and Frederick M. Ausubel. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114 USA.
A wilt disease of Arabidopsis thaliana can be instigated by pathogenic forms ofFusarium oxysporum. The formae speciales, conglutinans, raphani and matthioli, isolated from crucifer hosts are pathogenic to Arabidopsis as well. The susceptibility is specific because Arabidopsis is completely resistant to pathogenic forms from flax and tomato. Different ecotypes (or varieties) of Arabidopsis display varying degrees of natural resistance to the crucifer pathogens. We have begun to characterize the genetic interaction between Arabidopsis and Fusarium by examining the resistance and virulence, respectively, of both plant and fungal mutants. Our progress in screening for Fusarium mutants that have defects in virulence will be detailed.
392. Genes for pea pathogenicity and a gene for utilization of a specific pea root exudate are located on the same supernumerary chromosome in Nectria haematococca. Marianela C. Rodriguez and Hans D. VanEtten. Department of Plant Pathology, University of Arizona
Nectria haematococca can exist in a wide range of environments, although individual isolates have a limited habitat range. Previous research indicated that habitat diversity is partially due to the presence of supernumerary chromosomes containing host specifying genes. These chromosomes have been called "Conditionally Dispensable" (CD) since they are not needed for axenic growth, but they are important in expanding the habitat of individual isolates. One of these CD chromosomes carries a cluster of genes for peapathogenicity, called the PEP cluster. Preliminary work has suggested that the same CD chromosome that carries the PEP cluster, also carries genes for the utilization of homoserine (HS), which is a transitional amino acid present in large amounts in the root exudates of pea plants. Recent results have shown that isolates cured of the CD chromosome by benomyl treatment can not grow on HS. These results verify that genes for homoserine utilization (HUT) are located on the same CD chromosome as the PEP genes. Conventional genetics indicate that the HUT phenotype is encoded by more than one gene, and that at least one HUT gene is on a non-dispensable chromosome. We propose that HUT genes provide isolates carrying them a competitive advantage in the pea rhizosphere, prior to the establishment of a pathogenic association with the pea roots.
393. Saturation Mutagenesis of Magnaporthe grisea using DNA Insertions- a genomics approach. Sara L. Tucker1, Yan Meng2, Gayatri Patel2, Natalia Kaczkurkin1, Yong-Hwan Lee3, Nicole Donofrio3, M. Alejandra Mandel1, Collin LeMaster1, Melania Figueroa1, Brian King2, Love Gill2, Jordan Brock2, Ravi Rajagopalan3, Douglas Brown3, Thomas Mitchell3, Ralph Dean3, Mark Farman2, Marc Orbach1, 1University of Arizona, Tucson, AZ 85721,2University of Kentucky, Lexington, KY 40546, 3North Carolina State University, Raleigh, NC 27695 USA
Magnaporthe grisea represents a model organism for the study of fungal pathogenicity and growth. We are taking a functional genomics approach to study the infection of rice by M. grisea. As part of an NSF-funded project that involves collaboration between 9 research groups at 6 universities we are creating a collection of 50,000 DNA insertion lines in theM. grisea strain 70-15. Our goal is to disrupt all genes encoded in the M. griseagenome in order to determine which genes are important for pathogenicity on rice. The mutants are being generated using transforming constructs containing a Hygromycin resistance cassette flanked by promoterless fluorescent protein genes. Constructs differ in their end sequences (AT vs. GC rich ends) in an attempt to maximize genome coverage and to assess whether the ends target insertion into AT- or GC-rich regions. Standard and Agrobacterium tumefaciens-mediated transformation methods are being used to introduce the DNA intoM. grisea. All 50,000 insertion lines will be analyzed for defects in pathogenicity, metabolism and alterations in morphology. Such a project necessitates development of high throughput methods for the generation and screening of putative transformants. In addition, a database has been created for recording all of the data generated per transformant. The advances made so far and the optimization of techniques for genetic manipulation of this fungus will be discussed.
394. Identification of pathogenicity determinants in the interaction between the oomycete plant pathogen Peronospora parasitica and Arabidopsis. Jim Beynon, Rebecca Allen, Peter Bittner-Eddy, Laura Grenville and Anne Rehmany. Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK.
Peronospora parasitica is the causal agent of downy mildew on Arabidopsis and Brassica crops. We have cloned the Arabidopsis resistance genes RPP13 andRPP1 that recognise the downy mildew isolates Maks9 and Emoy2, respectively. These two interactions result in very different resistance phenotypes. RPP13 elicits a localised necrotic lesion whereas RPP1 results in a spreading necrotic lesion. In order to study the basis of these different interaction phenotypes we are cloning the corresponding avirulence genes, ATR13 and ATR1. We have used a map based cloning approach and located the genes to overlapping BAC contigs. The mapping cross has been shown to be segregating for up to 15 different avirulence genes. Using Suppression Subtractive Hybridisation we have identified a range of genes that are specifically up-regulated on infection of Arabidopsis. An analysis of their structure and their relationship to the avirulence genes will be presented.
395. Negative regulation of prb1 and ech42 from Trichoderma atroviride is driven by physical contact with the host. Carlos Cortes1,2, Vianey Olmedo-Monfil1, Alfredo Herrera-Estrella1. 1Plant Genetic Engineering Department. CINVESTAV-IPN. Irapuato, Guanajuato. Mexico. 2Escuela de Quimico-Farmacobiologia. UMSNH. Morelia, Michoacan. Mexico.
Interfungal relationships depend on recognition between the associated species.Trichoderma atroviride is able to establish necrotrophic associations with a wide range of fungi, most of them classified as phytopatogens. This ability has been successfully used in the biocontrol of several fungal pests. It has been proposed that the mycoparasitic activity in T. atroviride has limited specificity based on the fact that several fungal species are suceptible to Trichoderma attack. In this regard, the induction of hydrolytic enzymes play a key role in completing the parasitic phenomenum. Prb1 and Ech42 are normally induced whenTrichoderma is directly confrontated with a potential host even if physical contact is avoided. When Sclerotium rolfsii was used as a host the induction of prb1 andech42 was only detected when both host and parasite were physically separated by a cellophane membrane. On the contrary, transcriptional repression of both genes occurred when physical contact took place. Trichoderma virens, a closely related specie, showed induction of the homologous genes to prb1 and ech42 when confrontation was carried out with S. rolfsii demostrating a specie-specific response. Fusion of the prb1 promoter to the gfp reporter gene allowed the detection of the possible negative regulatory element.
396. New transformation system for the human pathogenic yeast, Candida parapsilosis, based on FLP-mediated site specific recombination. Attila Gacser1, Siegfried Salomon1, Joachim Morschhaeuser2 and Wilhelm Schaefer1. 1University of Hamburg, Institute of General Botany, Department of Molecular Phytopathology and Genetics (AMP III), Ohnhorststr. 18, D-22609 Hamburg, Germany. 2University of Wuerzburg, Institute for Molecular Biology of Infectious Diseases, Röntgenring 11, D-97070 Wuerzburg, Germany
The opportunistic human pathogen Candida parapsilosis causes superficial cutaneous infections and systemic candidosis. Here, we describe an efficient system for gene disruption in this fungus, using direct transformation and dominant selection of a clinical isolate. This omits the necessity to create auxotrophic mutants. The transformation system is based on the MPAR (mycophenolic acid) resistance marker and its subsequent deletion by FLP-recombinase mediated, site specific recombination. The MPAR cassette was used to generate C. parapsilosis mutants disrupted for one allele of either the lipase1 gene or lipase2 gene. This flipper system for genetic manipulations enhances the efficiency of molecular biological research in C. parapsilosis. Thus, it helps to improve our knowledge of its biology and virulence.
397. Downy Mildew Genomics and Plant Disease Resistance. Posthuma, K., Elberse, J., Weisbeek, P. and van den Ackerveken, G. Molecular Genetics. Utrecht University, Utrecht, The Netherlands.
Downy mildews infect many important crops grown in temperate climates. To protect crops from downy mildew disease, plant breeders have crossed in natural resistance genes. However, resistance is usually rapidly overcome by the pathogen. This project aims to identify new resistance genes that mediate the recognition of important pathogen proteins and may therefore be more durable. A genomics approach is used to identify downy mildew genes that encode secreted proteins and that are specifically expressed during the infection process. On the one hand, these genes can be used by plant breeders to select potential breeding lines. On the other hand, this project will enable us to study which downy mildew genes are required in the infection process. Two downy mildew – plant interactions are studied: Peronospora parasitica – Arabidopsis and Bremia lactucae – lettuce. Complementary DNA libraries of spores and downy mildew-infected plants have been constructed to collect a large number of Expressed Sequence Tags (ESTs). These ESTs will be screened for signal peptides and for similarity to genes or proteins in the public databases. Microarray technology will be used to study the expression of these genes during infection of the host. B. lactucae genes encoding extracellular proteins will be transiently expressed in lettuce, and lines reacting with a hypersensitive response will be tested further for downy mildew resistance. The selected lines used by lettuce breeders to obtain more durable resistance to downy mildew disease.
398. Stage-specific gene transcription in Phytophthora infestans prior to and during the early stages of potato infection. Anna O. Avrova, Eduard Venter*, Paul R. J. Birch, and Stephen C. Whisson. Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, U.K.*Department of Genetics, University of Pretoria, Pretoria 0002, South Africa.
Phytophthora infestans, the oomycete agent of potato and tomato late blight, produces several different cell types prior to and during the early stages of potato infection. All of these cell types can be easily generated and studied in the absence of the host plant and so form the basis for developmental stage-specific gene discovery. We have used amplified fragment length polymorphism (AFLP)-based mRNA fingerprinting (cDNA-AFLP) and suppression subtractive hybridisation (SSH) to identify transcripts that were up-regulated in germinating zoospore cysts, and germinating zoospore cysts with appressoria, but not in vegetative mycelium. These transcripts included representatives of most major classes of heat shock proteins:hsp60, hsp70, hsp90 and hsp100. Other stress-related, and potential pathogenicity genes were also identified. Real-time RT-PCR was used to quantify the expression of selected transcripts originating from germinating cysts, with and without appressoria, relative to the constitutively expressed actB gene, in vegetative mycelium, germinating cysts, and at three time points post-inoculation of potato cultivar Bintje (15, 48, and 72 hours). All of the transcripts were up-regulated in germinating cysts, and many were found also to be up-regulatedin planta.
399. Cloning avirulence genes from Phytophthora infestans. Miles Armstrong, Anna Avrova, Steve Whisson, Paul Birch. Plant Pathogen Interactions Scottish Crop Research Institute.
Phytophthora infestans is a hemi-biotrophic pathogen that exhibits gene-for-gene interactions with its host potato. Compared to bacterial pathogens, relatively few fungal avirulence genes have been cloned and only one from the oomycetes. We aim to clone and characterise avirulence genes from the oomycete pathogen P. infestans as a first step in studying the molecular basis of host/oomycete recognition and specificity. Two approaches are being followed: The Avr2 gene is being mapped in a segregating population of F1 individuals derived from a sexual cross. Secondly, a number of candidate genes identified in silico from ESTs are being amplified and sequenced from isolates in order to identify SNPs associated with specific avirulences. This approach has identified a strong candidate for the Avr3 gene.
400. Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic oomycete Phytophtora palmivora. Hoang Quoc-Khanh and Tran Hoang Ngoc Ai. Institute of Tropical Biology 1 Mac Dinh Chi St., Dist. 1, Ho Chi Minh City, VN-70000 VIETNAM
Agrobacterium tumefaciens-mediated transformation has been successfully applied to the phytopathogenic fungus Phytophtora palmivora. The transformants were resistance to hygromycin B as the selective trait, governed by Aspergillus nidulans gpd promoter andA. nidulans trpC terminator. The transformants appeared to be stable through mitotic and meiotic cell division. The presence of the hph gene was detected by PCR. Our findings indicate that the technique offers potential as an alternative tool to conventional transformation techniques and should address to the P. palmivora pathogenicity and decision-makes to disease management.