215 Definition of tissue-specific and general requirements for plant infection in a phytopathogenic fungus. Marie Dufresne, Andrew Foster and Anne Osbourn. Sainsbury Laboratory, John Innes Centre, Norwich, UK.
Surprisingly little is known about the factors that condition the ability of pathogens to colonize different plant tissues. Magnaporthe grisea has recently emerged as a paradigm for molecular genetic dissection of factors determining fungal pathogenicity to leaves, and mutational analyses have identified a number of genes required by M. grisea for pathogenic differentiation and colonisation of leaf tissue. We have shown that M. grisea can infect the roots of cereals, and that this fungus can be used for genetic analysis of factors determining root infection. Preliminary investigations using characterised M. grisea mutants from other laboratories have allowed us to identify mutants with leaf-specific, root-specific and general defects in ability to cause lesion formation on leaves and/or roots. For example, the melanin biosynthetic mutant alb1 is non-pathogenic to barley leaves, but is unimpaired in its ability to cause lesions on roots of barley or wheat. Conversely, a global regulator of nitrogen assimilation (NUT1) is essential for root infection but is dispensable for disease development on leaves, while the MAP-kinase PMK1 and the predicted ABC transporter ABC1 are required for infection of both tissues and so represent general disease determinants. Screening of REMI mutants is now being carried out to identify novel determinants of tissue specificity and general factors required for colonisation of plants in M. grisea. To date two REMI mutants have been identified that fail to give lesions on wheat and barley roots. Characterisation of the genetic defects in these mutants is underway.
216 A MAPK controls host root penetration and pathogenesis in Fusarium oxysporum. Antonio Di Pietro, F. Isabel García-Maceira, Emese Méglecz, and M. Isabel G. Roncero. Departamento de Genética, Universidad de Córdoba, Spain.
The soilborne vascular wilt fungus Fusarium oxysporum infects a wide variety of plant species by directly penetrating roots, invading the cortex and colonizing the vascular tissue. A mitogen-activated protein kinase (MAPK), FMK1, controls several key steps in pathogenesis of F. oxysporum. Targeted mutants carrying an inactivated copy of fmk1 show normal vegetative growth and conidiation in culture but are non-pathogenic on tomato plants. Conidia of fmk1 mutants germinating in the tomato rhizosphere fail to differentiate penetration hyphae resulting in strongly reduced root attachment. Additional defects in fmk1 mutants include impaired abilities to breach the liquid-air interface and to grow invasively on tomato fruit tissue. We are currently attempting to identify downstream effector genes whose expression is altered in F. oxysporum MAPK mutants. One such gene, pl1, shows drastically reduced transcript levels in fmk1 mutants and encodes the cell wall-degrading enzyme pectate lyase.
217 Functional analysis of two closely linked genes at the Tox1B Locus of Cochliobolus heterostrophus. Mark Rose2, Thipa Asvarak1, Shun-Wen Lu1, Xudong Zhu1, Olen Yoder1, GillianTurgeon1. 1Department of Plant Pathology, Cornell University, Ithaca, NY 14853 2Novartis Agribusiness Biotechnology Research, Research Triangle Park, NC 27709
Cochliobolus heterostrophus race T, causal agent of Southern Corn Leaf Blight, requires T-toxin for high virulence on T-cytoplasm corn. Production of the polyketide T-toxin is controlled by two unlinked loci, Tox1A and Tox1B carried on 1.2 Mb of DNA not found in race O, a mildly pathogenic form of the fungus, or in any other Cochliobolus spp. or relative. Horizontal acquisition of the 1.2 Mb of DNA is suggested. PKS1, a polyketide synthetase encoded at Tox1A, has been proven necessary for T-toxin production. To identify genes at Tox1B, a race T cDNA library was successively probed, first with a NotI fragment from a Tox1B deletion mutant, then with the corresponding wild type fragment, to screen for genes missing from Tox1B. Closely linked decarboxylase (DEC1) and reductase (RED1) genes were identified. Disruption of DEC1 in race T reduced both T-toxin production and virulence of the mutant on T- cytoplasm corn while disruption of RED1 affects neither. Transformation of a large deletion mutant at Tox1A or at Tox1B with PKS1 or DEC1/RED1 respectively, or race O with all three genes, fails to restore toxin production, suggesting a cluster of genes necessary for toxin biosynthesis at both loci.
218 A novel alcohol oxidase/RNA-binding protein with affinity for mycovirus double- stranded RNA from the plant pathogenic fungus Helminthosporium (cochliobolus) victoriae. S. A. Ghabrial, Plant Pathology Dept., University of Kentucky, Lexington, KY 40546 USA
Diseased isolates of the plant pathogenic fungus Helminthosporium (cochliobolus) victoriae harbor two isometric dsRNA viruses. The diseased isolates produce little or no victorin, and are hypovirulent. We have cloned and sequenced a novel alcohol oxidase (Hv-p68) from H. victoriae that co- purifies with mycoviral dsRNAs. Hv-p68 was also detected as a minor component of the virus capsid. Sequence analysis revealed that Hv-p68 belongs to the large family of FAD-dependent GMC oxidoreductases and that it shares significant sequence identity (>67%) with the alcohol oxidases of the methylotrophic yeasts. Unlike the intronless alcohol oxidases from methylotrophic yeasts, a genomic fragment of the Hv-p68 gene was found to contain four introns. Hv-p68, purified from fungal extracts, showed only limited methanol oxidizing activity and its expression was not induced in cultures supplemented with methanol as the sole carbon source. Northern hybridization analysis suggested that Hv-p68 expression is induced by virus infection since significantly higher Hv-p68 mRNA levels (10-20 fold) were detected in virus-infected isolates compared to virus- free ones. We demonstrated by gel mobility assays and Northwestern blot analysis that Hv-p68 exhibits RNA-binding activity and presented evidence that the RNA- binding domain is localized within the N-terminal region that contains a typical ADP-binding beta-alpha-beta-fold motif. In addition to RNA-binding activity, Hv- p68 also exhibits phosphotransferase/kinase activities, and is a candidate for the virion-associated kinase activity responsible for phosphorylation of the capsid protein. Hv-p68 is proposed to play a role in viral RNA packaging/ replication and in regulating viral pathogenesis.
219 Cloning and characterisation of avirulence gene Avr2 of Cladosporium fulvum. Rianne Luderer, Frank L.W. Takken, Pierre J.G.M. de Wit and Matthieu H.A.J. Joosten. Laboratory of Phytopathology, Wageningen University and Research Centre, Binnenhaven 9, 6709 PD Wageningen, NL
The interaction between tomato and the biotrophic fungus Cladosporium fulvum complies with the gene-for-gene model. The tomato resistance locus Cf-2 contains two homologous genes, Cf-2.1 and Cf-2.2. Both genes confer a hypersensitive response (HR)-mediated resistance to isolates of C. fulvum producing the matching elicitor. Attempts to clone the Avr2 gene by reverse genetics have not been successful. Therefore, a PVX-based binary expression vector was used to allow Agrobacterium tumefaciens-delivered functional expression of a cDNA library of C. fulvum in tomato plants (Takken et al., Plant J., 24(2), 2000). Upon toothpick inoculation of Agrobacterium colonies onto tomato leaves, five independent clones, containing an identical open reading frame (ORF), were identified that gave Cf-2-specific HR. Avr2 encodes a cysteine-rich protein of 78 amino acids (AA), with a predicted signal peptide for extracellular targeting of 20 AA. Tobacco lines expressing either Cf-2.1 or Cf-2.2 responded with a HR upon AVR2, indicating that both Cf-2 genes confer AVR2 recognition. Strains of C. fulvum virulent on Cf2 tomato plants circumvent recognition by various single mutations in the ORF of the Avr2 gene, that either result in a frameshift or in the insertion of a stopcodon. To prove that Avr2 is indeed responsible for avirulence of C. fulvum on Cf2 plants, a strain virulent on Cf2 plants will be transformed with the Avr2 gene.
220 Sequence analysis and expression studies of Erysiphe graminis cDNAs from epiphytic mycelium. S. Eckert1, M. Both1, A. DeBruijn2, E. Mueller2 and P. Spanu1. 1Imperial College of Science Technology and Medicine, London, UK 2Syngenta, Jealott's Hill Research Station, Berks, UK
The ascomycete fungus Erysiphe graminis f.sp. hordei (Egh) is an obligate biotrophic parasite which is the causal agent of barley powdery mildew. We have extracted RNA from epiphytic Egh mycelium 3 days after infection i.e.prior to "en masse" sporulation. This RNA was then used to construct a cDNA library in the phagemid vector lambdaTriplEx2. We have analysed the sequences of about 450 randomly selected clones (Expressed Sequence Tags -ESTs). From the pool of sequences analysed, 349 clones (78%) are over 200 nt. Of these sequences, 191 identify homologs with significant BLAST matches, of which 167 have known functions. About half of the sequences analysed are unique. Of the sequences over 200 bp, the most abundant group with known or deduced function are transposons and retroelements, followed by ESTs encoding proteins involved in translation, and those involved in signalling. The library also contains ESTs encoding deduced proteins ("hypothetical proteins") with similarity to 15 different unknown ORFs in other organisms. ESTs with no known homologies are 45%. We also present a comparison of the results with Egh ESTs from a library of germinating spores (Oliver et al. www/crc.dk/phys/blumeria). A few clones were selected and used to probe RNA blots to assess expression during different stages of development and infection (spores, pre-sporulating epiphytic mycelium and infected barley leaves with heavily sporulating colonies). The results also enable us to assess whether the abundance of these clones in the cDNA library reflects the level of expression in the mycelium. This study is the starting point for a global expression study on a Egh using micro-arrays.
221 Activation of a plant secondary metabolite by Botrytis cinerea: Resveratrol acts as a profungicide. Alexander Schouten, Lia Wagemakers, Francesca L. Stefanato, Rachel M. van der Kaaij & Jan A.L. van Kan. Department of Phytopathology, Wageningen University, PO Box 8125, 6700 EE Wageningen, The Netherlands.
In grape (Vitis) the secondary metabolite resveratrol is considered a phytoalexin, which protects it from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonisation of grape. We initiated a functional molecular-genetic analysis of B. cinerea laccases by characterising laccase gene(s) and evaluating the phenotype of targeted gene replacement mutants. Two different laccase genes from B. cinerea were characterised, Bclcc1 and Bclcc2. Only Bclcc2 was strongly induced in liquid cultures by either resveratrol or tannins. This suggested that Bclcc2, but not Bclcc1 plays an active role in the oxidation of both resveratrol and tannins. Analysis of both Bclcc1 and Bclcc2 replacement mutants confirmed this. Only Bclcc2 replacement mutants were not capable of converting both resveratrol and tannins. When growing on resveratrol, both the wild type and the Bclcc1 mutant showed an inhibited growth and Bclcc2 replacement mutants were unaffected. Thus, contrary to the current theory, BCLCC2 does not detoxify resveratrol but converts it into compounds that are more toxic for the fungus itself. Consquently, rather than a virulence factor BCLCC2 can be considered an avirulence factor. The activation of a plant secondary metabolite by a pathogen introduces a new dimension to plant-pathogen interactions and the phytoalexin concept.
222 Molecular identification of a sexual interloper: Venturia pirina, reproduces sexually on its nonhost, apple. Kim M. Plummer1, Christiane Stehmann2 and Shaun Pennycook2. 1The University of Auckland, School of Biological Sciences, Private Bag 92019, Auckland, New Zealand. 2HortResearch, Mt Albert Research Centre, 120 Mt Albert Rd, Private Bag 92169, Auckland, New Zealand
Venturia inaequalis causes apple black spot (apple scab), the most economically important disease of apple world wide. Venturia spp. are pathogens on Rosaceae and are commonly identified on the basis of morphological characters and host specificity. Morphological characters are sometimes absent or ambiguous and so DNA sequence analysis was used to aid identification. rDNA sequence variations, between different Venturia spp., were utilised to design species-specific primers. rDNA sequencing was used to identify Venturia spp. from apple leaf litter in New Zealand (spring, 1998). Venturia pirina (pear scab pathogen) and Venturia inaequalis (apple scab pathogen) were detected as ascospores discharged from apple leaf litter. Pseudothecia of both species were located on the dead apple leaves, however only those of V. inaequalis were associated with scab lesions. V. pirina was identified using rDNA sequence analyses, as morphological characters could not distinguish this fungus from V. asperata (a rare saprophyte on apple) and other Venturia spp. pathogenic on rosaceous fruit trees. V. pirina field isolates were pathogenic on pear, but only weak saprophytes on apple. In rare instances, when appressoria of V. pirina appeared to penetrate the cuticle of apple leaves, epidermal cells responded with a localised hypersensitive response (HR). This is the first report of HR-like events induced by V. pirina on its nonhost, apple. This is also the first record of sexual reproduction of V. pirina on apple. It is assumed that V. pirina pseudothecia formed in senescing tissue, when defence mechanisms such as HR were no longer active.
223 Genome organization at the site of integration for a horizontally transmitted element in a Leptosphaeria species. Janet L. Taylor NRC Plant Biotechnology Institute, Saskatoon, SK Canada
Several Leptosphaeria species cause disease on cruciferous plants. These species are distinguished by the phytotoxins that they produce and their hosts. One of these Leptosphaeria species makes polyketide toxins and attacks Japanese horseradish. However, we found two isolates of this species that have a repetitive element from the related species, L. maculans. These two isolates have an expanded host range, now attacking brown and yellow mustard. Their metabolite profiles have changed with no production of polyketide toxins. Now they make a host-selective toxin that is a tricyclic sesquiterpene. We have isolated two cosmid clones covering the region of element insertion. Initial sequence analysis by blast search has revealed the following similarities to known proteins. An area with high similarity to a phosphatidylinositol glycan class O protein of mouse. These proteins are involved in transfer of phosphoethanolamine to the GPI anchors that attach proteins to the cell surface membrane. Another area has low similarity to reverse transcriptase genes found in retrotransposons. This sequence is separated from the inserted repetitive element, LMR1. Most interestingly, we have sequence that translates to a protein with a conserved motif of sesquiterpene synthases. This motif is the only region of significant similarity among the synthases. Further details of the genome organization will be presented.
224 Biocontrol of Fusarium oxysporum infection via protoplast fusants possessing genes producing disease-suppressive agents. Kamel A. Ahmed (1), Mohamed H. Hamoda (1), Mohamed S. Abd Elsalam (2) and Ola I. El-Hamshary (2) 1- Genetics Dept., Fac. Agric., Cairo Univ. 2- Microbial Genetics Dept., NRC., Egypt.
The capacity of some microbial strains to produce biocontrol agents such as antimicrobial substances and iron chelates was determined. Pair-wise protoplastfusants between these strains e.g. Pseudomonas fluorescens, P. aurogenosa, Entrobacter cloacae and Trichoderma harzianum were carried out. Efficiency of these fusants on infectivity of Fusarium oxyspoum will be discussed.
225 Xylanases of Magnaporthe grisea: differential requirement for pathogenicity. Shengcheng Wu, Zhiying Zhao, Alan G Darvill, and Peter Albersheim. University of Georgia, Complex Carbohydrate Research Center, Athens, Georgia, USA
Phytopathogenic fungi that infect Gramineae tend to secrete copious amount of arabinoxylan-degrading enzymes. These enzymes are proposed to be pathogenic factors for they help in breaking down the cell wall barrier and obtaining nutrients so that the fungi can enter and flourish inside plants. Magnaporthe grisea, the rice blast fungus, produces multiple isoforms of endo-beta-1,4-D-xylanases under various growing conditions. We have cloned a total of six xylanase genes that are differentially expressed both in fungal culture and in infected rice seedlings. For example, two (Xyl4 and Xyl5) are expressed exclusively in infected rice leaves. Infection assays of knockout mutants indicate significant reduction in pathogenicity of the mutants that lack anyone of Xyl3, Xyl4 and Xyl5 genes. Additional multiple knockout mutants (xyl3xyl4, xyl3xyl5, xyl4xyl5 and xyl3xyl4xyl5) will be generated, and their phenotype and pathogenicity will be examined and presented. Interestingly, careful infection analysis of the previously described xyl2 mutants on various ages of rice seedlings reveals convincing increase in virulence. Thus, it is possible that either Xyl2 or the hydrolytic products of Xyl2 is recognized by rice as elicitor. (This work was supported by U. S. Department of Energy grant DE-FG05-93ER20114 and the DOE-funded (DE-FG05-93ER20097) Center for Plant and Microbial Complex Carbohydrates.)
226 Cross-infection and survival of Colletotrichum acutatum from strawberry in strawberry and nonspecific hosts using transformants containing GFP constructs. S. Freeman1, S. Horowitz 1,2, and A. Sharon 3. 1ARO, The Volcani Center, Bet Dagan 50250, and 2Tel Aviv University, Tel Aviv 69978, Israel
Colletotrichum acutatum, the cause of strawberry anthracnose, is one of the most important fungal pathogens of this crop worldwide. Cross-infection from additional hosts may exist since many botanical species are hosts to the pathogen and can serve as a source for primary inoculum. Host range, specificity and survival of C. acutatum from strawberry was examined with pepper, eggplant, tomato, bean and strawberry. Colonization and survival was confirmed by PCR-specific primer amplification for C. acutatum. Elucidation of the mode of infection and colonization of the pathogen in host and nonhost tissues was achieved using transgenic isolates. Transformants of C. acutatum, expressing the UidA (GUS) or SGFP-TYG reporter genes were obtained by electroporation with constructs containing a homologous DNA fragment. Major differences between pathogen interaction with the host and nonhost were observed 50hr post-inoculation. In strawberry, highly asymmetric appressoria developed due the formation of a penetration pore compared to symmetric structures in nonhost tissues lacking the pore. The strategy of invasion in strawberry included a short biotrophic phase in which the hyphae were mainly restricted to the intercellular space between the epidermal cells. Four days after symptom development, a large amount of intercellular and intracellular fluorescence accompanied by hyphal growth were observed with the GFP transformants. This marked the necrotrophic phase responsible for anthracnose and blight symptoms. While no visible disease symptoms were observed in the nonhost plants, extensive hyphal growth and appressoria were present on the leaf surfaces but were restricted to the intercellular spaces. Whether this pathogen can adapt as an epiphyte or possess an asymptomatic endophytic lifestyle in nonhost plants should be considered.
227 Identification of gene products involved in plant pathogen interactions using proteomics. Fitzgerald, A.M.2, Stehmann, C.1, Greenwood, D.R.1, Plummer, K.M.2 . 1 HortResearch, Mt Albert Research Centre, Private Bag 92 169, Auckland, NZ. 2 School of Biological Sciences, Auckland University, Private Bag 92 019, Auckland, NZ
Apple scab is caused by the ascomycete fungus Venturia inaequalis. In contrast to many fungal plant pathogens, V. inaequalis grows between the cuticle and epidermal cells during its biotrophic phase of growth. At this stage, the pathogen does not ingress into or disrupt plant cells. Consequently, the intercellular space (apoplast) is the plant/pathogen interface and should contain plant and pathogen gene products that are responsible for the maintenance and specificity of the disease interaction.
The aim of this project is to identify the fungal proteins that confer avirulence and pathogenicity in addition to plant disease response proteins from apoplastic fluids of scab infected apple. These apoplastic fluids contain only a limited number of proteins and about 30-40 bands are visible on coomassie stained SDS-PAGE. Therefore, proteomics is a realistic approach to identify the proteins secreted into the apoplast during infection.
Proteins have been extracted from the apoplast of infected and uninfected apple seedlings. Protein extracts were heavily contaminated with phenolics and other low MW contaminants and methods had to be developed to remove these for successful two-dimensional (2D) electrophoresis. 2D electrophoresis separated approximately 200 apoplastic proteins of healthy seedling leaves, which are mainly acidic and of relatively low MW (20-40 kD). In infected leaves, apoplastic proteins appeared to be degraded to peptides of MW 3-6 kD. Therefore, it is not possible to identify additional secreted plant and/or fungal proteins, specific to this disease, by comparison of infected vs. uninfected tissue. The mechanisms involved in this proteolytic breakdown are under current investigation. Apoplastic proteins of infected and uninfected tissue are being analysed using MALDI TOF, ESI-MS/MS and Edman chemistry.
228 ATP-binding cassette (ABC) transporters from Botrytis cinerea. Maarten A. De Waard1, Keisuke Hayashi2, Henk-jan Schoonbeek1, Tycho Vermeulen1. 1Wageningen University, Laboratory of Phytopathology, Wageningen, The Netherlands. 2Ube Research Laboratory, Ube, Yamaguchi, Japan.
Botrytis cinerea is a pathogen with a broad host range. Host plants are known to produce plant defense compounds, implying that the pathogen is exposed to a variety of fungitoxic compounds. Obviously, the pathogen developed mechanisms to cope with plant defense compounds during evolution. One mechanism can be activity of ABC transporters, which reduce the accumulation of toxic compounds in mycelium. These transporters may also account for development of resistance to fungicides used to control diseases incited by the pathogen. To test these hypotheses, we have studied ten ABC genes (BcatrA-N) from B. cinerea. Expression of BcatrB is upregulated by the grapevine phytoalexin resveratrol. BcatrB replacement mutants show an increased sensitivity to resveratrol and are less virulent on grapevine leaves. The same mutants have an increased sensitivity to phenazine antibiotics produced by Pseudomonas and the phenylpyrrole fungicide fludioxonil. These results demonstrate that BcatrB provides protection against natural toxic compounds and, at the same time, is a determinant in fungicide activity. Fungicides from different chemical classes not only induce transcription of BcatrB but of other Bcatr genes as well, suggesting that several ABC transporters effect sensitivity of B. cinerea to these products. Laboratory-generated mutants of B. cinerea resistant to azole fungicides display a decreased accumulation of azoles and an increased basal level of BcatrD expression. Transcript levels induced by azoles in azole-resistant mutants correlate with resistance levels as well. Therefore, we propose that BcatrD is the major ABC transporter involved in activity of azoles. Currently, we test this by phenotyping the sensitivity of disruption and overexpression mutants of BcatrD to azoles.
229 ATP-binding cassette (ABC) transporters from the wheat pathogen Mycosphaerella graminicola. Maarten A. De Waard, Marco M.C. Gielkens, Stephen D. Goodall, Ioannis Stergiopoulos, Koen Venema, and Lute H. Zwiers. Wageningen University, Laboratory of Phytopathology, Wageningen, The Netherlands.
The fungus Mycosphaerella graminicola (anamorph state: Septoria tritici) is the causal agent of septoria tritici leaf blotch, one of the most important diseases of wheat. Little is known about mechanisms of pathogenesis and fungicide resistance in this pathogen. ABC transporters may play a role in these processes by providing protection against plant defense compounds and fungicides, respectively, or by secretion of virulence and mating type factors. This hypothesis forms the basis of our research. Five single copy genes encoding ABC transporters, designated Mgatr1-5, have been characterized. All genes have the [NBF-TMD6]2 configuration and are highly homologous to other fungal and yeast ABC transporters. Northern blot analysis revealed that they display distinct expression profiles when treated with secondary plant metabolites and fungicides. Expression of these genes was different in mycelium and yeast-like spores of this dimorphic fungus. Heterologous expression of the Mgatr genes in a strain of Saccharomyces cerevisiae with multiple disrupted ABC genes showed that the encoded proteins of most Mgatr genes can transport a range of chemically unrelated compounds, such as fungicides, mycotoxins, and secondary plant metabolites. The function of the Mgatr genes is also studied in disruption mutants of M. graminicola. These mutants were made by Agrobacterium tumefaciens-mediated transformation. We have developed this technique as an efficient tool to generate targeted gene disruption. At present, we are studying the phenotype of the disruption mutants with respect to sensitivity to toxicants and virulence.
230 A MAP kinase module regulating filamentous growth, mating and pathogenic development in Ustilago maydis. Philip Mueller, Michael Feldbruegge and Regine Kahmann. Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse D- 35043 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 pheromone response characterized by conjugation tube formation and pheromone-responsive gene expression. It was proposed that the pheromone signal is transmitted via a MAP kinase cascade to the transcription factor Prf1. Three putative components of this MAP kinase cascade have been described, kpp2 (ubc2)encoding a MAP kinase, fuz7 encoding a MAPK kinase and kpp4 (ubc4)encoding a MAPKK kinase. Deletion of either kpp2,fuz7 or kpp4 results in loss of conjugation tube formation and attenuates mating. While fuz7 and kpp4deletion mutants exhibit a complete block in pathogenic development, deltakpp2 mutants retain residual pathogenic potential. To analyze the connection between these signaling components in more detail we have generated constitutively active alleles of fuz7 (fuz7DD) and kpp4 (kpp4-2). Expression of kpp4-2 leads to elevated pheromone gene transcription and to conjugation tube formation in wild type cells, but not in deltakpp2 or in deltafuz7 strains suggesting that Kpp4 acts upstream of Fuz7 and Kpp2. Consistently, fuz7DD induces the same phenotype in a kpp2- dependent, but kpp4-independent manner. In addition we show that prf1 is dispensable for this morphological transition. We conclude that Kpp4, Fuz7 and Kpp2 are members of one MAPK cascade that regulates Prf1 and yet unidentified target proteins triggering morphological changes.
231 A gene directly regulated by the b mating type locus of Ustilago maydis is involved in pathogenic development. Gerhard Weinzierl1, Ralph Bohlmann2, Andreas Brachmann3, Tobias Hindemitt1, Regine Kahmann1 and Joerg Kaemper1. 1Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch- Str., D-35043 Marburg, 2Aventis Crop Science, 14 -20 Rue Pierre Baizet, B.P. 9163, F-69009 Lyon Cedex 09, 3Institute of Genetics and Microbiology, Ludwig-Maximilians University, Maria-Ward-Str. 1a, D-80638 Muenchen
In Ustilago maydis, the multiallelic b mating type locus provides the central regulatory instance for the establishment of the pathogenic stage during its life cycle. This developmental control is achieved via the b- encoded bE and bW homeodomain proteins. The bE and bW proteins can form a heterodimer that is thought to function as transcriptional regulator for genes that play a crucial role during differentiation processes linked to pathogenicity. To get an insight into these processes, our aim was to isolate direct target genes of the bW/bE heterodimeric complex. Using differential display we have isolated the b-dependently expressed gene dik6, encoding a putative seven transmembrane receptor. The promoter elements essential for b-mediated regulation were identified by deletion analysis. Subsequent electrophoretic mobility shift experiments and DNAse protection assays using a bE-bW fusion protein substantiated that dik6 is a direct target gene of the bE/bW complex. Deletion mutants of dik6 show only an attenuation of pathogenicity, i.e. 36% of the wild type infection rate. However, the search of the U. maydis genomic sequence revealed another potential transmembrane receptor with 36% identity to Dik6. Interestingly, the respective gene, dkh6, exhibits a b- dependent expression pattern identical to dik6, suggesting for both genes a possible function during the pathogenic development. We will discuss these results with respect to potential functions of Dik6 and Dkh6 during the infection process.
232 Genetics and ecology of fumonisin toxins. Anne E. Desjardins, Ronald D. Plattner, Gary P. Munkvold, and Robert H. Proctor. USDA/ARS, National Center for Agricultural Utilization Research, Peoria, IL and Department of Plant Pathology, Iowa State University, Ames, IA
Fumonisins are a family of toxic fungal metabolites produced by Fusarium verticillioides (synonyms F. moniliforme and Gibberella fujikuroi mating population A) and related species. F. verticillioides is not only the most common fungal pathogen of maize, but also is among the most common fungi found in symptomless maize plants. The recent cloning of fumonisin biosynthetic genes and development of techniques for gene disruption now allow critical analysis of the importance of fumonisins in fungal infection and disease. A polyketide synthase gene, designated FUM5, that is required for fumonisin biosynthesis has been cloned from F. verticillioides. Disruption of FUM5 in fumonisin-producing strains reduced fumonisin production by more than 99%. Two fumonisin- nonproducing (FUM5-) strains that were generated by FUM5 disruption and the two fumonisin-producing (FUM5+) strains from which they were derived were tested for their ability to infect maize ears and to cause ear rot following four application methods. In field tests in Illinois and Iowa in 1999 and in Illinois in 2000, fungal spores were applied to ears by silk-channel injection and by silk-spray, to stalks by injection, and to seeds by planting next to a fungal-infested toothpick. Disease severity on harvested ears was evaluated by visible symptoms and by the relative weight of symptomatic and nonsymptomatic kernels. Fumonisin levels in kernels were determined by HPLC. Presence of applied strains in kernels was determined by analysis of recovered strains for fumonisin production by HPLC and for FUM5 alleles by PCR. FUM5- strains were able to infect ears following all application methods. Following silk-channel application, FUM5- strains infected the kernels, caused ear rot, and inhibited the accumulation of fumonisins in the kernels. Application of FUM5- strains by silk-spray, stalk injection, or seed treatment increased the frequency of these strains in kernels, but did not increase levels of ear rot or affect the concentrations of fumonisins in the kernels. These results show that production of fumonisins is not required for F. verticillioides to cause ear rot following silk-channel injection, or to infect maize ears following silk-spray inoculation, stalk injection, or seed treatment. Thus, the importance of fumonisins in the ecology of F. verticilliodes remains unclear.
233 Identification and characterization of pathogenicity mutants of Colletotrichum lagenarium by insertional mutagenesis. Naoyuki Takayanagi1, Yoshitaka Takano2, and Tetsuro Okuno1. 1Kyoto University, Graduate School of Agriculture, Kyoto, Japan. 2NC State University, Plant Pathology, Raleigh NC USA
Colletotrichum lagenarium is the causal agent of anthracnose of cucumber. We have initiated a mutational analysis of fungal pathogenicity in C. lagenarium using restriction enzyme-mediated integration (REMI) mutagenesis. Here we present characterization of three pathogenicity mutants of C. lagenarium. Mutants KE51 and KH190 showed normal growth, conidiaion, germination, and appressorium formation. However, melanized appressoria formed by both mutants failed to penetrate into the host epidermal cells although they were able to penetrate cellulose membranes, which suggested that genes required for penetration into the host plant were impaired in these mutants. Mutant KH151 showed reduction in both growth and conidiation on nutrient agar, and failed to form appressoria. Furthermore, KH151 formed no lesions on cucumber leaves even when inoculated through wounded sites, suggesting lack of an ability to grow invasively inside the plant. Genomic DNA flanking the integrated plasmid was recovered from the mutant KE51. Using the recovered DNA as a probe, a genomic clone of the wild-type strain was isolated. The 4 kb HindIII fragment corresponding to the tagged site complemented the pathogenicity of KE51. These results demonstrate that this fragment contains a pathogenicity gene impaired in KE51. We determined DNA sequences of the plasmid insertion region in the complementing fragment, and found a single open reading frame that exhibited homology to genes encoding a methyltransferase-like protein identified in several organisms.
234 Effects of double-stranded RNA on protease production and virulence of Metarhizium flavoviride against the grasshopper Rhammatocerus schistocercoides . Maria Helena P. Fungaro1, Mayra Kassawara Martins1, Patrícia Vieira Tiago1, Marcos Rodrigues Faria2, Márcia Cristina Furlaneto1.1Universidade Estadual de Londrina, Londrina, Pr, Brazil. 2Cenargen, Embrapa, Bras lia, DF, Brazil
Bands of dsRNA were detected in five out of seven isolates of the entomopathogenic fungus Metarhizium flavoviride. In order to study the effects of dsRNA on protease production and virulence against the grasshopper, many methods were applied as attempts to obtain isogenic strains, differing by the presence or absence of dsRNA. Attempts to obtain isogenic strains by curing were unsuccessful. An alternative approach, i.e. the transfer of dsRNA via forced heterokaryosis was successfully conducted. The nuclear contents of the recipient colonies were confirmed to be identical to the parental strain by RAPD. The production of proteases determines virulence towards their hosts. So, the wild and isogenic strains (CG422 met and CG442 met dsRNA, without and with dsRNA, respectively) were analysed for Pr1 production and virulence against grasshopper Rhammatocerus schistocercoides. The results of Pr1 analyses showed no statistical differences among isolates with and without dsRNA. Data from bioassays also showed no statistical differences among isolates. We conclude that these dsRNA fragments do not cause hypovirulence to M. flavoviride .
235 Genetic variation in the cuticle-degrading protease activity of the entomopathogen Metarhizium flavoviride. Furlaneto, Marcia Cristina; Fungaro, Maria Helena Pelegrinelli; Pinto, Fabiana Gisele da Silva; Tiago, Patricia Vieira. University of Londrina, Microbiology, Londrina, Parana, Brazil
Extracellular proteases have been shown to be virulence factors in fungal entomopathogenicity. We examined the production of the cuticle-degrading extracellular proteases, subtilisin-like (Pr1) and trypsin-like (Pr2) in isolates of the fungus Metarhizium flavoviride. Fungal growth was in mineral-medium (MM) containing nitrate, and in MM supplemented with either cuticle from Rhammatocerus schistocercoides or with the non-cuticular substrate casein. The substrates used for growth influenced the expression of both proteases analyzed, and for nearly all isolates, the highest protease activities were observed in medium containing insect cuticle, with more Pr1 being produced than Pr2. There was natural variability in the production of cuticle-degrading proteases among isolates, although this was less evident for Pr2.
236 The compatible interaction between potato and Phytophthora infestans. Katinka Beyer1, Andres Binder2, Thomas Boller3, and Margaret A Collinge4. 1Friedrich Miescher-Institut, Basel, Switzerland. 2Syngenta, Basel Switzerland. 3Botanical Institut, Basel Switzerland . 4University of Zuerich, Plant Biology, Zuerich Switzerland
Phytophthora infestans causes late blight disease on potato and tomato, which is the most important disease of potato worldwide. P. infestans is notorious for causing the Irish Potato Famine shortly after it first appeared in Europe in the 1840s. Despite the fact that it is known since a long time, we still know little about the molecular processes involved in infection and resistance. In this work we are examining the interaction between potato and P.infestans with a genomics approach, looking at the genes induced during infection in both, host and pathogen. To screen for induced genes we used suppression subtractive hybridization (SSH), comparing different stages of the infection process. Screening of the subtracted libraries is still in progress, but we have already isolated a number of potato genes and a few P. infestans genes. For a subset of the plant genes differential expression has been confirmed on Northern Blots. Some of the plant genes have not been reported to be pathogen induced before. In contrast to many other studies, we are concentrating on the compatible interaction. The induced genes are interesting at both the scientific and the biotechnological level. The plant genes may include classic PR genes or genes involved in tolerance and in late-expressed resistance. The P.infestans genes induced during a compatible infection could tell us more about the interaction at the molecular level and may include pathogenicity or virulence factors. The information we acquire about this interaction could be valuable in the develpment of protection strategies against late blight disease.
237 Avirulence in Venturia inaequalis Win, J.2, Stehmann, C.1, and Plummer, K.2. 1HortResearch, Mt Albert Research Center, Auckland 2Department of Biological Sciences, University of Auckland, Auckland, New Zealand
Venturia inaequalis is a fungus that causes scab disease in
apple (Malus). We are investigating the gene-for-gene interaction between the
resistance gene Vm originally from Malus micromalus and the
corresponding avirulence gene avrVm from V. inaequalis. Host
differential reactions are readily distinguished by a hypersensitive response (HR) in host
differential h5 and large sporulating lesions (susceptibility) in host differential h1. It has been
thought that the recognition event between VM and AvrVM leads to HR and hence the
resistance. Our objective is to isolate and characterize avrVm using a reverse
genetics approach. HR is induced when the cell free culture supernatant (CFCS) of V.
inaequalis is infiltrated into leaves of h5 but not on the susceptible host h1. The
HR-inducing activity is greatly reduced by proteinase K digestion but resistant to boiling.
Ultrafiltration of the CFCS shows most activity is present in a fraction between 3 kDa and 30
kDa molecular weights and is contained in the supernatant after precipitation with 60% acetone.
The activity has also been localized to a fraction eluting from EconoPac-Q ion-exchange column.
Isoelectric focussing (IEF) gel electrophoresis shows that there are three predominant protein
species present in this fraction and they have very low pI (approximately between
3.0 and 3.4). The N-terminal amino acid sequences have been obtained from two of the three
proteins. Both show identical sequences. A degenerate primer has been designed to isolate the
cDNA encoding for the protein(s) using a PCR-based strategy. A genomic library will be
screened with this cDNA to isolate a full length putative avrVm gene. The identity
of the putative avrVm will be confirmed by complementation experiments.
238 The avirulence gene ACE1 (AVR1-IRAT7) of the rice blast fungus Magnaporthe grisea encodes a polyketide synthase. Heidi U. Böhnert, Isabelle Fudal, Waly Dioh, Didier Tharreau*, Jean- Loup Notteghem* and Marc-Henri Lebrun Physiologie Cellulaire Végétale, UMR 1932 CNRS/Biotechnologies - Aventis CropScience, 14 rue Pierre Baizet, 69009 Lyon, France. *UR-PHYMA, CIRAD-CA, Montpellier.
Resistance of rice to the rice blast fungus Magnaporthe grisea is governed by specific interactions between fungal avirulence genes and corresponding plant resistance genes. Genetic studies using the rice pathogenic M. grisea isolates Guy11 and ML25 which were fertile in crosses led to the identification of the avirulence gene AVR1-IRAT7 (1) that interacts with the rice resistance gene Pi11-t. AVR1-IRAT7 maps to chromosome one at 30cM from AVR1 CO39 (2). The gene was isolated by positional cloning and complementation analysis. A genomic fragment of 15.5 kb able to confer avirulence to virulent strains was found to contain only one large ORF termed ACE1. Disruption of ACE1 renders the avirulent parent Guy11 virulent. In the virulent parent, insertion of a retroposon has occurred at the ACE1 locus. Sequence analysis revealed that ACE1 encodes a large multifunctional enzyme possessing several domains characteristic of polyketide synthases. Expression of ACE1 is specific for the penetration of leaves and artificial membranes. We propose that it is not the ACE1 gene product itself, but a secondary metabolite which serves as the signal which is recognized by resistant host plants. ACE1 therefore represents a new class of fungal avirulence genes. 1. Silue D. et al. 1992. Phytopathology 82: 1462-1467. 2. Dioh W. et al. 1999. Molecular Plant-Microbe Interactions. 13: 317-327.
239 Vitamin Deficient Mutants of the Fungal Wheat-Pathogen, Mycosphaerella graminicola. Joanne Ayriss1, John Hargreaves1 and Andy Corran2. 1IACR-Long Ashton Research Station 2syngenta, Jealott's Hill Research Station, Bracknell, Berkshire, RG42 6EY
The fungal pathogen Mycosphaerella graminicola
(Fuckel) J. Schröt. in Cohn, (anamorph: Septoria tritici Rob. Ex Desm)
causes leaf blotch in Wheat. It is an important economic disease causing significant yield loss
throughout the World. The pathogen is mainly controlled by the application of protectant and
eradicant fungicides, therefore it is important to develop new fungicides with novel modes of
action to help manage and reduce the risk of a build up of resistant strains.
Enzymes of the thiamine biosynthetic pathway could provide new potential fungicide targets to be developed against crop diseases such as M. graminicola. The aim of this project is to validate thiamine biosynthesis as a potential target for fungicide intervention by determining whether the genes of the thiamine biosynthetic pathway are important pathogenicity factors in M. graminicola.
240 Expression of the cercosporin resistance gene crg1 as a dicistronic unit with a functionally unrelated gene in the phytopathogenic fungus Cercospora nicotianea. Kuang-Ren Chung1, 2, Marilyn Ehrenshaft1 and Margaret E. Daub1. 1Department of Plant Pathology, North Carolina State University, Raleigh, NC 2Current address: Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, FL
The C. nicotianae crg1 gene was identified as a gene required by the fungus for resistance to a perylenequinone toxin, cercosporin. Northern analysis identified transcripts of two sizes (4.5 and 2.6 kb) using crg1 probes, suggesting that crg1 was co-transcribed with an upstream or downstream gene. The 2.6 kb transcript (expected size for crg1) was present in very low amounts. Sequence analysis identified two ORFs flanking crg1. Using the upstream gene as a probe, we identified transcripts of 2.2 and 1.8 kb. However, two transcripts of 4.5 and 2.0 kb were identified when the downstream gene was used as a probe, suggesting that the 4.5-kb transcript is likely a co-transcribed mRNA. The downstream gene displays strong amino acid homology to a uracil transporter in yeast, and is therefore designated put1 (putative uracil transporter). The 4.5-kb transcript could be identified using either a crg1 or put1 gene probe, and was constitutively expressed in different media. The 2.6- kb transcript, also expressed constitutively, was identified only by the crg1 probe. The 2.0-kb transcript was identified only by the put1 probe and was not expressed in complete medium or V8 medium. The cDNA fragments composed of crg1 and put1 were amplified by RT-PCR directly from the 4.5-kb mRNA using one primer from the crg1 sequence and a second primer from the put1 sequence. Four cDNA clones derived from RT-PCR were sequenced, and revealed that a 64-bp fragment was spliced out in the junction of crg1 and put1. Northern analysis of three crg1-disruption mutants using the crg1 probe indicated that both the 4.5 and 2.6-kb transcripts were missing. Using the put1 probe, the 2.0-kb transcript, but not the 4.5-kb transcript, was detected in the three crg1- disrupted mutants. Taken together, we conclude that the 4.5-kb transcript is a dicistronic mRNA of both crg1 and put1. To our knowledge, this is the first reported case of a dicistronic transcript identified from filamentous fungi. Both genes can also be expressed independently as individual genes and are regulated differently. Whether or not the 4.5-kb message is translated into a fusion protein has not yet been determined.
241 Identification of pathogenicity genes in Heterobasidion annosum using expressed sequence tags (ESTs). Magnus Karlsson1, Åke Olson1 and Jan Stenlid1. 1Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden
The basidiomycete fungus Heterobasidion annosum is the causal agent of annosum root rot and is economically the most important disease of coniferous forests in northern temperate regions. H. annosum is a wood-decaying pathogen which can utilize a variety of carbon sources, such as starch, cellulose and other glucans, pectin, lignin and various phenolic compounds. It is known to secrete a wide range of extracellular enzymes and toxins. The purpose of this research project is to investigate the pathogenicity of H. annosum and to identify key factors that enables the fungus to infect and cause disease. A cDNA-library was constructed from H. annosum mycelia, where pathogenicity genes were induced by Pinus sylvestris seedling roots. Partial sequencing of individual cDNA-fragments yields expressed sequence tags (ESTs) and these tags are then used in homology searches against available sequence information in public databases. This will give an idea of the genes expressed during the infection process. In order to identify genes that are important for the pathogenicity of the fungus the cDNA-fragments will be screened for differential expression, comparing induced and control mycelia. A further study of putative pathogenicity genes will include extensive expression profiling. The results from the first sequencing indicate that the cDNA-library is of good quality and we plan to sequence at least 1000 cDNA clones. In the first 200 clones fragment lengths are spanning from 350 bp to 2500 bp, with an average of 900 bp. So far 35 % of the sequences does not show any significant homology to characterized genes, while 34 % seem to be involved in protein synthesis and 31 % show significant homology to other genes.
242 cAMP signaling and sporulation in Ustilago maydis. Katherine Wake and Jim Kronstad. Biotechnology Laboratory, University of British Columbia, Vancouver, B.C., V6T 1Z3, Canada.
The basidiomycete U. maydis switches from a non-pathogenic haploid yeast- like phase to a pathogenic filamentous dikaryon as a result of mating between two compatible haploid cells. Dikaryotic cells are able to proliferate in the host plant, initiate tumor formation and undergo another morphological change that results in the formation of melanized, diploid teliospores.
In general, it has not been possible to obtain teliospore formation of U. maydis outside the host plant and the signals that control sporulation are not well characterized. Kusch and Schauz (Crypt. Bot 1989: 1: 230-235) reported that U. maydis is also able to produce a haploid spore upon growth in medium with a reduced level of nitrogen. These spores have been called chlamydospores and they appear similar to teliospores in that they are rounded and melanized. However, unlike the other morphological stages of the U. maydis life cycle, chlamydospores are poorly understood and it is not known whether the mating loci or the cAMP /PKA pathway are involved in this morphological switch. We have found that a process which resembles chlamydospore formation in U. maydis can be triggered by the addition of the solvent dimethyl formamide (DMF) to cultures. Using DMF, cells with mutations in components of the cAMP pathway were examined for their ability to develop chlamydospore like cells. Cells defective in ubc1 (the regulatory subunit of PKA) were unable to develop chlamydospores in response to DMF. Cells with defects in both ubc1 and adr1 (a catalytic subunit of PKA) respond to DMF by the production of chlamydospores suggesting that adr1 functions in a pathway which inhibits chlamydospore development. Understanding chlamydospore formation may provide a model to explore the genetics of U. maydis sporulation, a late but crucial stage of disease development.
243 PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea. Mathieu Gourgues, Joaquim Cots, Pierre-Henri Clergeot, Marie-Pascale Latorse, Francoise Laurans , Régis Pépin* and Marc-Henri Lebrun UMR 1932 CNRS-Aventis and Fungicides Dpt, Aventis CropScience, Lyon, France. *Universit Claude Bernard, Lyon, France.
Unravelling functions implicated in the infection process of plant pathogenic fungi is an important challenge for crop protection. We are using as a model the fungal plant pathogen Magnaporthe grisea responsible for a major disease of rice. In order to identify functions required for the infection process, we have studied non-pathogenic mutants obtained by plasmid-mediated insertional mutagenesis. Among 3000 REMI transformants, we recovered 32 mutants either non-pathogenic (n: 10), or significantly reduced in their pathogenicity (n: 22). Only 30% of these mutants were tagged by the plasmid. We studied one of these mutants called punchless. This mutant formed melanized appressoria that failed to breach either leaf epidermis or artificial membranes such as cellophane. punchless appressoria did not differentiate penetration pegs, but had a cellular ultrastructure, turgor and glycogen content similar to wild type appressoria before penetration. punchless also failed to invade wounded or infiltrated leaves. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 amino acids (Pls1p) related to the tetraspanin superfamily. These animal proteins are components of membrane signaling complexes involving integrins that control cell differentiation, motility and adhesion. Therefore, this type of signaling pathway could be involved in penetration peg formation. The expression pattern and the intracellular localization of Pls1p were assessed to understand the role of tetraspanins in appressorium function. Pls1p was only detected in appressorium during the penetration process. Localization of Pls1p and biochemical characterization of this protein will be presented.
244 WdChs2p, a class I chitin synthase, together with WdChs3p (Class III), contributes to virulence in Wangiella (Exophiala) dermatitidis, a model black pathogen of humans. Z. Wang1, L. Zheng1, H. Liu1, M. Hauser2, S. Kauffman2, J. M. Becker2, and P. J. Szaniszlo1. The University of Texas at Austin, TX1, The University of Tennessee, Knoxville, TN2.
We have previously reported that W. dermatitidis has at least four chitin synthase genes (WdCHS), which we designated WdCHS1 (class II), WdCHS2 (class I), WdCHS3 (class III) and WdCHS4 (class IV). After each was cloned and characterized, they were also disrupted singly and in many combinations using various strategies. In this report, we compare the effects of disrupting WdCHS2 and WdCHS3 singly and together on the viability, growth, morphology, chitin synthase (WdChs) activities and virulence of this polymorphic agent of phaeohyphomycosis. As with the wdchs2 and wdchs3 single mutants, the wdchs2wdchs3 double mutant cultured at 25 C or 37 C grew at wild- type rates and showed no morphological defects when grown as yeasts, hyphae or sclerotic bodies. However, assays showed that the same disruption strains were reduced in WdChs activity, with the double mutant having the least activity. These assays, together with those of a wdchs1wdchs3wdchs4 triple disruption mutant that allowed WdChs2p to be evaluated alone, also showed that WdChs2p contributed the majority of the WdChs activity. Although no loss of virulence was detected when the single disruption strains were tested in mice, the wdchs2wdchs3 double mutants were significantly less virulent. The importance of the WdChs2p and WdChs3p isozymes to virulence was finally confirmed by reintroducing either WdCHS2 or WdCHS3 into the double mutant and reconstituting wild-type virulence.
245 Characterisation of a large polygalacturonase gene family in the oomycete plant pathogen Phytophthora cinnamomi. Arvid Goetesson, Jerry S Marshall, David A Jones and Adrienne R Hardham. Plant Cell Biology, Research School of Biological Sciences, Australian National University, Canberra, Australia.
Polygalacturonases (PGs) are secreted by filamentous plant pathogens at early stages of plant infection, and their pectinolytic activity is believed to play a major role in tissue invasion and maceration. Oomycete plant pathogens are economically and ecologically important but little is known about the biochemistry and molecular biology of oomycete pathogenesis. PG activity was demonstrated in culture filtrates of the oomycete Phytophthora cinnamomi. A polygalacturonase gene family in P. cinnamomi was partially isolated and characterized. Sixteen complete pg genes and three incomplete sequences were isolated. Thirteen of the genes occurred in three clusters. Since P. cinnamomi is a diploid organism, some pg sequences which are > 99% identical could be alleles, but this is unlikely since the sequence context is different for all isolated pg sequences. DNA gel blots indicated that there may be more than 19 pg genes in the P. cinnamomi genome. Many of the predicted mature PGs had N-terminal extensions which contained repeats but were dissimilar in length and sequence. Predicted pI values varied between 4.1 and 9.3. Ten PGs had none or one potential N-glycosylation sites, while five had 10 to 14 potential N-glycosylation sites. Two pg genes were expressed in yeast and shown to encode functional PGs. Oomycetes are phylogenetically distinct from true fungi and more closely related to brown algae. However, phylogenetic analysis of PGs positioned P. cinnamomi PGs close to fungal endoPGs and distant to plant PGs. Three amino acid residues which have been subject to diversifying selection in the P. cinnamomi PG protein were identified.
246 Highly conserved virulence-related CPS1homologs from plant and human pathogenic fungi. Shun-Wen Lu, B. Gillian Turgeon, and Olen C. Yoder. Department of Plant Pathology, Cornell University, Ithaca, NY 14853.
The Cochliobolus heterostrophus gene CPS1 encodes a protein with similarity to nonribosomal peptide synthetases. We have identified CPS1 homologs in 34 fungal species belonging to 16 genera, closely or distantly related to Cochliobolus. These include both plant pathogens of economic importance (e. g., Fusarium graminearum, Magnaporthe grisea) and human pathogens of medical importance (e. g., Candida albicans and Coccidioides immitis). Four CPS1 homologues were cloned; three from phytopathogenic fungi, including the wheat head scab fungus Fusarium graminearum (FgCPS1, 6003 bp), the potato early blight fungus Alternaria solani (AsCPS1, 2369bp) and the barley net blotch fungus Pyrenophora teres (PtCPS1, 2306 bp). The fourth was cloned from the human pathogenic fungus Coccidioides immitis (CiCPS1, 2435 bp). Sequence analyses indicated that these genes share at least 80 % amino acid similarity and contain a highly conserved modular structure as do nonribosomal peptide synthetases. Targeted gene disruptions have confirmed that CPS1 is required for pathogenesis of at least three different plant pathogens i.e., C. heterostrophus to corn, C. victoriae to oats and Fusarium graminearum to wheat. We propose that CPS1 controls biosynthesis of a general fungal virulence factor (yet unidentified), which is required for pathogenesis by a wide array of fungi, including both plant and human pathogens.
247 Interaction of the triazole fungicide, flutriafol, with Leptosphaeria maculans, the cause of blackleg disease in oilseed Brassicas. Katherine M. Adams, Barbara Howlett, Anthony Bacic, Philip Salisbury. University of Melbourne, School of Botany, Melbourne, Australia
Leptosphaeria maculans, (anamorph Phoma lingum) is an ascomycete which causes blackleg disease in oilseed Brassicas. In Australia the triazole fungicide flutriafol, which acts by inhibiting 14 alpha - demethylation in ergosterol biosynthesis, is the only fungicide registered for control of this disease. The purpose of this work is to examine the biochemical interaction between the fungicide and fungus, and to elucidate genetic mechanisms which may confer triazole resistance. Seven L. maculans mutants with flutriafol resistance have been produced by insertional mutagenesis (REMI) or by in vitro conditioning on fungicide amended media. Ergosterol biosynthesis and gene expression is being compared among these mutants and wild-type L. maculans. Fragments of the L. maculans homologues of five genes implicated in triazole resistance in other fungi have been obtained. These encode regions of 14alpha- demethylase (ERG11), delta 5,6 desaturase (ERG3) and three ABC-transporter proteins. RT-PCR and Northern analysis are being used to study the expression of these genes in all isolates following exposure to flutriafol. Ergosterol and its sterol precursors are being separated and identified by gas chromatography ûû mass spectrometry. Analysis has revealed ergosterol is the most abundant sterol in L. maculans, with four ergosterol precursors present in lower abundance. Exposure of wild-type L. maculans to flutriafol causes a decrease in total ergosterol and an accumulation of the sterol substrate of 14 alpha-demethylase. Kate Adams is a recipient of the Nancy Millis award, provided by the Victorian State Department of Natural Resources and Environment.
248 Visualization of AOS accumulation induced by Botrytis cinerea infecting tomato and bean. Klaus B. Tenberge, Marcus Beckedorf, Britta Hoppe, and Martina Solf. Institut fur Botanik und Botanischer Garten, Westfalische Wilhelms-Universitat Munster, Schloßgarten 3, D-48149 Munster, Germany
Botrytis cinerea causes serious diseases, called grey mould, in at least 235 plant species, including important crops. The pathogen is a typical necrotroph, inducing host cell death before invasion. Active oxygen species (AOS) have been shown to be involved in infection . In addition to the induced generation of AOS during the host oxidative burst, the pathogen might produce AOS itself. The aim of the presented work is to gain insight in role and source of AOS by visualizing the spatio-temporal occurrence of AOS during pathogenesis. Spore adhesion, germination and swellings of germ tube tips attached to the surface by a matrix material as well as penetration and colonization of host leaf tissues was documented on tomato and bean. Using LM, the chloronaphthol staining and the NBT technique were applied to visualize H2O2 and O2-. Using TEM, the cerium chloride technique specific for H2O2 resulted in electron dense precipitate of cerium perhydroxide at the interface of B. cinerea and host cells in infected tomato and bean leaves . H2O2 was present in the periplasmic space, in the host cell wall and at the outer surface of the host cell as well as at the outside of the fungal wall. Specific precipitate was observed inside fungal cells that appeared vital. The H2O2 generating system was analyzed using specific inhibitors, suggesting that fungal superoxide dismutase contributes to the production of AOS.  von Tiedemann A 1997. Physiol. Mol. Plant Pathol. 50, 151-166.  Prins TW, Tudzynski P, von Tiedemann A, Tudzynski B, ten Have A, Hansen ME, Tenberge KB, van Kan JAL 2000. In: Fungal Pathology, pp. 33-63, Kronstad JW, ed., Kluwer Academic Publishers, Dordrecht. Our research is funded by the EC in the EU-FAIR project "Oxidative attack by necrotrophic pathogens - New approaches for an innovative and non-biocidal control of plant disease" (AOS PLANT).
249 New Restriction Fragment Length Polymorphism (RFLP) analysis markers for typing Aspergillus fumigatus strains. Camile Pizeta Semighini, Steven Park1, David S. Perlin1, and Gustavo H. Goldman. Faculdade de Ciencias Farmaceuticas de Ribeirão Preto, Universidade de São Paulo, Brazil and 1Public Health Research Institute, USA.
Aspergillus fumigatus is an opportunistic fungus causing
several respiratory diseases, such as allergic bronchopulmonary aspergillogis, aspergilloma and
invasive aspergillosis. The later is presently a major cause of death amongst
immunocompromised patients, associated with a high mortality rate (85%) even when
appropriate treatment is used. The incidence of aspergillosis has increased significantly over the
past two decades in parallel with the number of imunocompromised patients. Although many
typing approaches have been proposed, an ideal epidemiological typing technique is not
available that is applicable to a wide range of A. fumigatus isolates. In this study,
we isolated and tested Restriction Fragment Length Polymorphism (RFLP) markers for A.
fumigatus based on PCR-products amplified by the Random Amplified Polymorphic
DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A were amplified.
The fragments Afd and Af5 were 85 % and 88 % identical at the DNA level to the Afut1
retrotransposon from A. fumigatus. Fragment Af4A is a duplication of the fragment
Af4 and both showed similarity at the amino acid level with endonucleases from other fungal
retrotransposons. We have used both RAPD with the primer R108 and RFLP assays with Afut1,
Afd, and Af4A as hybridization probes to determine the genetic relatedness of clinical isolates of
A. fumigatus isolated sequentially from four cancer patients with recurrent
aspergillosis following treatment with amphotericin B. Genetic relatedness was determined by
using the Coefficient of Dice. The combination of these different methods was used to
demonstrate that the isolates infecting the four patients were not identical. This approach should
be valuable for molecular epidemiological investigations of Aspergillus infections,
which should facilitate the development of preventive measures for patient management.
Financial support: CNPq and FAPESP, Brazil.
250 A pharmacological and molecular approach to the study of signal transduction in the barley powdery mildew fungus. Gemma Priddey, Ziguo Zhang, Pushpa Chaure, Alison Hall, Emma Perfect, Sarah Gurr. Department of Plant Sciences, University of Oxford, OX1 3RB, UK.
Blumeria graminis f.sp. hordei is the causal agent of barley powdery mildew disease. Infection is spread by asexual conidia, which, on contact with the leaf surface, undergo a highly regulated and complex programme of development. As an obligate biotroph B. graminis cannot be grown axenically and so tissue for experiments is limiting. We have employed and described a range of techniques to assess how B. graminis perceives, integrates and relays signals for morphogenesis up to the point of penetration. We have demonstrated that both physical properties of the leaf surface, such as hydrophobicity, and cuticle-derived chemicals, such as cutin monomers and cellulose, promote B. graminis differentiation. 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.
Furthermore, we have identified several component genes of signal transduction and cell integrity pathways in B. graminis, including two PKC genes, two MAPK genes and two chitin synthase genes. Their expression profiles show that they are regulated differentially during conidia germination and appressorial differentiation. They putatively play important roles in host penetration and pathogenicity. We aim to ascribe functions to these genes, by using our recently-described stable DNA transformation technique and also to study the interplay between the PKA, PKC and MAPK signal transduction pathways.
251 Molecular genetics of plant infection by the rice blast fungus Magnaporthe grisea. Nicholas J. Talbot, Eckhard Thines, Andrew J. Foster, Roland W.S. Weber, Pascale V. Balhadere, Virginie Colas . School of Biological Sciences, University of Exeter,Washington Singer Laboratories, Perry Road, Exeter, EX4 4QG.
The rice blast fungus elaborates a specialized infection structure called an appressorium in order to infect rice leaves. The appressorium is a dome-shaped cell which differentiates from a germ tube, shortly after conidial germination. M. grisea appressoria develop turgor pressure which is translated into mechanical force to breach the plant cuticle. This allows a narrow penetration peg to enter the leaf epidermis and colonise the tissue, later forming large bulbous infection hyphae. We are investigating the process of appressorium-mediated infection in M. grisea and in particular the mechanism by which turgor is generated. M. grisea appressoria accumulate very high concentrations of glycerol which acts as an osmolyte, allowing the cell to take up water and develop hydrostatic turgor. Appressoria form in water on the leaf surface and therefore glycerol is synthesised from storage products in the spore. M. grisea conidia contain a number of storage compounds including glycogen, lipid and trehalose. We have used a combination of genetic, biochemical and cell biological methods to study the relative contribution of each storage product to glycerol generation (Thines et al., 2000 Plant Cell 12, 1703-1718). Trehalose degradation occurs rapidly during conidial germination. Trehalose is synthesised in M. grisea by trehalose-6-phosphate synthase encoded by the TPS1 gene. Dtps1 mutants are extremely reduced in pathogenicity, due to a defect in cuticle penetration. The degradation of trehalose meanwhile appears to occur due to the activity of at least two trehalases, encoded by NTH1 and TRE1. Dtre1 and Dnth1 mutants are also affected in their ability to cause disease symptoms, although to a lesser extent than in Dtps1. Genetic control of trehalose metabolism appears to be, at least in part, due to the action of cAMP-dependent protein kinase A (PKA), based on enzymatic assays. Glycogen and lipid stores are also degraded during conidial germination and can be observed accumulating in appressoria during their formation, before disappearing as turgor is generated. The movement of these reserves is controlled by the PMK1 MAP kinase pathway and degradation in the appressorium is regulated by PKA. We are currently identifying and characterising genes encoding enzymes involved in lipid and glycogen degradation in appressoria with the aim of determining the basis of turgor generation and ap
252 Identification and characterisation of two metallothionein-encoding genes from the rice blast fungus, Magnaporthe grisea. Sara L. Tucker and Nicholas J. Talbot. School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, EX4 4QG
Magnaporthe grisea is the causal agent of rice blast disease. Considerable research has led to our current understanding of this pathogen however little emphasis has been placed on identifying genes specifically involved in plant tissue colonisation and growth of the fungus in planta. I this project we have adopted tow strategies to study plant tissue colonisation by M. grisea. The first strategy involved differential cDNA screening to isolate transcripts expressed in the wild-type strain of M. grisea, Guy-11 but not in a non-pathogenic MAP kinase mutant Dpmk1. Secondly, a candidate gene approach was used to identify a homologus of a gene identified in Uromyces fabae called PIG11. Because characterisation of PIG11 in this obligate biotrophic fungus is difficult, the presence of a homologue in the more experimentally amenable fungus M. grisea is significant. Using these approaches two metallothionein (MT)-encoding genes have been identified called MMT1 (the PIG11 homologue) and MMT2. Metaolothioneins are ubiquitous proteins with metal-binding propteries, although their function is somewhat elusive. Preliminary characterisation of the two MTs carried out to date will be presented, revealing their relatedness to other MTs, their patterns of expression in developmental mutants of M. grisea and the possible functions they may carry out during growth of the
253 Characterization of the MPG1 hydrophobin-encoding gene from the rice blast fungus Magnaporthe grisea. Michael J. Kershaw, Darren Soanes and Nicholas J. Talbot. School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, EX4 4 QG, United Kingdom
The rice blast fungus Magnaporthe grisea infects its host
by elaboration of a specialised infection structure known as an appressorium. This single-celled
structure forms in response to the hard, hydrophobic rice leaf surface and brings about infection
due to generation of hydrostatic pressure (3). M. grisea appressoria are
melanin-pigmented cells with a thickened cell wall that allows turgor to develop within the cell
due to accumulation of glycerol and the subsequent influx of water. Mechanical rupture of the
plant leaf cuticle occurs and a narrow penetration peg enters the leaf epidermis, providing the
route for fungal colonization of the plant leaf tissue (3).
During appressorium development the MPG1 hydrophobin gene is highly expressed and secretion of the class I hydrophobin is important for efficient elaboration of appressoria (1, 2). A targeted gene replacement of MPG1 reduces the ability of M. grisea to elaborate appressoria and mutants are consequently reduced in their ability to cause disease symptoms (1). Dmpg1 mutants also show reduced surface hydrophobicity and lack a conidial rodlet layer, indicating that MPG1 performs a number of roles in the developmental biology of M. grisea. The importance of differential expression of MPG1 to M. grisea can be clearly seen by the fact that numerous class I hydrophobin genes are able to functionally complement Dmpg1 mutants, restoring pathogenicity and appressorium development, but only if expressed under control of the MPG1 promoter (4). This suggests that class I hydrophobins, which are widely distributed among fungi and may perform orthologous functions (5, 6). The close functional relationship of hydrophobins is somewhat surprising considering the low level of amino acid homology among the group and emphasises the role of differential gene expression in determining hydrophobin function during fungal morphogenesis.
Regulation of MPG1 expression occurs due to the action of a number of cis-acting sequences and trans-acting factors which control MPG1 transcript levels. The latter include the NPR1 and NPR2 gene products which regulate pathogenicity of M grisea and the starvation stress response (5). The significance of MPG1 regulation and the role of specific amino acid residues in the function of the MPG1 hydrophobin will be discussed.
1. Talbot et al. 1993 Plant Cell 5:1575-90 2. Talbot et al. 1996 Plant Cell 8: 985-999 3. De Jong et al. 1997 Nature 389: 244-245 4. Kershaw et al. 1998 EMBO J. 17:3838-3849 5. Kershaw and Talbot, 1998. Fungal Genet. Biol 23: 18-33 6. Tal
254 Characterization of pathogenicity factors of Fusarium oxysporum f.sp. lycopersici. Roselinde G E Duyvesteijn, Yvonne W Boer, Menno van der Voort, Ben J.C. Cornelissen, Michel A Haring. University of Amsterdam, Plant Pathology, Amsterdam, Noord-Holland, The Netherlands
Fusarium oxysporum is a soilborne plant pathogenic fungus which can cause considerable economical loss on a farmers crop. Fusarium oxysporum forma specialis lycopersici(Fol) infects only tomato plants. Until now there are three different races of Fol known which are all pathogenic on tomato. During the infection Fusariumuses pathogenicity genes which are necessary to penetrate, invade and colonize the tomato roots. Characterizing the pathogenicity factors could give a deeper understanding of the infection process of the fungi. In order to characterize these genes insertional mutagenesis will be performed. However, by using a promoterless construct it is possible to select planta induced genes which are the most likely pathogenicity factors. Therefore, a promoterless -glucuronidase ( GUS) gene was cloned into a plasmid containing the hygromycin resistance selection marker. The new plasmid was transformed into the Fol race 2. In total 216 transformants were selected for their ability to grow on medium with hygromycin. Bioassays identified19 putative mutants which had reduced pathogenicity. Southern blots will be done to analyze the number of copies of the inserted plasmid and PCR experiments can be used to confirm the presence of the hygromycin and GUS genes. The inserted plasmid and its flanking DNA regions will be cloned of the non-pathogenic transformants. The involvement of the flanking DNA in pathogenesis will be established by making knock-out mutants.
255 Signaling and pathogenicity in the gray mold Botrytis cinerea. Christian Schulze Gronover, Annett Klimpel, Daniela Kasulke and Bettina Tudzynski.Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schlossgarten 3, 48149 Münster, Germany
Botrytis cinerea is the causal agent of grey mould diseases of many economically important fruits, vegetables and flowers. Our main interest is the study of the genes involved in the interaction process between the fungus and its host plant. As a necrotrophic fungus, B. cinerea induces host cell death after penetrating the plant tissue. Like other fungal pathogens, B. cinerea senses the presence of plant surfaces and triggers the synthesis of several specific gene products in response. We suggest that several conserved signal transduction pathways such as cAMP signaling are required for infection. Therefore, we work on cloning and characterization of genes encoding members of signal transduction pathways: genes coding for G-alpha proteins, adenylate cyclase and protein kinases. Heterotrimeric G proteins play an important role in transducing several extracellular signals from activated transmembrane receptors of the cell surface to a variety of intracellular targets via a cascade of interacting proteins, such as cAMP cyclase and protein kinases. Recently, we cloned two different G-alpha protein-encoding genes, bcg1 and bcg2. A 180 bp PCR fragment, obviousely coding for a third G-alpha protein, (bcg3), was also amplified. BCG1 showed a high degree of identity with CPG-1 from Cryphonectria parasitica and MAGB from Magnaporthe grisea and belongs to the Gai class (inhibitory G-alpha). BCG2 was grouped into the same family as MAGC from M. grisea and GPA-3 from Ustilago maydis which are not involved in the induction of infection structures. Interestingly, bcg1 and bcg2 are expressed in planta at a very early stage of infection (12 hours after drop infection). For both genes, bcg1 and bcg2, knock out mutants were isolated. bcg1 knock out-transformants showed a colony morphology clearly different from that of the wild-type. The colony margins were compact and sharply defined. Tomato and bean leaves inoculated with conidial suspensions from bcg1- and bcg2-mutants as well as from the wild-type caused similar primary necrosis lesions in the first hours. However, after two days no further increase of the diameter was observed for the lesions caused by the bcg1 conidia compared with the fast developing wild-type and bcg-2 necrosis spots. Interestingly, bcg1-mutants do not produce extracellular proteases anymore. Further infection studies and physiological tests are on the way.
In some other fungal pathogens it could be clearly demonstrated that cAMP plays an important role for successful plant infection. Therefore, a B. cinerea adenylate cyclase gene (bcac1) and two catalytic subunit-encoding genes of two putative protein kinase A (bc-PKAC1 and bc-PKAC2) were cloned. For the bcac1 gene, a gene replacement vector was constructed and knock out-transformants were isolated. They show reduced conidia formation and vegetative growth. The effect of the gene inactivation on the infection process on bean and tomato leaves is under investigation. The results will be discussed in the background of the role of signaling processes for the development of diseases caused by a broad spectrum of fungi.
256 Cochliobolus carbonum mutants lacking various classes of cell-wall-degrading enzymes. John S. Scott-Craig and Jonathan D. Walton. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824 U.S.A.
The maize pathogen Cochliobolus carbonum secretes a large number of plant-cell-wall-degrading enzymes when grown with maize cells walls as the sole carbon source. Xylan-containing polymers constitute almost 50% of the dry weight of the maize cell wall, non-cellulosic glucans about 8% and pectin about 6%. At least five xylan-degrading, four glucan-degrading and three pectin-degrading enzymes are produced by the fungus. Genes encoding four endo-xylanases (XYL1,2,3,4), one -xylosidase (XYP1), two exo-glucanases (EXG1,2), two mixed-link glucanases (MLG1,2), an endo-polygalacturonase (PGN1), an exo-polygalacturonase (PGX1) and a pectin methylesterase (PME1) have been cloned and used to create strains containing deletions of each gene. These deletions, singly and in combination, are being examined for their effects on the growth of the fungus in vitro on xylan, -glucan or pectin and on the ability of the fungus to infect maize.
257 Agrobacterium T-DNA tagging of pathogenicity genes in Fusarium circinatum. Sarah F. Covert, Mei-Ho Lee, and William S. Bowers. Warnell School of Forest Resources, University of Georgia, Athens, GA, USA.
Fusarium circinatum (teleomorph: Gibberella circinata) causes pitch canker disease on many species of pine. In particular, the native stands of Monterey pine (Pinus radiata) in California are severely affected by this disease. We are using Agrobacterium tumefaciens T-DNA gene tagging to identify the genes that make F. circinatum pathogenic on Monterey pine. A. tumefaciens strain AGL1(pPK2) transforms F. circinatum with an efficiency of 2 - 150 transformants/105 conidia. The T-DNA is integrated into the genome and is stable through mitotic and meiotic cell divisions. Seventy-five percent of the transformants contain a single T-DNA copy. The remaining 25% contain two copies of the T-DNA. We are currently screening hundreds of transformants for their ability to cause necrotic lesions on Monterey pine. If mutants lacking pathogenicity contain more than one T-DNA tag, we will use genetic segregation to isolate the tag causing the mutation of interest. Inverse PCR will be used to recover the F. circinatum DNA flanking the T-DNA. This approach should allow us to identify F. circinatum genes contributing to pitch canker symptom development.
258 In planta expressed genes in the interaction between Gaeumannomyces graminis and cereals. Morgane Guilleroux and Anne Osbourn., Sainsbury Laboratory
Suppression subtractive hybridization (SSH) has been used to generate a cDNA library enriched for sequences that are differentially expressed during infection of wheat roots by Gaeumannomyces graminis. This library has been assessed to confirm that representative constitutively expressed plant and fungal sequences (alpha-tubulin and actin, respectively) have been subtracted and XYL1 (a xylanase that is known to be expressed during infection)of GgA is expressed. A pilot study of the subtracted library has been carried out on 215 clones. These clones have been sequenced to check the quality of the library and subjected to a BLASTX search. Of these 215 clones, 150 reliable DNA sequences were obtained, 7 of which showed significant homology with fungal gene sequences available in the databases. However, the small average insert size (200bp) impairs both reliable homology search and hybridizations in Southern and northern blot experiments. A cDNA library has therefore been constructed from mRNA from infected roots, and is being used to isolate full-length cDNAs corresponding to subtracted clones of interest. Larger cDNAs fragments of two of the SSH clones have been used as probes on Northern blots and shown to be upregulated during infection. These clones are both of plant origin. An arrayed genomic DNA library of Gaeumannomyces graminis has been constructed and is being screened with the SSH library and other complex probes to gain a better understanding of the metabolic requirements of this root pathogen during the infection process. Gene function will be tested by gene disruption in related fungus Magnaporthe.
The Sainsbury laboratory is funded by the Gatsby Charitable Foundation
259 Active oxygen species in the Claviceps purpurea/rye interaction Sabine Moore, Suchitra Joshi, Birgitt Oeser and Paul Tudzynski. Institut fuer Botanik, Westf. Wilhelms-Universitaet, Schlossgarten 3, Muenster D-4814, Germany.
The ascomycete Claviceps purpurea is a biotrophic cereal pathogen which colonises young ovaries, drawing nutrients from the vascular tissue located at the ovary base, and forming a stable host-pathogen interface there. Both normal ontogenesis as well as the defence response involve production of active oxygen species (AOS) by the rye ovary; our research focusses on the ability of the fungus to overcome oxidative stress during colonisation and the importance of these mechanisms for pathogenicity. IEF gel analysis of axenic and pathogenic cultures show that the fungus contains at least three distinct catalases (1) and one Cu,Zn superoxide dismutase. Single mutants deficient in the major secreted catalase CATC/D (2) or the copper-induced, cell wall associated Cu,Zn SOD (SODA) show no significant reduction in pathogenicity; a double mutant lacking both enzymes shows in planta only a slight delay in conidiospore production. A second catalase gene has been cloned and deleted, analysis of the mutant will show if this gene corresponds to the second secreted catalase. We are searching for further AOS induced genes using copper which acts as a fenton catalyst and thus a stable source of AOS. Using differential screening with and without copper, we have isolated several interesting genes, most of which are also induced by hydrogen peroxide. In order to better understand the mechanisms of oxidative gene induction, we have cloned a gene which encodes a transcription factor induced by hydrogen peroxide; functional analysis of this gene is underway. 1. Garre V, Tenberge KB, Eising R: Phytopathol. 88. 744-753 (1998) 2. Garre V, M ller U, Tudzynski P: MPMI. 11, 772-783 (1998)
260 A histone deacetylase gene related to yeast HOS2 is necessary for extracellular depolymerase expression and virulence in the fungus Cochliobolus carbonum. Dipnath Baidyaroya, Gerald Broschb, J-H Ahna, Sigrun Wegenera, Stefan Graessleb, Oscar Caballeroa, Peter Loidlb, and Jonathan Waltona. aDepartment of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, and bDepartment of Microbiology, University of Innsbruck, Medical School, A-6020 Innsbruck, Austria
The gene ccHOS2, encoding a putative histone deacetylase (HDAC) related to yeast HOS2, was isolated from the filamentous fungus Cochliobolus carbonum, a pathogen of maize that makes the HDAC inhibitor HC-toxin. Conidia of engineered cchos2 mutants were smaller and less septate. HDAC activity in the mutant was decreased by ~50%. Growth of the cchos2 mutant in vitro was normal on glucose and sucrose but was reduced on arabinose, xylose, xylan, pectin, and maize cell walls. The mutant produced less extracellular depolymerase activity and induction of the corresponding genes was also reduced. The cchos2 mutant also produced fewer lesions than the wild type on susceptible maize due to reduced penetration efficiency. Except for altered conidial morphology, these phenotypes were similar to those of a C. carbonum strain mutated in the ccSNF1 gene encoding a glucose-regulated protein kinase. The results show that (1) an HDAC gene can have multiple functions in a filamentous fungus, (2) HDAC genes can be required for gene induction as well as repression, (3) some HDAC genes can have specific functions, and (4) ccHOS2 is required for virulence of C. carbonum on maize.
261 Genetic and physical mapping of the Avr1a avirulence gene in Phytophthora sojae. Terry MacGregor1, Madan Bhattacharyya2, Brett Tyler3 and Mark Gijzen1. 1Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, ON, Canada. 2G303 Agronomy Hall, Iowa State University Ames, Iowa, USA, 3Department of Plant Pathology, The University of California, Davis, CA
Compatibility between soybean and the oomycete phytopathogen Phytophthora sojae is controlled by host resistance (R) genes and pathogen avirulence (Avr) genes. The segregation of the Avr1a avirulence gene in P. sojae F2 populations is consistent with that of a dominant allele at a single locus. By using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and bulked segregant analysis, the Avr1a locus was mapped in two F2 populations derived from four different parental isolates of P. sojae. Four RAPD and nine AFLP markers linked to Avr1a were identified from an estimated 2100 RAPD and 40,000 AFLP loci screened. Segregation analysis of all 13 markers in one F2 population of 90 progeny generated a map of 113.2 cM encompassing Avr1a, with the closest marker co-segregating with the trait. Chromosome walking was initiated using the co-segregating marker as a probe to screen a P. sojae bacterial artificial chromosome (BAC) library, and a contig of eight clones, spanning 170 kb, was constructed. Three markers derived from the contig, covering a distance of 120 kb, co-segregated with Avr1a in an enlarged F2 population of 199 progeny. Thus, the ratio of physical distance to genetic distance appears greater than 120 kb/cM in this region of the P. sojae genome. Analysis of P. sojae genomic DNA blots, using the markers as hybridization probes, revealed that all of the markers tested represent single or low copy sequences and showed a high level of conservation around the Avr1a locus in all four races studied, plus nine other races. This study has laid the foundation for a map-based cloning strategy for isolating Avr1a.
262 The pisatin demethylases from Nectria haematococa MPVI and Fusarium oxysporum f.sp. pisi are highly similar. Catherine C. Wasmann1, Kevin McCluskey2 and Hans D. VanEtten1. 1Department of Plant Pathology, Room 204 Forbes Building, University of Arizona, Tucson, Arizona 85721 U.S.A. 2Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City, Kansas 66160.
Several fungal pathogens of pea are able to degrade the pea phytoalexin pisatin thus detoxifying it. This detoxification is best characterized in the fungus Nectria haematococca where it has been shown that isolates pathogenic on pea can detoxify pisatin via a cytochrome P450-mediated demethylation. Seven genes (PDA) for pisatin demethylase have been identified in N. haematococca by classical genetics and three of these genes have been sequenced. Whereas the gene (PDA) for pisatin demethylase from N. haematococca is highly specific for pisatin as an inducer and repressed by glucose, the PDA genes from other fungal pathogens are, in general, neither specific for pisatin nor strongly repressed by glucose. A PDA gene from another fungal pathogen of pea, Fusarium oxysporum f.sp. pisi isolate T415, has been sequenced and its deduced amino acid sequence found to be 89% similar to that of the PDA1 gene of N. haematococca. The ability of pisatin and several related compounds to induce the gene from F. oxysporum has been tested in vivo and, like the PDA1 gene from N. haematococca, the gene from F. oxysporum appears to be specific for pisatin. However, in contrast to the PDA1 gene from N. haematococca, induction of the F. oxysporum gene by pisatin is not repressed by glucose and, in this respect, the PDA gene from T415 appears similar to the pisatin demethylases of other pea pathogens.
263 MAP kinases in signal transduction pathways: cloning and functional analyses of PMK1 and MPS1 homologues from Claviceps purpurea. G. Vautard-Mey1, B. Oeser1, M. H. Lebrun2 and P. Tudzynski1. 1 Westfaelische Wilhelms-Universitaet Muenster, Institut fuer Botanik, Schlossgarten 3, 48149 Muenster, Germany. 2 UMR 1932 CNRS-Aventis, Physiologie Cellulaire V g tale, 14 rue P. Baizet, 69009 Lyon, France.
The early infection process of phytopathogenic fungi may involve both cell wall components able to recognise the host surface as well as signal chain elements triggering differentiation of infection structures and subsequent tissue colonisation. MAP kinases play a central role in sensing and transducing extracellular signals important for the regulation of gene expression. Claviceps purpurea, a common biotrophic pathogen of cereals responsible for the ergot disease, is used as a model to identify components of pathogenicity-related signalling pathways. Degenerate primers were used to amplify two genomic fragments whose deduced peptide sequences are homologous respectively to Pmk1 and Mps1, two MAP kinases essential for the pathogenicity of M. grisea. Both PCR probes were used to screen a C. purpurea genomic library. Cpmk1 and Cpmk2 (Claviceps purpurea MAP kinase genes 1 and 2) were subsequently isolated and sequenced. Their deduced peptide sequences show 95 % and 84 % identity to Pmk1 and Mps1, respectively. In order to determine the importance of both kinases for the pathogenicity of C. purpurea, two replacement vectors were constructed and used to transform the wild type strain. Knock-out mutants were obtained for both genes. Determination of their pathogenicity is now in progress. The expression of Cpmk1 and Cpmk2 during infection of rye is also being studied using RT-PCR. In order to determine whether Cpmk1 is able to restore appressoria formation in the M. grisea deltapmk1 mutant strain, complementation experiments are being performed. Similar experiments will be carried out using Cpmk2 and the M. grisea deltamps1 mutant which is affected in secondary hyphae formation. As C. purpurea doesnÆÆt form special infection structures, such experiments could provide insight on the functional homology of kinases conserved in pathogenic fungi which have evolved different modes of infection.
264 Colletotrichum graminicola pathogenicity mutants identified using restriction enzyme mediated integration (REMI). Michael R Thon, Etta Nuckles and Lisa J. Vaillancourt. University of Kentucky, Department of Plant Pathology, Lexington KY, USA. 40546-0091.
A collection of approximately 1600 REMI transformants of the plant-pathogenic fungus Colletotrichum graminicola was screened for mutants that were reduced in pathogenicity to maize stems and leaves. Three non-pathogenic mutants have been identified as well as six mutants with reduced pathogenicity. Many transformants were reduced in growth rate and spore germination rate however these factors were not correlated with pathogenicity. The mutants contain defects in both pre- and post-infection stages of pathogenesis, including spore germination, appressorium formation, and host tissue colonization. One non- pathogenic mutant is able to complete host infection but is unable to colonize host tissue. Genomic DNA flanking the plasmid integration site of this mutant was used to identify corresponding clones in a wild-type genomic library. Pathogenicity of this mutant was restored when it was transformed with a 6 kb subcloned DNA fragment. Sequence analysis indicates that this strain contains a mutation in a putative homologue of the yeast SPC3 gene, which encodes a component of the yeast signal peptidase, involved in processing of protein secretion signals.
265 Structural and functional analysis of an oligomeric hydrophobin gene in Claviceps purpurea. G. Vautard-Mey1, B. Oeser1, T. Correia1, M. J. Kershaw2, N. J. Talbot2 and P. Tudzynski1. 1 Westfaelische Wilhelms-Universitaet Muenster, Institut fuer Botanik, Schlossgarten 3, 48149 Muenster, Germany. 2 School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Exeter EX4 4QG, United Kingdom.
Hydrophobins are small secreted proteins ubiquitous to filamentous fungi. Fungal aerial structures such as aerial hyphae, conidia and fruiting bodies are coated with an amphipathic layer, resulting from the self-assembly of hydrophobins on the outer cell wall. Among the many functions they may have, hydrophobins were shown to be implicated in aerial hyphae emergence, adhesion to hydrophobic surfaces and interaction of phytopathogens with their host. An unusual gene, encoding a modular protein consisting of five hydrophobin units separated by GN-repeats, was identified in C. purpurea, a biotrophic fungus responsible for the ergot disease of cereals. Until now, only the C. fusiformis trihydrophobin shows a comparable structure (1). The three internal units share more than 90 % identity at the nucleotidic level, suggesting that the protein might result from recent duplication events which occurred in a tripartite precursor. Sequencing of cDNA clones revealed that the main pentahydrophobin transcript codes for the full- length protein. Hydrophobins can be divided into two classes of different biochemical properties, according to the spacing of eight conserved cysteine residues. Due to its modular structure, the C. purpurea pentahydrophobin, composed of five class II units, could have unusual biochemical properties. Complementation experiments are being performed, using the Magnaporthe grisea deltampg1 mutant strain lacking a monomeric class I hydrophobin. Mgp1 is until now the sole hydrophobin which has been shown to be required for pathogenicity. The pentahydrophobin gene (Cpph) was shown to be expressed during infection of rye (2). In order to determine its importance for the pathogenicity of C. purpurea, a replacement vector was constructed and used to transform the wild type strain. Mutants lacking cpph were isolated and analysis of their pathogenicity is in progress.
(1) de Vries, O. M. H., Moore, S., Arntz, C., Wessels, J. G. H. and Tudzynki, P. (1999) Eur. J. Biochem. 262: 377-385. (2) Tenberge, K. B., Stellamanns, P., Plenz, G. and Robenek, H. (1998) Eur. J. Cell Biol. 75: 265-272.
266 Cloning and characterization of two subtilisin-like protease genes from the fungal endophyte Neotyphodium lolii. Michelle McGill and Barry Scott. Institute of Molecular BioSciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand
Subtilisin-like proteases play important roles in fungal nutrition and pathogenicity. Although the role of these proteases is well established in fungal interactions with insects, nematodes and other fungi, little is known about their role in plant-fungus interactions. The plant-fungal interactions where these proteases have been detected include the endophyte Epichlo typhinaand its host grass Poa ampla,and the grass pathogen Magnaporthe grisea and its host, Poa pratensis.In both cases the fungus produces high levels of subtilisin-like proteases suggesting that these enzyes have an important biological role. We have cloned two subtilisin-like protease genes from N. lolii,a fungal endophyte of perennial ryegrass. The prt1gene encodes a protein with 49% identity to proteases from Aspergillusspecies. Downstream of prt1is a 360 base pair minisatellite that consists of forty copies of a 9 base pair motif. The prt2gene encodes a protein with 56% identity to proteases from entomopathogenic fungi. The subtilisin-like proteases in this group act as virulence factors by breaking down the physical barriers to fungal infection and also provide a source of nutrition. RT-PCR has been used to analyze expression of prt1under a range of nutritional conditions. Results show that under conditions of high nutrient availability prtgenes may not be expressed, while when preferred carbon or nitrogen sources are lacking expression of the prtgenes is derepressed.
267 Cloning a protein kinase from Cryphonectria parasitica and its transcriptional regulation by hypovirus. Jin-Won Choi, Myoung-Ju Kim, Seung-Moon Park, Moon-Sik Yang, and Dae-Hyuk Kim. Chonbuk National University, Biological Sciences, Chonju, Chonbuk, Korea
The gene encoding Ser/Thr protein kinase (cpk-1) was isolated from chestnut blight fungus, Cryphonectria parasitica. Degenerated primers for cppk-1 was designed based on conserved catalytic domain of many fungal PK. The expected size of 350-bp amplicon was obtained by using PCR and cloned into plasmid vector. Sequence comparison of the cloned fragment showed the highest similarity to ptk1, Ser/Thr protein kinase from Trichoderma reesei, with 82% identity. The cpk-1 consisted of three exons with two intervening sequences of 67 bp and 71 bp in size, and the deduced cppk-1 protein product, CPK-1, had an estimated molecular mass of 70.5 kDa and a pI of 7.45. Primer extension experiment revealed the major transcription initiation site located at -49 bp from the translation start codon and the sequencing of cDNA clone indicated the poly(A) occurred at 569 bp down-stream of the stop codon. Northern blot analysis and RT-PCR were conducted to examine the expression pattern of cpk-1 from virus-free C. parasitica strain EP155/2 as well as its isogenic hypovirulent strain UEP1. The cpk-1 expressed at low level at both 1 and 5 day after the liquid culture while the cpk-1 expression increased considerably at 5 day by the presence of the hypovirus CHV1-713. This indicates the implication of hypovirus in the signal transduction pathway of C. parasitica through a modulation of cppk-1 expression and it is one of the first evidence showing that the hypovirus disturbs fungal signal transduction pathway at the transcriptional level. Correlation of an aberrant expression of cpk-1 to the specific viral symptoms is under investigation. M-J Kim was supported by Brain Korea 21 program from the Korean Ministry of Education.
268 Use of Ac/Dsbased gene traps for identification and characterization of pathogenicity factors from the rice-blast fungus, Magnaporthe grisea Naweed I. Naqvi.,1 Shanthi, S.,1 Weil, C.,2 Kunze, R.,3 Sundaresan V 1. 1Institute of Molecular Agrobiology, 1 Research Link, National University of Singapore, Singapore 117604, 2Dept. of Biological Sciences, University of Idaho, Moscow, ID83844, USA; 3Institut fur Genetik und Mikrobiologie, Universitat Munchen, Maria-Ward-Strasse 1a, 80638 Munich, Germany
In Magnaporthe grisea, the causal ascomycete of blast disease on rice, the pathogen cycle involves the following developmental sequence: deposition of a conidium on the rice leaf, germination of the conidium to form a germ tube, differentiation of the germ tube into a specialized infection structure called appressorium, penetration of the leaf surface by the melanized appressorium via a penetration peg, differentiation of the penetration peg into secondary hyphae leading to subsequent colonization of plant tissue. In this developmental sequence, only the melanization and pressurization of the appressorium is well understood (Annu. Rev. Microbiol., 50:491-512, 1996). The molecular basis of the infection-related development of a plant pathogenic fungus has not been fully explored, and elucidation of the underlying basis of this stimulus-response relationship at the structural and regulatory level would help us gain insights into mechanisms controlling the infection process and would lead to development of novel disease control strategies. We are using a novel insertional mutagenesis approach to explore the molecular basis of infection-related development in M. grisea. Towards this end, we have developed a system in which transposition of the maizeAc/ Dselements occurs in M. grisea. Evidence for successful Ac/Ds transpositions in the blast fungus shall be presented alongwith strategies for their use as valuable insertional-mutagenesis tools in identifying genes involved in pathogenicity of the rice-blast fungus. Preliminary data on the use of Agrobacterium T-DNA mediated random insertions in Magnaporthe shall also be discussed.
269 Virulence factors and defense proteins in tomato xylem sap during colonization by Fusarium oxysporum. Martijn Rep, Petra Houterman and Ben J. Cornelissen. University of Amsterdam, Plant Pathology, Amsterdam, The Netherlands.
Fusarium oxysporum colonizes tomato plants through invasion of xylem vessels. Whether the fungus will colonize the entire plant, resulting in wilt disease, or is restricted to one or only a few vessels, is determined by recognition events taking place within vessels . Proteins secreted by the fungus in xylem sap are likely to play a crucial role here. On the one hand, they can serve to promote fungal colonization, for instance through degradation of plant cell walls or suppression of plant defense mechanisms. On the other hand, some of these proteins may in fact induce plant defense reponses when they are recognized by plant cells. Such elicitors of plant defense responses have not yet been identified for Fusarium or any other fungal vascular pathogen.
In order to better understand the molecular basis of Fusarium pathogenicity, we aim to identify the full spectrum of proteins excreted by the fungus in tomato xylem vessels. In addition, we wish to identify plant proteins that are produced during disease development, in order to have a view on the strategies employed by the plant to suppress colonization. We have recently undertaken steps towards this goal. Xylem sap protein patterns from healthy and Fusarium-infected plants were compared by SDS-PAGE, revealing candidates for race-specific Fusarium proteins as well as plant-produced 'defense' proteins. Identification of these proteins with Mass Spectrometry is in progress.
270 Gene expression during pre-symbiotic development of arbuscular mycorrhizal fungi M'Barek Tamasloukht, Guillaume Becard* and Franken Philipp. Max-Planck-Institut fur terrestrische Microbiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Germany *Equipe de Mycologie Vegetale, UMR5546 CNRS/Universite Paul Sabatier, Pole de Biotechnologie Vegetale; 24, chemin de Borde-Rouge BP Auzeville, France
Arbuscular mycorrhizas (AM) are symbiotic associations formed between vascular land plants and fungi of the order Glomales (Zygomycota). These associations can be found on the majority of vascular plant species and represent therefore key elements of most terrestrial ecosystems. Unfortunately, AM fungi cannot be propagated in pure culture, since they are obligate symbionts and need to colonise roots to fullfill their life cycle. Nevertheless, they germinate without the host and show limited growth of hyphae during presymbiosis. This growth arrests after approximately two weeks, but hyphal elongation and branching can be induced by addition of root exudates. In order to investigate the molecular basis of presymbiotic hyphal development, we are analysing gene expression in the AM fungi Gigaspora rosea and Gig. Gigantea by two methods, differential RNA display and suppressive substractive hybridisation. Up to now, more than 600 clones were obtained which contain cDNA fragments with an average size between 150 and 1500 bp. Randomly selected clones were screened by reverse Northern blot, in order to identify genes upregulated by the addition of root exudates. Differential expression of selected genes mainly involved in signal transduction processes and in respiration has been verified by RT-PCR. In parallel, cytological investigations are carried out to prove the hypotheses, which were drawn from sequence and expression data.
271 Mapping avirulence genes by para-sexual fusion of different Fusarium oxysporum f. sp. lycopersici races. Hedwich A.S. Teunissen, Jurriaan J. Mes, Jurgo Verkooijen, Ben J.C. Cornelissen and Michel A. Haring. Plant Pathology, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, the Netherlands.
Resistance of tomato to Fusarium oxysporum f. sp. lycopersici (Fol) is monogenic and dominant. Fol race 1 isolates, containing the avirulence gene 1 (A-1), are avirulent on tomato lines harbouring the corresponding resistance gene (I-1). Fol race 3 isolates contain the avirulence gene 3 (A-3) and are therefore avirulent on I-3 plant lines. For the isolation of either the Fol A-1 gene and the A-3 gene a genetic approach was chosen. Since no sexual stage of this fungus is known, para-sexual crosses were used for mapping studies. A race 1 isolate (putative genotype A1a2a3) and a race 3 (a1a2A3) isolate were transformed with either a phleomycin or a hygromycin resistance gene containing plasmid. Transformants with a single insertion that have retained their pathogenic characteristics were selected. Protoplast fusion of a phleomycin resistant race 1 transformant and a hygromycin resistant race 3 transformant resulted in progeny resistant against both antibiotics. 31 Fusionproducts originating from seven different parental combinations were identified. Pathogenicity tests were performed with the stable fusionproducts using tomato lines carrying either an I-1 or I-3 resistance gene. 28 Fusionproducts behaved in a race 1-like manner (A-1 present; A-3 absent). Only one fusionproduct was able to overcome I-1 resistance (A-1 absent; A-3 present). 2 Fusionproducts showed an "avirulence-recombinant" phenotype suggesting the presence of both A-1 and A-3. Retrotransposon based "Foxy"-AFLP analysis showed exchange of parental DNA in all fusionproducts. Based on 100 polymorphisms between race 1 and race 3, the race 1 genetic contribution in the fusionproducts ranged from 26-96%. One interesting polymorphic fragment was identified showing a 100% correlation with the race 1 phenotype. This fragment will be cloned, analysed and used in further finemapping of the Fol avirulence gene A-1.
272 Analysis of the expression of the virulence factor-encoding gene pksP of Aspergillus fumigatus using the green fluorescent protein Kim Langfelder1, Bruno Philippe2, Bernhard Jahn3, Jean-Paul Latg 2, and Axel A. Brakhage1. 1Institut fur Mikrobiologie und Genetik, Technische Universitat Darmstadt, Schnittspahnstrasse 10, D-64287 Darmstadt, F.R.G.; 2Institut Pasteur, Laboratoire des Aspergillus, 25 Rue du Docteur Roux, F-75015 Paris, France; 3Institut fur Medizinische Mikrobiologie und Hygiene, Universitat Mainz, D-55101 Mainz, F.R.G.
Aspergillus fumigatus is an important pathogen of the immunocompromised host causing pneumonia and invasive disseminated disease with high mortality. In order to analyse putative virulence factor-encoding genes in vivo, the enhanced green fluorescent protein (EGFP) was established as a reporter. Two promoters of A. fumigatus were fused with the egfp gene. The promoter of the pyrG gene, encoding orotidine-5'-phosphate decarboxylase, and that of the pksP gene. pksP encodes a polyketide synthase involved in both pigment biosynthesis and virulence. EGFP production was analysed by fluorescence spectrometry, Western blot analysis and fluorescence microscopy. PYRG-EGFP derived fluorescence was detected during all fungal developmental stages. In addition, pyrG-egfp expression was detected in germinating conidia when isolated from the lungs of immunosuppressed mice. By contrast, PKSP-EGFP derived fluorescence was found only in phialides and conidia under standard conditions, indicating a developmentally controlled expression of the gene. Interestingly, PKSP-EGFP derived fluorescence was also detected in vivo in hyphae of germinating conidia when isolated from the lungs of immunosuppressed mice. This finding suggests that the pksP gene is also expressed in hyphae during invasive growth of the fungus. 1. Jahn et al. (1997) IAI 65:5110 2. Langfelder et al. (1998) Med Microbiol Immunol 187:79 3. Brakhage et al. (1999) Contrib to Microbiol, Vol. 2:205 4. Jahn et al. (2000) IAI 68:3736
273 Visualization of interactions between Pseudomonas biocontrol bacteria and Fusarium oxysporum f. sp. radicis-lycopersici on tomato roots using autofluorescent proteins. A. Lagopodi1, G. M. Bloemberg1, A.F.J. Ram1,2, A. Wijfjes1, C.A.M.J.J. van den Hondel1,2 and E.J.J. Lugtenberg1. 1Leiden University, Inst. Mol. Plant Sciences, Leiden, The Netherlands,2 Department of Appl. Microbiol. Gene Techn., TNO- Nutrition, Zeist, The Netherlands.
Biological control of soil-borne plant pathogenic fungi with rhizobacteria forms an alternative to the use of chemical pesticides. In order to improve the efficacy of biocontrol fundamental knowledge of the interactions between plants, biocontrol bacteria and phytopathogenic fungi is required. Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391 have been shown to efficiently suppress crown and root rot disease of tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici (F.o.r.l.) in greenhouse experiments (1, 2). Biocontrol of tomato crown and root rot strains was successfully reproduced in a gnotobiotic sand system after inoculations of tomato seedlings with F.o.r.l and the biocontrol Pseudomonas strains. In this system where the host, the pathogen and the biocontrol agent are present as the only biotic factors, biocontol can be studied in detail. Recently F.o.r.l. was successfully transformed with gfp (green fluorescent protein) and gfp expression was stable after several generations on nutrient media as well as on tomato. At present different autofluorescent proteins are used to mark biocontrol Pseudomonas strains. The use of different autofluorescent proteins will give the opportunity to visualize the interactions between the plant, the pythopathogenic fungus and the biocontrol bacterium simultaneously, and to study the nature of the biocontrol effect. Unraveling the interactions of biocontrol bacteria and pathogenic fungi on tomato roots will contribute to development of more efficient methods for biocontrol.
1. Dekkers L.C. 1997. PhD Thesis, Leiden University, The Netherlands. 2. Chin-A-Woeng T. F. C. et al., 1998. Mol. Plant Microbe Interact.11: 1069- 1077.
274 Involvement of a pacC homolog from Sclerotinia sclerotiorum in sclerotial development and virulence. Jeffrey A. Rollins. University of Florida, Plant Pathology, Gainesville, FL
The broad phytopathogenic potential of Sclerotinia sclerotiorum has been attributed to the production and synergistic activities of oxalic acid and cell wall degrading enzymes. The acid environment created by oxalic acid secretion increases hydrolytic enzyme activity, stimulates sclerotial initiation, and inhibits further oxalic acid production. The role of ambient pH in the regulation of these processes at the gene-expression level is being investigated. A homolog of the Aspergillus nidulans pH-responsive transcription factor, pacC, has been cloned from S. sclerotiorum. This gene, pac1 , can functionally substitute for pacC in A. nidulans. Expression of pac1 in S. sclerotiorum is dramatically increased under alkaline growth conditions and within aerial hyphae participating in sclerotial development. Targeted disruption of the pac1 gene was accomplished. Cultures of pac1 null strains grown without exogenous control of pH were reduced in oxalic acid accumulation by approximately fifty percent. The growth rate and gross hyphal morphology, however, appeared unchanged. These strains were also able to initiate sclerotial morphogenesis, but did not form mature, fully developed sclerotia. Virulence of null pac1 strains was also reduced. Whether this attenuated virulence is the direct result of reduced oxalic acid production is being investigated. These results indicate that pac1 is not essential for oxalic acid production but may play a role in regulating the levels of this and possibly other factors necessary for full virulence and sclerotial development. Gene expression and phenotypic characterization of null pac1 strains in response to varying ambient pH conditions is underway.
275 A web-based resource for information on vegetative compatibility groups of Fusarium oxysporum. Talma Katan and H. Corby Kistler. The Volcani Center, Bet Dagan, ISRAEL and USDA ARS Cereal Disease Laboratory and Plant Pathology Department, University of Minnesota, St. Paul, USA.
Fusarium oxysporum is a ubiquitous soilborne species that includes saprophytes as well as important plant pathogens. Information on vegetative compatibility (the ability of closely related strains to form heterokaryons in laboratory tests) has been used to study and catagorize genetic diversity in this fungus. As a species, F. oxysporum affects over 100 host species, but individual strains are highly host specific. Strains also have been categorized by host specificity into groups known as formae speciales. A vegetative compatibility group (VCG) usually includes only strains from a single forma specialis whereas formae speciales may be comprised of one or more VCG. The correspondence of VCG with host specificity has been useful in assigning strains to host specificity and in estimating the diversity of strains causing disease on a particular host. A numbering system has been established in which formae speciales and VCGs have been assigned numerical codes. Each code number is composed of a 3-digit forma specialis code and a 1-2 digit serial number for individual VCGs within a forma specialis. To date 48 formae speciales have been subjected to VCG analysis and the number of VCGs in a forma specialis ranged from 1 to 24, averaging four VCGs per forma specialis. A web site has been established that compiles information on VCGs of Fusarium oxysporum and can be accessed at www.cdl.umn.edu//scab/vcg.html. Formae speciales are tabulated by their numerical codes, host plants and the VCGs identified for them. A comprehensive list of 193 references is also included.
276 Virulence in Mycosphaerella graminicola (anamorph Septoria tritici) is associated with deletions in a putative avirulence locus. Gert Kema, Els Verstappen, Odette Mendes, Ineke de Vries and Cees Waalwijk. Plant Research International, Wageningen, The Netherlands
Gene-for-gene interactions may play a role in structuring Mycosphaerella graminicola populations. We generated segregating populations by crossing two Dutch field isolates, IPO323 (avirulent) and IPO94269 (virulent) and studied F1, BC1 and F2 populations by inoculation assays on five wheat cultivars. Avirulence inherited as a monogenic character. A genetic linkage map was generated and the putative avr locus as well as three co-segregating AFLP markers were mapped. These markers were used to select specific BAC clones from both parents. Sequence analyses of corresponding contigs of both parents revealed an ORF coding for a putative protein carrying a signal peptide that was absent in the virulent parent. The flanking regions of this 840 bp deletion showed a strong homology, which enabled us to design primers that produced either a 1500 bp or a 650 bp fragment. The frequency of this deletion in several natural M. graminicola populations ranged from 80 to 95%. This suggests that the deletion occurred only once, which is unexpected if this locus would be under selection. The frequency of avirulent phenotypes in natural populations not always correlated with the presence of the ORF, which is an indication that more cultivar specific avirulence loci may play a role in this pathosystem. The functional analysis of this avr locus is under way.
277 EST data mining: Novel extracellular proteins from the oomycete plant pathogen Phytophthora infestans. Trudy Torto, Allison Styer, Sophien Kamoun. Department of Plant Pathology, The Ohio State University-OARDC,Wooster, OH
Interactions between plants and microbial pathogens involve complex signal exchanges at the plant surface and intercellular space interface. Surface components of a pathogen must play important roles in the development of a complete infection cycle and recognition by resistant plants. We aim at identifying extracellular proteins fromPhytophthora infestans, an economically important oomycete pathogen. Targeting extracellular proteins will increase the probability of identifying proteins essential for virulence and survival of the pathogen. We developed and validated an algorithm (PexFinder V1.0) for automated identification of secreted and membrane proteins from expressed sequence tag (EST) data sets. The program integrates a series of sequence analysis scripts with signal peptide predictions based on SignalP V2.0 (http://www.cbs.dtu.dk/services/SignalP-2.0/). Analysis of 2,147 ESTs from P. infestans using PexFinderidentified 261 ESTs (12.2%) corresponding to a set of 145 nonredundant Pex (Phytophthora extracellular proteins) genes. Of these, 85 (59%) Pex genes are novel with no significant matches in public databases. The algorithm was validated using a number of methods. For example, PexFinder identified numerous genes with significant matches to known extracellular proteins, as well as all previously characterized extracellular proteins from Phytophthora that were represented in the EST data set. Functional genetic assays, such as high throughput virus and Agrobacterium-based expression systems, are being applied to the novel Pex genes to determine their role in virulence/avirulence. This approach led to the discovery of novel classes of extracellular signal molecules from
278 Comparison of compatible interactions between tomato and Phytophthora infestans. Christine D. Smart1, Kevin L. Myers1, Mark D. D Ascenzo2, Paul P. Debbie2,3, Gregory B. Martin1,2, and William E. Fry1. 1Department of Plant Pathology, Cornell University, 2The Boyce Thompson Institute at Cornell University, 3BTI Center for Gene Expression Profiling at Cornell University, Ithaca, NY
Tomato-specialization in the oomycete pathogen Phytophthora infestans is characterized by a biotrophic growth habit and increased pathogenicity on tomato. To investigate the mechanism of this specialization, we compared tomato host responses to a specialized isolate, to those of a non-specialized (more necrotrophic and less aggressive) isolate. As expected, we saw induction of the hypersensitive response (HR) more quickly in tomato plants inoculated with the non-specialized isolate. Induction of pathogenesis-related (PR) genes known to be markers for the salicylic acid or jasmonic acid signaling pathway was similar in plants inoculated with specialized or non-specialized isolates. However, basic PR genes known be ethylene-regulated were induced more quickly in tomato plants inoculated with the specialized (biotrophic, more aggressive) isolate than in plants inoculated with the non-specialized isolate. These findings support the hypothesis that while HR is necessary for a reduction in aggressiveness, induction of the 10 PR genes tested is not responsible for the difference between highly pathogenic (tomato-specialized) and moderately pathogenic (non-specialized) isolates of P. infestans. We are currently using microarray analysis to identify differences in gene expression between these two compatible interactions.
279 Identification of differentially expressed genes involved in the symbiosis between Neotyphodium coenophialum and tall fescue by suppression subtractive hybridization analysis. L.J. Johnson1, R.D. Johnson1, C.L. Schardl2, and D.G. Panaccione1. 1, West Virginia University, Morgantown; 2, University of Kentucky, Lexington.
Tall fescue (Festuca arundinaceae) is typically infected with Neotyphodium coenophialum, an asexual, endophytic fungus. Colonization is completely internal in host tissue with no visible symptoms, suggesting that the plant does not respond to endophyte colonization. This fungal endophyte provides many benefits to its plant host, such as increased biomass and seed production, protection from insects, drought tolerance and resistance to nematodes and some fungal pathogens. We hypothesize that the underlying physiology of the plant-endophyte interaction is the result of changes in gene expression. Our research focuses on identifying genes that have altered expression in the plant/fungal symbiosis. Using suppression subtractive hybridization (SSH), a PCR-based cDNA subtraction technique, we have currently identified at least 23 differentially expressed genes. Up-regulated plant genes of particular interest encoded a PDR5-like ABC transporter, an Omega-3 fatty acid desaturase, and a heat shock protein (hsp70). In addition, genes suppressed in the plant due to the presence of endophyte were also identified. These included genes for an aminopeptidase N, a chlorophyll A-B binding protein, methionine associated enzymes, and a pathogenesis-related protein (PR- 10). Interestingly, several of these suppressed plant genes have been reported to be induced in other plants by fungal pathogens. Overall these results suggest that tall fescue is an active participant in the endophyte symbiosis and that the endophyte may be suppressing plant defense genes. Further analysis and identification of genes with altered expression in the plant fungus symbiosis may elucidate mechanisms responsible for the benefits associated with endophyte infection of tall fescue.
280 Characterization of an ABC transporter from Phytophthora sojae. Paul F. Morris, Mary S.Connolly. Bowling Green State University, Biological Sciences, Bowling Green, OH, USA
ABC transporters are members of a large protein super family involved in both the uptake and efflux of compounds across membranes. These transporters have been identified in both prokaryotic and eukaryotic organisms. A 1300bp fragment from the 3' end of ABC transporter has been identified in both zoospores and Phytophthora. sojae -infected soybean hypocotyls. Sequence analysis suggests that this gene is part of the pleiotropic drug resistance family of ABC transporters, many of which are up-regulated by compounds that also serve as substrates for the transporter. Homologues of the Phytophthora gene are present in Arabidopsis and Spirodella, and the Spirodella gene is known to be up-regulated in response to cold temperatures, osmotic stress, and abscisic acid. The response of the P. sojae gene to a variety of environmental stimuli will be presented.
281 Detection of plant genes activated by Ustilago maydis infection in an alternate host. Cristina G. Reynaga-Peña1, Jos Ruiz-Herrera1 and Patricia S. Springer2 . 1 Depto. de Ingenieria Genética, CINVESTAV Unidad Irapuato, Irapuato, Gto., México and 2 Department of Botany and Plant Sciences, University of California, Riverside, Riverside, CA, U.S.A.
In this work we are using a novel approach towards the understanding of molecular signals involved in plant-pathogen interactions. It is known that Ustilago maydis, the pathogenic fungus responsible for corn smut, has a complex life cycle that can only be completed upon invasion of the host plant. It has been proposed that a close relationship between plant and pathogen must exist during Ustilago infection, where communication between both organisms is necessary. Previously, our lab has started to use tissue cultures and alternative host plants to study Ustilago-plant interactions. We are now characterizing a novel infection model system using Arabidopsis thaliana as a host plant, and have observed mycelial growth of Ustilago on several plant tissues. The development of this model system permitted us the use of an enhancer/gene trap transposant system that has been developed in Arabidopsis to identify genes that show changes in expression during Ustilago infection. At this time, preliminary results indicate that several transposants show differential expression of the reporter gene upon infection. These transposants are now in the process of identification.
282 Identification of genes involved in pathogenicity of the fungal common bean pathogen Colletotrichum lindemuthianum. R. Lauge , D. Parisot, A.-L. Pellier, C. Veneault, M. Dufresne and T. Langin. Laboratoire de Phytopathologie Moléculaire, Institut de Biotechnologies des Plantes, batiment 630, Université Paris-Sud, 91405 Orsay Cedex, France.
Insertional mutagenesis using the plasmid pAN7-1 was done on the fungal pathogen Colletotrichum lindemuthianum. 1100 independant transformants were tested for impaired pathogenicity on common bean. 11 mutants were identified, of which most are blocked during the early stages of the infection cycle: at the penetration step or during the biotrophic phase. A fine cytological characterization of the phenotype and the level of blockage of the various mutants is underway. Molecular analyses and demonstration of the link between the insertion event and the pathogenicity phenotype have been achieved for three mutants and all of them are tagged. The inability of two mutants to penetrate (although they produce appressoria) is due to disruption of genes encoding a serine/threonine kinase (clk1) and a copper ATPase transporter (clap1) respectively. We are currently investigating the precise role of these two genes in appressorium functionality. A third mutant is blocked at the end of the biotrophic phase after the differentiation of the infection vesicle and of the primary hyphae. The disrupted gene (clta1) encodes a putative transcriptional activator of the GAL4 family. A search for the sequences that are under the control of this activator has been initiated using a one-hybrid derived strategy. The genes that will be obtained should provide us with information on the type of function that is regulated by CLTA1. Of the remaining mutants, six that display a clear phenotype are under molecular analysis. Preliminary results show that insertion flanking sequences display strong similarities with a hexose transporter and a regulator of inorganic phosphate transport for at least two of these mutants. Latest data will be presented and discussed.
283 Characterization of the ToxB gene from Pyrenophora tritici-repentis. J. Patrick Martinez, Sean A. Ottum, and Lynda M. Ciuffetti. Department of Botany and Plant Pathology. Oregon State University, Corvallis 97331 U.S.A.
Race 5 isolates of Pyrenophora tritici-repentis, causal agent of tan spot of wheat, are characterized by the production of lesions with spreading chlorosis on wheat cultivar Katepwa. This host-specific chlorosis has been correlated with a 6.61 kDa host-specific toxin (HST), Ptr ToxB (Strelkov et al., 1999, MPMI 12:728-732). A 261 bp gene, ToxB, was cloned and characterized from a race 5 isolate of P. tritici-repentis. ToxB encodes a putative 23 amino acid signal peptide and a 64 amino acid HST, Ptr ToxB. The ToxB gene appears to be present in multiple copies in race 5 isolates. Non-pathogenic isolates of this fungus contain a single copy gene (toxb) that shows 86 % identity at the nucleotide level when compared to ToxB. Analysis of Ptr ToxB from heterologous expression in Pichia pastoris confirms that ToxB encodes a HST. Current research will attempt to determine the function of these "additional" ToxB genes, and their relationship to the toxb gene in non-pathogenic isolates.
284 Transformation of Pythium aphanidermatum to geneticin resistance. John J. Weiland. Sugarbeet and Potato Research Unit, USDA-Agricultural Research Service, Northern Crop Science Laboratory, Fargo, N.D. 58105-5677
Conditions for the production of protoplasts and gene transfer in Pythium aphanidermatum were investigated. Efficient protoplast generation was possible after culture of mycelium in potato dextrose broth followed by digestion with 0.5% (w/v) each of cellulase and beta-D-glucanase. Plasmid pHAMT35N/SK encoding the nptII gene under control of the Ham34 promoter from the oomycete Bremia lactucae was used to define electroporation parameters for gene transfer. A square-wave electroporation pulse of 2500V/cm at 50 microfarad capacitance reproducibly produced transformants, albeit at low efficiency (0.1-0.4 transformants from ~100,000 regenerable protoplasts per microgram of DNA). Twenty seven independant transformants exhibited wild-type growth on potato dextrose agar amended with geneticin at 50 microgram per ml, a concentration that near completely inhibited the growth of untransformed fungus. Southern blot analysis indicated that transforming DNA was integrated into the fungal genome as a tandem array of plasmid monomers. Co-electroporation of of pHAMT35N/SK with pEGFP encoding enhanced green fluorescent protein (EGFP) under the control of the immediate early promoter from the mammalian cytomegalovirus produced transient expression of blue-green fluorescence. Application of the technique to studies on the biochemical basis for pathogenesis in this agriculturally-important group of fungi are discussed.
285 Multidrug resistance proteins of Cochliobolus heterostrophus. Uvini P. Gunawardena, Olen C Yoder, Gillian Turgeon. NADII, Plant Health, San Diego, CA
Multi drug resistance (MDR) proteins in plant pathogenic fungi are of interest for their potential to mediate interactions between hosts and pathogens, as well as for the opportunities they offer to genetically engineer plants for resistance to microbial virulence factors such as mycotoxins. In plant pathogenic fungi, two major groups of MDR proteins, ABC (ATP Binding Cassette) and MFS (Major Facilitator Superfamily) transport proteins, have been shown to play important roles during fungal interactions with host plants. We have mined the genomic sequence of Cochliobolus heterostrophus, a pathogen of corn, and found 51 putative ABC and more than 20 putative MFS proteins. The genes encoding these proteins are being targeted for deletion and the resulting mutants tested for viability for alteration of their virulence to corn, and for their sensitivity to plant antimicrobial compounds.
286 Calcineurin (CN) affects morphology, mating and pathogenicity in Ustilago maydis . J. Duick Egan and S.E. Gold. Dept. of Plant Pathology, Univ. of Georgia, Athens, GA 30602.
Ustilago maydis is a dimorphic basidiomycete and the causal agent of corn smut disease. Dimorphism is controlled through signal transduction pathways involving cAMP and the mitogen-activated protein (MAP) kinase cascade. Protein phosphatases reverse the effects of protein kinase phosphorylation. Calcineurin (CN, also known as protein phosphatase 2B, PP2B) is a serine/threonine protein phosphatase and, in other systems, has a role in reversing the phosphorylation of the substrates of cAMP-dependent protein kinase. Therefore, we hypothesize that CN plays an important role in dimorphism, and mutants would have enhance budding growth. Mutants in the U. maydis CN catalytic subunit, ucn1, are viable and indeed have a multiple budding phenotype. Mating reactions and pathogenicity of these mutants are severely reduced and galls never developed in inoculated maize plants. These results suggest that CN plays an important role in morphogenesis, mating, and pathogenicity in U. maydis.
287 Progress towards cloning avirulence genes in the wheat stem rust fungus, Puccinia graminis. Les J. Szabo and Alexi Balmuth. Cereal Disease Laboratory, ARS, USDA, St. Paul, Minnesota, U.S.A.
Puccinia graminis is a heteroecious rust fungus, with uredinial (asexual) and telial stages on cereal and forage grass species and the pycnial and aecial stages on members of the barberry family. Genetics has demonstrated that infection of wheat by P. graminis f.sp. tritici follows a gene-for-gene interaction involving resistance genes in the host and avirulence genes in the rust pathogen. In order to better understand this interaction, we have undertaken the cloning of several avirulence genes from P. graminis f.sp. tritici. A mapping population of P. graminis has been developed in which eight single dominant avirulence genes are segregating (Zambino et.al., 2000). DNA markers (AFLPs and RAPDs) have been used to construct a partial genetic map in which these avirulence genes have been mapped to seven linkage groups. Cloning and characterization of DNA markers linked to avirulence genes will be presented.
288 A Class I hydrophobin from Cochliobolus heterostrophus. Scott E. Baker, Olen Yoder and Gillian Turgeon. Plant Health Department, Novartis Agricultural Discovery Institute, 3115 Merryfield Row, San Diego, CA, 92121
A gene encoding a class I hydrophobin (CHH1) was identified in the corn pathogen Cochliobolus heterostrophus. CHH1 encodes a putative protein (CHH1p) of 116 amino acids that has 31% identity with the Magnaporthe grisea MPG1 protein, also a class I hydrophobin. We have generated deletions of CHH1 and our phenotypic analysis indicates that appressorium formation, conidiation and virulence are unaffected by loss of this protein, in contrast to MPG1 deletions which show reduced conidiation, appressorium formation and virulence. These data are consistent with the prior observation that neither melanin production nor appressoria are necessary for full virulence of C. heterostrophus. M. grisea requires both melanin and functional appressoria for infectivity. Moreover, a G alpha protein (CGA1 in C. heterostrophus, MAGB in M. grisea) is important for infection by M. grisea but not C. heterostrophus (Horwitz et al. FG&B 1999 26:19).
289 Cloning of a functional avirulence gene homolog from a Magnaporthe grisea strain that is a pathogen of perennial ryegrass. Rebecca Peyyala and Mark Farman. Department of Plant Pathology, University of Kentucky, USA
Magnaporthe grisea (anamorph = Pyricularia grisea) causes a serious disease of perennial ryegrass (Lolium perenne) called gray leaf spot. Perennial ryegrass appears to have little or no natural resistance to this disease. Rice is resistant to strains of M. grisea that cause gray leaf spot indicating that it may be a good source of resistance to this disease. Southern hybridization analysis of M. grisea strains infecting perennial ryegrass revealed that they have a homolog of the AVR1-CO39 avirulence gene, which prevents M. grisea from infecting rice cultivar CO39 due to the presence of the corresponding resistance gene Pi-CO39(t). The homolog, which we refer to as AVR1-CO39Lp, was amplified by PCR, cloned and sequenced. It differed from AVR1-CO39 by three point mutations and 2 frame shifts but these did appear to affect the presumed AVR1-CO39 transcription unit. The AVR1-CO39Lp gene was then transformed into fungal strain ML33-2 that is virulent on CO39. Transformants were tested for the presence of AVR1-CO39Lp by Southern hybridization and the function of the gene was tested by inoculation on CO39. ML33-2 transformants carrying AVR1-CO39Lp were avirulent on CO39 but were unaffected in their ability to infect 51583, a cultivar that lacks Pi-CO39(t). Therefore, AVR1-CO39Lp is a functional avirulence gene that likely prevents the perennial ryegrass pathogens from infecting CO39 rice, due to recognition by the Pi-CO39(t) resistance gene. Experiments are in progress to determine if Pi-CO39(t) will also function in perennial ryegrass to confer resistance to gray leaf spot disease.
290 Identification of a Cys(6)-Zn(2) transcription factor that binds the pisatin-responsive region of PDA1 in Nectria haematococca. Rana Khan, Reynold Tan and David Straney. Dept. Cell Biol. & Molec. Genetics, University of Maryland, College Park, MD. 20742
The pea pathogen Nectria haematococca MP VI (Fusarium solani) provides a genetic model system for soil borne pathogens. One of the intriguing aspects of the interaction of this fungus with its host, garden pea (Pisum sativum), is its use of host-specific compounds to coordinate both developmental and gene-specific responses in pathogenesis. Pisatin, the isoflavonoid phytoalexin produced by pea, provides a host-specific cue for both of these responses. Expression of the PDA1 gene, which encodes a cytochrome P450, is highly induced by exposure of mycelium to pisatin. Pisatin appears to be the major inducer for this gene during pathogenesis as well. PDA1 regulation provides a system to identify fungal components that allow a pathogen to recognize and respond to host-specific cues to coordinate pathogenesis. We have defined a 40 bp region in the PDA1 promoter that mediates pisatin-responsive transcription in vivo. A DNA-binding factor was found to bind this 40 bp region in mycelial extracts. The yeast one-hybrid system was used to clone the gene encoding the protein which binds this 40 bp region. Sequence motifs from the cloned gene suggest that it is a Cys(6)-Zn(2) binuclear cluster factor. The cloning of this factor will allow testing the possibility that it may bind pisatin as well and so function as a nuclear receptor, similar to the mechanism regulating many mammalian cytochrome P450 genes.
291 High frequency mitotic gene conversion across the genome of the oomycete , Phytophthora sojae. Jureerat Chamnanpunt and Brett M Tyler. Department of Plant Pathology, University of California, Davis,USA.
Microbial populations depend on genetic variation to respond to novel environmental challenges. Plant pathogens are notorious for their ability to overcome pesticides and host resistance genes as a result of genetic changes. We show here that in Phytophthora sojae , an oomycete pathogen of soybean, high frequency of mitotic gene conversion rapidly converts heterozygous loci to homozygosity, resulting in heterokaryons containing a highly diverse population of nuclei. We examined gene conversion within a selected linkage group, and in unlinked markers throughout the genome. Loci as close as 0.7 kb apart underwent independent conversion at frequencies of up to 80%. In many cases, conversion was highly polar, proceeding in only one direction, suggesting that conversion was initiated by allele-specific double stranded breaks. High frequency conversions were initiated when certain strains of P.sojae were mated to produce F1 hybrids. They continued throughout vegetative growth and were stimulated by further sexual reproduction. Pedigree analysis indicated that individual loci undergo multiple independent conversions within the nuclei of a vegetative clone, and that conversion may be preceded by a heritable "activation" state that commits the direction of conversion. The high polarity of gene conversion was determined in cis by the sequence polymorphism at the site of conversion. The conversion frequency is strongly affected by genetic background of some particular strain (P7076). We proposed the hypothesis that the double stranded breaks were stimulated by P7076 genome or interaction with P7076 genome.
292 Maize ribosome-inactivating protein has antifungal activity against Aspergillus species. Kirsten Nielsen and Gary A. Payne. NC State University, Plant Pathology, Raleigh, NC
The abundant maize kernel ribosome-inactivating protein (RIP) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans were treated with RIP protein. RIP treatment of conidia from A. flavus resulted in increased hyphal branching. These results indicate that both fungi were affected by RIP treatment but the toxicity seen with treatment of A. nidulans is apparently avoided by A. flavus. Even though conidia were treated with RIP prior to germination, a developmental time-course revealed that changes in fungal growth for both species were not seen until the post-divisional growth stage. The inhibitory activity of RIP against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion. To determine whether or not the antifungal effect of RIP was due to its enzymatic activity, we constructed expression plasmids with point mutations at active site residues. The resulting proteins had no effect on the growth of A. nidulans and no ribosome-inactivating activity in vitro. These results indicate that the antifungal activity of RIP requires the enzymatic ribosome- inactivating activity. However, these results also lead to the question ûû How does RIP1 enter the fungal cell to inactivate the ribosomes? At 25 kD, maize RIP would appear to be too large to cross the fungal cell membrane. We are investigating the means by which RIP exerts its antifungal effect by localization of fluorescently labeled RIP or control proteins during their interaction with A. nidulans.
293 Characterizing the PMK1 Pathway in Magnaporthe grisea. Jin-Rong Xu, Li Zheng, Marie Nishimura, Gyungsoon Park, Chaoyang Xue, Stephen Lam. Purdue University, Botany & Plant Pathology.
Rice blast, caused by Magnaporthe grisea, is one of the most severe fungal diseases on rice throughout the world. Like many other fungal pathogens, M. grisea develops specialized structures (appressoria) to invade its hosts. We have identified a MAP kinase PMK1 (Pathogenicity MAP kinase 1) that is essential for appressorium formation and infectious growth in M. grisea. To understand how the PMK1 MAP kinase is activated, we have isolated several putative components of the PMK1 pathway including homologues of budding yeast Ste4, Ste20, Ste11 and Ste12. Gene replacement mutants of these M. grisea genes will be isolated to determine their roles in the PMK1 signal transduction pathway. A yeast two-hybrid library has been constructed to identify genes interacting with PMK1. Data from the preliminary screening will be presented. In addition, we have constructed a subtractive library enriched for genes regulated by PMK1. Three genes identified in this library have been selected for further characterization.
294 Development of selectable markers in the human-pathogenic fungus Histoplasma capsulatum. Adam K. Bahrami and Anita Sil. Department of Microbiology & Immunology, University of California-San Francisco
Genetic approaches to studying the human-pathogenic fungus Histoplasma capsulatum are limited by the lack of sufficient marker genes to select for the uptake of transforming DNA. A H. capsulatum cDNA library was screened for clones that complement auxotrophic mutations of Saccharomyces cerevisiae. We identified cDNA clones that complement S. cerevisiae ade2, leu2, or his3 mutations and are now disrupting the corresponding genomic loci. Additional work aims to identify a conditional promoter for use in the study of this organism.
295 Analyses of a ketoreductase ORF at the AVR1-MARA locus of Magnaporthe grisea. Michael W. Harding, M. Alejandra Mandel and Marc J. Orbach. Department of Plant Pathology, University of Arizona, Tucson, AZ.
We are working to characterize the gene AVR1-MARA from the rice blast fungus Magnaporthe grisea. AVR1-MARA is a stable avirulence gene that segregates as a single locus in strain 4224-7-8, conditioning an avirulent response on the rice cultivar Maratelli. A map-based cloning approach has been taken to clone AVR1-MARA. An open reading frame with significant homology to a ketoreductase gene from Streptomyces hygroscopicus was found in DNA sequence linked to this locus. The ketoreductase in S. hygroscopicus is part of the rapamycin polyketide biosynthetic cluster. We are examining expression of the ketoreductase mRNA under a variety of growth conditions. Expression is also being examined in infected rice plants. Total RNA from M. grisea cultured in media with complete or limiting nutritional components or infected rice leaves will be isolated and analyzed by northern analysis. To determine whether ketoreductase function is necessary for avirulence, we are currently working to disrupt the gene by targeted gene replacement. The ketoreductase in 4224-7-8 will be replaced with a selectable resistance marker and the mutant strain tested for virulence on 'Maratelli'. The current data on ketoreductase expression and function will be discussed.
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