86. Constructing a BAC contig of the Phytophthora sojae genome by repetitive sequence fingerprinting.
Felipe Arredondo, Alan Chan and Brett M. Tyler, Department of Plant Pathology, University of California, Davis, CA95616.
The more than 40 species of the oomycete Phytophthora cause serious diseases of a huge range of crop and ornamental plants. We are characterizing genes that control recognition, host specificity and pathogenicity in the soybean pathogen Phytophthora sojae. To facilitate isolation of such genes
from P. sojae by map-based cloning, we are constructing a BAC contig of the entire genome of this
organism. We have ordered a library of 7680 BACs of average size 55kb, spanning the 62 Mb
genome 7 times. We are hybridizing the BACs with unique mixtures of random probes, most of them
repetitive. We will use the subset of probes hybridizing to each BAC to identify overlapping BACS.
Computer software has been developed to collect, simulate and analyze the data. We estimate that
about 120 probe mixtures, each hybridizing to around 700 BACS, will be sufficient to enable us to
quickly assemble the BACs into less than 100 contigs spanning >95% of the genome. More detailed
restriction analysis will be used to consolidate the initial set of contigs. Correlation of the BAC
contigs with the genetic map using PCR will enable rapid cloning of any genes placed on the genetic
map.
87. Functional analysis of the Magnaporthe grisea avirulence gene AVR2-YAMO.
Gregory T. Bryan1, Leonard Farralll, Marc Orbach2, Seogchan Kang3, James A. Sweigardl, and Barbara Valentl. l DuPont Science and Engineering Laboratories, Wilmington, DE 19880-0402 2Department of Plant Pathology, University of Arizona AZ 85721. 3Department of Biology, University of New Mexico, Albuquerque NM 97131.
The ascomycete Magnaporthe grisea causes disease on a wide variety of graminaceous hosts,
although individual strains of the fungus are limited to infecting one or two grass species. Some
examples of host-pathogen specificity in this system appear to fit the classical gene-for-gene
hypothesis. The avirulence gene AVR2-YAMO confers avirulence towards the rice cultivar Yashiro-mochi, and the telomeric location of the AVR2-YAMO gene in some rice pathogens appears to
account for frequent spontaneous mutation to virulence towards Yashiro-mochi. Transformation of
virulent strains of M. grisea with a functional copy of the cloned AVR2-YAMO gene confers
avirulence on Yashiro-mochi. Functional AVR2-YAMO genes have also been cloned from strains
of M. grisea that infect Digitaria spp. or Pennisetum spp. and not rice. The cloned gene encodes a
protein with a predicted molecular weight of 26 kDa that has some sequence similarities to neutral
zinc metalloproteases. Mutation of specific amino acid residues in the putative zinc-binding region
removes the ability to confer avirulence. Sequence comparisons with non-functional avr2-yamo
homologs from rice pathogens indicate that maintenance of the putative zinc protease motif is
essential. The temporal expression of the AVR2-YAMO gene is being studied by fusion of the AVR2-YAMO promoter to both the -glucuronidase gene and the green fluorescent protein gene. Initial
experiments suggest AVR2-YAMO is expressed during infection suggesting a role in pathogenicity.
Anti-AVR2-YAMO antisera has been raised in rabbits using a recombinant AVR2-YAMO protein
antigen. It is unknown whether the AVR2-YAMO protein is secreted or remains within the fungus
so both intracellular and secreted proteins are being analyzed. It is hoped to use this polyclonal anti-AVR2-YAMO antisera to localize expression.
88. Characterization of a new colonial mutant of Neurospora crassa .
Katrina D. Campsall, Paul Sallmen, and P. J. Vierula, Department of Biology, Carleton University, Ottawa, Ontario. KIS 586.
Sbr (small brown) is a new colonial mutant of Neurospora crassa which was recovered in a screen
of transformants harbouring ectopic integrations of a hygromycin phosphotransferase (Hygr) gene
construct. Insertional inactivation by a single copy of this drug resistance marker appears to be
responsible for the mutant morphology. The sbr strain forms slow-growing compact colonies which
become dark brown with age. Instead of elongate tubular hyphae, sbr grows by forming spherical
buds. Each of these spherical compartments range from approximately 3 um to 20 um in diameter
and are densely packed with nuclei. Immunofluorescence microscopy with an anti-tubulin monoclonal
antibody revealed a scattered arrangement of microtubules. We have cloned sbr genomic DNA into
a Lambda bacteriophage vector and have identified several putative clones from this genomic library
that carry the Hygr gene construct. Sbr DNA flanking the marker has been further subcioned into a
Bluescript plasmid. The DNA sequence is currently being analyzed.
89. Chitin biosynthesis in the phytopathogenes Magnaporthe grisea and Botrytis cinerea.
Florence Chappeland, Anne Vidal-Cros, Martine Boccara. Universite Pierre et Marie Curie, Paris, FRANCE
Two different approaches were used to study the relationshipbetween chitin synthase expression and virulence in the two filamentous phytopathogenes: M. grisea and B. cinerea. In the first approach, chitin synthase genes (chs or csd) fragments were amplified by PCR in M. grisea and B. cinerea. Amplified fragments corresponding to four different genes in M. grisea and three genes in B.cinerea. were characterized. The importance of each chs or csd gene during the vegetative cycle as well as during the infectious process is studied through transcription analysis and construction of disrupted mutants. In a second approach, a B. cinerea mutant affected in chitin biosynthesis was isolated by selection against Calcofluor. This mutant, which grows normally in culture, exhibits decreased virulence on grape and produces four times more elicitors of plant defence reactions than wild type strain. Preliminary biochemical characterization is in favor of a Csd3 phenotype(regulation of chitin synthase 3). The results of these two appoaches will be presented and discussed.
90. The Ustilago maydis ubc1 gene is required for gall formation in maize.
Scott E. Gold1, Steven Brogdon1, James W. Kronstad2, Franz Durrenberger2 and Maria E. Mayorga1. 1Department of Plant Pathology, University of Georgia, Athens, GA 30602-7274; 2Biotechnology Laboratory, University of British Columbia, 6174 University Boulevard, Vancouver, B.C. V6T 1Z3, Canada.
Ustilago maydis, the causal agent of corn smut disease, is a semi-obligate plant pathogen. The
haploid grows as saprophytic budding yeast cells on culture medium while the dikaryon formed upon
mating of compatible haploid strains is filamentous and obligately pathogenic. We are interested in
understanding the underlying genetic mechanisms involved in the dimorphic transition and their
impact on pathogenesis. Specifically, we have focused on genes, which when mutated, affect the
ability of the fungus to express the filamentous phenotype. We employ, as our starting material,
haploid strains disrupted in the adenylate cyclase gene (uac1) and therefore express a consitutively
filamentous phenotype. Matings of two uac1 mutant strains were previously shown to be unable to
generate pathogenic dikaryons. Ultraviolet light induced and spontaneous second site suppressor
mutants that have yeast colony morphology on solid media have been generated in a uac1 disruption
background. Earlier, we complemented one of these mutants and found that the corrective gene
encoded the regulatory subunit of cAMP dependent protein kinase (ubc1). We have analyzed the
dramatically reduced virulence of ubc1 mutants. These mutants colonize the plant causing chlorotic
symptoms but are unable to induce tumor formation in the host. The results taken together with past
analysis of the uac1 pathogenicity, suggest that the regulation of the cAMP pathway is critical for
normal disease development. Our interpretation is that cyclic AMP dependent protein kinase activity
is necessary during initial infection but should be reduced or eliminated for progression to gall
formation.
91. Gene expression of the blackleg fungus Leptosphaeria maculans in the presence of alkenyl glucosinolates.
Adrienne Sexton and Barbara Howlett, Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville 3052 VIC. Australia.
Blackleg caused by the ascomycete Leptosphaeria maculans is the most economically important disease of oilseed Brassicas worldwide. Generally Indian mustard (B. juncea) is resistant to the blackleg fungus and consequently has been used as a source of resistance in canola (B. napus) breeding strategies. Indian mustard has high levels of alkenyl glucosinolates, sulphur-containing compounds that give mustard a pungent taste. Upon wounding of the plant, these compounds are cleaved by the plant enzyme myrosinase into glucose and gases such as isothiocyanates, which are toxic to many organisms including L. maculans.
Recently blackleg isolates that can attack some Indian mustard varieties have been found in Australia.
These isolates may be able to tolerate or detoxify hydrolysis products of glucosinolates in the plant.
We are using differential display to identify genes expressed by L. maculans isolates in the presence
of glucosinolates. Such genes may be involved in detoxification of glucosinolates or evasion of their
effects. A 1 kb mRNA is expressed only in the presence of 2-propenyl glucosinolate (5 and 10
microgram/ml) and myrosinase (5 microgram/ml). This message has a high degree of sequence
similarity to a yeast 5S ribosomal DNA binding protein, which is transcribed at high levels as a
response to growth in high concentrations of glucose. Other differentially expressed genes of L.
maculans are being sought.
92. Genetic analysis of the dimorphic transition in Ustilago maydis.
Maria E. Mayorga and Scott E. Gold. Department of Plant Pathology, University of Georgia, Athens, GA 30602-7274
We are interested in understanding the genetic and biochemical mechanisms controlling fungal
dimorphism. Ustilago maydis, the causal agent of corn smut disease, alternates between a unicellular,
non-pathogenic yeast-like form and a dikaryotic, filamentous, pathogenic form. Because of well
developed molecular genetic techniques, Ustilago can be used as a model fungal system to study the
process of yeast-hypha transition and its importance in pathogenicity. Previously we obtained a
mutant strain that exhibits constitutively filamentous growth in its haploid form. Complementation
of this mutant led to the isolation of the gene encoding adenylate cyclase, uac1. Additional
mutagenesis of this constitutive filamentous strain allowed the isolation of a large number of
suppressor mutants lacking the filamentous phenotype, termed ubc, for Ustilago bypass of cyclase.
Analysis of one of these supressor mutants led to the identification of ubc1, encoding the regulatory
subunit of cAMP-dependent protein kinase. These genes are important not only for dimorphic
switching but also in pathogenicity: mutations in uac1 and ubc1 appear to affect tumor formation in
the plant. We are currently analyzing additional suppressor mutations in order to identify new genes
in the cAMP signal transduction pathway involved in dimorphic growth in U. maydis. Microscopic
analysis is being used to determine the effect of the mutations on cell growth and morphology.
Characterization of some ubc class of suppressor mutants is described.
93. Insertional Mutagenesis, cloning and characterization of the col 1 gene of the dimorphic fungal pathogen Ophiostoma ulmi.
V. Pereira, J.C. Royer, W.E. Hinz, C. Bowden, K. Kokurewicz, D. Field, M.Hubbes and P.A. Horgen, University of Toronto, Canada.
A colony mutant col1, of Ophiostoma ulmi was produced by insertional mutagenesis, which has a
dramatically reduced mycelial growth rate and a near-normal budding cell growth rate. Genomic
DNA flanking the inserted transforming DNA was recovered and used to isolate the uninterrupted
DNA from a wild-type genomic library. The uninterrupted gene was isolated, and the sequence
determined. The isolated col 1 gene when transformed back into the yeast-like mutant restored the
filamentous growth pattern. Analyses and comparison of the translated nucleotide sequence would
suggest that the unique COL 1 protein shows some similarity to RNA and poly (A) binding proteins.
The 600 amino acids at the carboxy terminus of the COL 1 protein align very well with the PRP24
(spliceosomal) protein of Saccharomyces cerevisiae. Attempts to complement the col1 mutant with
the Prp24 gene, and to complement the yeast Prp24 mutant with the col 1 gene will be described.
The open reading frame of the col 1 gene was fused into the plasmid vector pJC40 for expression in
Escherichia coli. A histidine-tagged protein was purified by affininty chromatography. This protein
was used to prepare polyclonal antibodies against the COL 1 protein.
94. Cloning a gene which encodes a glycoprotein present at the fungal-plant interface formed in the Colletotrichum-bean interaction.
Sarah Perfect1, J. Green1, R.O'Connell2. 1University of Birmingham, UK., 2IACR-Long Ashton, UK.
Colletotrichum is a large genus of plant pathogenic fungi causing anthracnose on a wide range of crops. C. lindemuthianum is a hemibiotrophic species which causes anthracnose of bean, Phaseolus vulgaris. During the inital biotrophic stage of infection, the fungus differentiates infection vesicles and primary hyphae within host epidermal cells. These specialised intracellular hyphae invaginate the host plasma membrane, from which they are separated by a matrix layer. Monoclonal antibodies (MAbs) raised to isolated infection structures have been used to identify proteins present at the fungal-plant interface.
One of these MAbs, designated UB25, recognises a protein epitope in a 40kDa N-linked glycoprotein specific to intracellular hyphae. Indirect immunofluorescence and EM-immunogold labelling show that the glycoprotein is present in the infection peg, and the fungal walls and matrix surrounding the intracellular hyphae. However, it is not present in secondary necrotrophic hyphae, which suggests that it is specific to biotrophic infection structures. The glycoprotein may therefore be involved in the establishment and maintenance of biotrophy.
A cDNA library has been constructed from total RNA isolated from infected bean hypocotyl
epidermis. The MAb UB25 has been used to immunoscreen the library and positive clones have been
isolated and sequenced. Analysis of the deduced amino acid sequence revealed the presence of two
distinct domains, one of which is proline rich and contains short repetitive motifs. The functional
significance of this will be discussed. In addition, Southern analysis indicates that the glycoprotein
recognised by UB25 is fungally encoded and is present in several Colletotrichum species.
95. Genetic and molecular evidence for regulation of genes involved in HC-toxin biosynthesis in Cochliobolus carbonum
race 1.
John W. Pitkin, Anastasia N. Nikolskaya, Joong-Hoon Ahn and Jonathan D. Walton. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824.
The Tox2 chromosome of C. carbonum race 1 (Tox2+) contains race 1-unique genes required for
HC-toxin biosynthesis and efflux. Crosses between different race 1 field isolates and between race
1 and race 2 (Tox2-) isolates occasionally result in progeny with an altered virulence phenotype on
maize. These strains have a "slow" virulence phenotype on maize due to a severely reduced ability
to produce HC-toxin. Analyses of the progeny indicate that the new pathogenicity phenotype may
be due to the loss of a gene responsible for the activation of at least two race 1-specific genes: HTS1,
which encodes HTS, a cyclic peptide synthetase required for HC-toxin biosynthesis, and TOXA,
which encodes a putative HC-toxin efflux pump. Comparisons of Tox2 chromosomal structure from
various cross progeny has led to the preliminary identification of a 20-100 kb region as the site of the
activator gene. We also have cloned a race 1-specific gene, TOXE, with sequence similarity to known
fungal activator genes. Strains with TOXE gene disruptions have a Tox2- virulence phenotype on
maize. Unlike strains with the slow virulence phenotype, strains with TOXE disrupted have wild-type
HTS activity yet make no detectable HC-toxin. The TOXE gene product may control race 1-specific
genes other than HTS1 and TOXA, such as those required for the synthesis of AEO, a novel amino
acid in HC-toxin. The TOXE gene is present in the slow virulence progeny, thus there is evidence for
at least two race 1-specific regulatory genes that affect HC-toxin biosynthesis.
96. Cytokines produced in mice infected by a virulent Paracoccidioides brasiliensis.
Januario,A.; Pietro,R.C.L.R.; Rodrigues,E.L.L. and Silva,C.L.. Departament of Parasitology, Microbiology and Immunology. School of Medicine of Ribeirao Preto, University of Sao Paulo.
Paracoccidioides brasiliensis is a pathogenic dimorphic fungus that reaches the lung of host through inhalation of conidia which can be destroyed by the host immune cells or transform in yeasts, that multiply and disseminate to other organs. The clinical symptoms of disease are characterized by a cronic granulomatous reaction, with empairment of the cell mediated immune response and exacerbate humoral immunity. The aim of this study was to asses the production of cytokines during the paracoccidioidomycosis evolution in several organs of P. brasiliensis infected mice by RNAm detection. In the first week of infection, we verified an accented increase in mRNA production for IL-2, IL4, IL-10 and IFN-, comparing with uninfected mice. We observed a decline of all cytokines at the fifth week, but this was always higher than control. In tenth week of infection we obtained an elevation in the levels of EL-10 mRNA, correlated with the number of fungi in organs. Through this study we suggest that occurs the activation of both T CD4 cells populations and possibly interleukin-10 is involved with cellular immunity reduced in paracoccidioidomycosis, by its immunosupressive properties, that favour the fungi multiplication. These results can contribute to a better understanding of immunopathogenesis of paracoccidioidomycosis.
Financial Support: CAPES, CNPQ, FINEP, FAPESP
97. Biochemical and molecular characterization of non-pathogenicity in the Glomerella magna-cucurbit system.
Regina Redman and Rusty Rodriguez, NBSC, Biological Resources Division, United States Geological Survey, Seattle, WA 98115
Biochemical and molecular analyses were performed on watermelon and cucumber plants subjected to four different conditions: 1- water controls, 2- inoculated with conidia from the non-pathogenic mutant (path-1), 3- inoculated with conidia from the wild-type (L2.5), and 4- inoculated under cross protection conditions. Cross protection involved exposure of plants to path-1 conidia for 48 hours followed by exposure to a lethal concentration of L2.5 conidia. The deposition of lignin, peroxidase activity, and specific plant defense related gene expression indicated that the host defense system did not activate for 3 - 4 days after exposure to L2.5. In contrast, little to no defense system activation occurred in water control plants, plants colonized with path-1, or cross protected plants.
The genetic complexity of non-pathogenicity was assessed by the isolation of 20 non-pathogenic
REMI mutants. Although the REMI mutants were capable of protecting plants against wild type
disease, they varied in the efficiency of cross protection. Southern blot analysis of the REMI mutants
indicated that both single and multiple integration events occurred, and that integrations occurred at
several genomic locations. The phenotypic and genotypic variation between REMI mutants suggests
that non-pathogenicity is genetically complex.
98. The production of auxin in transgenic fungi and its effect on fungal virulence and host specificity.
Hagit Levi-Kedmi, Rudi Maor and Amir Sharon, Department of Plant Science, Tel Aviv University, Tel Aviv 69978 Israel.
As natural plant pathogens, fungi hold potential as biocontrol agents of weeds. Highly virulent fungal pathogens may be capable of eliminating large weed populations, while natural host specificity ensures that cultivar plants are left untouched. Most weed pathogens, however, are either not specific or not virulent enough to be effective as biocontrol agents. The insertion and high-level expression of virulence genes in fungi may be an effective means of elevating the virulence of weed-specific pathogens, rendering many weed pathogens better biocontrol agents.
In this study we tested the potential of the plant hormone indole-3-acetic acid (IAA) to enhance
fungal virulence. The fungus Cochliobolus heterostrophus was transformed with the IAA
biosynthesis genes iaaM and iaaH of the bacterium Pseudomonas savastanoi. Transgenic fungal
isolates of opposite mating type containing either iaaM or iaaH were crossed and progeny containing
both genes were obtained. Transformants that produce either indole-3-acetamide (IAM), IAA, or
both were identified and their pathogenicity to host (corn) and non-host (pea) plants was tested.
Isolates that contained both genes and produced IAA showed increased virulence on corn, while IAA
had no effect on the pathogenicity of the fungus to pea plants. These results indicate that fungal
virulence may be enhanced without affecting host specificity. This approach may open new ways for
engineering superior biocontrol agents with the desired level of virulence and host range.
99. HCf-1 and HCf-2: two hydrophobins of Cladosporium fulvum.
Pietro D Spanu, Department of Plant Sciences, University of Oxford, UK
Fungal hydrophobins play a role in some plant-fungus interactions. They have been proposed to aid attachment to the host, to mediate specialised morphogenesis and have toxin activity. Their synthesis is induced during infection in ectomycorrhizal fungi. These proteins appear to be ubiquitous in higher fungi and may have diverse functions in different organisms. They are characterised by eight cysteins at conserved positions and by having similar patterns of hydrophobicity; however sequence homology between different hydrophobins is very low, even from those of the same organism. C. fulvum is a hemibiotrophic pathogen of tomatoes and successful infection relies on the ability of the fungus to avoid perception by the host. Hydrophobins have been proposed to assist in this purpose. In this poster I am testing this hypothesis. Two hydrophobins (HCf-1 and HCf-2) are identified, partially purified, N-term sequenced and cloned. I shall present data on expression in vitro and in planta as well as preliminary data on the phenotype of the mutants which lack either of the two hydrophobins.
100. Protease and carbohydrase strategies of Aspergillus fumigatus, phytopathogenic and entomopathogenic fungi.
Raymond J. St. Leger and Lokesh Joshi. The Boyce Thompson Institute at Cornell University, Tower Road, Ithaca, NY 14853, USA
We compared saprophytes (Neurospora crassa, Aspergillus nidulans), an opportunistic human
pathogen (A. fumigatus), an opportunistic insect pathogen (A. flavus), plant pathogens (Verticillium
albo-atrum, V. dahliae, Cochliobolus victoriae, Colletotrichum spp., Magnaporthe grisea, Nectria
haematococca), a mushroom pathogen (V. fungicola) and entomopathogens (V. lecanii, Nomuraea
rileyi, Beauveria bassiana, Metarhiziwn anisopliae) in their abilities to degrade and utilize host-derived macromolecules (horse lung polymers, porcine mucin, hyaluronic acid, plant cell walls and
insect cuticle).The major class of protease produced in most media by most plant pathogens and N.
rileyi were broad spectrum basic trypsins; analogous peptidases produced by insect pathogens and
Aspergillus spp. were specific for Phe-Val-Arg-X. Most plant pathogens produced additional very
low levels of subtilisin-like proteases. Analogous enzymes comprised the major protease component
secreted by Aspergillus spp., and entomopathogens. This provided them with much greater activity
as cf. plant pathogens against elastin, mucin and insect cuticle. Plant pathogens and Aspergillus spp
also produced high levels of several glycosidic enzymes on mucin and plant cell walls, which contain
inductive carbohydrate substrates. Growth of A. fumigatus on mucin degraded mucin carbohydrates
and mucin proteins by 40% and 75%, respectively. The residual mucin resisted further degradation
because of the inability of the pathogens enzymes to remove bound sialic acid. Although they lack
hyaluronidase, most of the fungi secreted a range of other enzymes on host-derived macromolecules
e.g. phospholipases that are common components of bacteria as well as reptilian and invertebrate
venoms. The wide distribution of these enzymes may help explain the pathogenicity of opportunists
such as A. fumigatus, which are not subject to selection of specific virulence genes.
101. Differential gene expression in galled and asymptomatic tissues of loblolly pine infected with fusiform rust. Jaimie M. Warren, Alan L. Zwart, and Sarah F. Covert. The University of Georgia, Athens.
Cronartium quercuum f. sp. fusiforme is an obligate pathogen of oak (Quercus spp.) and pine (Pinus
spp.). We hypothesize C.q. fusiforme causes gall on infected pine trees by altering the expression of
pine genes that regulate cellular proliferation. The goal of this study is to identify transcripts which
are differentially expressed in asymptomatic and galled tissues of infected pine. We have approached
this problem using RNA fingerprinting, a modification of the differential display technique originally
described by Liang and Pardee in 1992. To date, preliminary screens have identified 30 differentially
expressed cDNAs. Ongoing work focuses on screening for additional cDNAs, verifying differential
expression, and determining genome of origin.
102. Transformation may enhance the virulence of Nectria haematococca on pea.
Catherine C. Wasmann and Hans D. VanEtten. Department of Plant Pathology, University of Arizona, Tucson, Arizona 85721.
Isolates of the fungus Nectria haematococca pathogenic on pea are able to detoxify the phytoalexin
pisatin via a cytochrome P450-mediated demethylation. To examine the role of pisatin demethylating
ability (Pda) in pathogenicity, mutants deficient in Pda were created by transformation-mediated gene
disruption. All eleven of the Pda- transformants were reduced in virulence on pea and a Pda+
transformant used as a control had virulence equivalent to the Pda+ recipients (N. haematococca
isolates 77-13-4, 77-13-5, and 77-13-7). We now have begun to examine the virulence of 120
transformants in which integration of the transforming DNA did not disrupt the PDA gene locus.
Many of these transformants produced lesions which were longer than those produced by the
corresponding recipient isolates 77-13-5 and 77-13-7. In repeat assays of 49 of the putatively
hypervirulent transformants 30% continued to produce lesions that were at least 30% longer than
the wild type isolates. To test whether N. haematococca DNA sequences in the disruption vector
pKOI had an impact on the transformants virulence, pCWHyg2, which lacks these sequences, was
used to transform 77-13-7. In repeat assays some of these transformants also consistently produced
lesions that were 30% longer than those made by 77-13-7. The enhanced virulence observed in these
experiments may be isolate-dependent as the virulence of N. haematococca isolate 94-6-1
transformed with pCWHyg2 was the same as untransformed 94-6-1.
103. MAP Kinase Pathways in Magnaporthe grisea.
Jin-Rong Xu and John E. Hamer, Purdue University, West Lafayette, IN 47907.
Many plant pathogenic fungi, including Magnaporthe grisea -the causative agent of the rice blast disease, develop specialized structures to invade their hosts and undergo dramatical morphological changes to grow invasively in plants. Our research objective is to study genetic mechanisms regulating this plant infection-related morphogenesis. We have isolated a MAP kinase PMK1 (Pathogenicity MAP Kinase 1) from M. grisea which is essential for appressorium formation and invasive growth in plants. Further characterization of the PMK1 MAP kinase pathway is in progress and will be presented. In addition to PMK1, we isolated two other M. grisea MAP kinases. PMK2 is 83% similar to S. cerevisiae HOGl gene, and 93% similar to S. pombe styl gene. PMK3 is 85% similar to S. cerevisiae SLT2 MAP kinase gene. Five PMK3 gene disruption mutants were isolated. pmk3 mutants are nonpathogenic on rice plants, but make melanized appressoria on Teflon membranes or slideglass with the addition of 10 mM cAMP. On onion epidermis, pmk3 appressoria fail to penetrate, but elicit autofluorescence and papilla formation in onion epidermal cells. pmk3 mutants are also dramatically reduced in conidiation, however, there is no obvious growth defect as measured by colony diameter on a variety of media. pmk3 mutants are not temperature sensitive or infertile. It appears that PMK3 regulates infection processes downstream of PMK1, and may play important roles in penetration, invasive hyphae differentiation and conidiation.
104. Comparative genetics of polyketide toxin production by Mycosphaerella zeae-maydis and Cochliobolus heterostrophus.
Sung-Hwan Yun, B.G. Turgeon, and O.C. Yoder. Department of Plant Pathology, Cornell University, Ithaca, NY.
M. zeae-maydis and C. heterostrophus race T are not closely related, however both appeared suddenly in the field in 1970, both are highly virulent to corn containing Texas male sterile cytoplasm (T) and both produce linear polyketides (PM- and T-toxin, respectively) which have similar structures and specifically affect T-corn. Molecular genetic analysis has shown that production of T-toxin requires a polyketide synthase (ChPKS1) and a decarboxylase (DECl). Genes encoding these enzymes are on different chromosomes and ChPKS1 is flanked by A+T rich, repeated, noncoding DNA. To compare biosynthesis of PM-toxin with that of T-toxin, a PM-toxin-encoding gene (MzmPKS1) was cloned by PCR and compared to ChPKS1. The genes are similar in that six enzymatic domains and four introns are conserved. They differ in that they share only 62 % amino acid identity and there is no A+T rich DNA flanking MzmPKS1. Moreover, an additional ORF (RED l) located 5' of MzmPKSl is similar to reductases associated with bacterial and plant PKSs. These differences suggest that the two fungi did not appear suddenly as a result of acquiring the same gene(s) for polyketide production. Further, a gene cluster may be involved in PM-toxin biosynthesis. Targeted disruption of either MzmPKS1 or RED1 causes loss of ability to produce PM-toxin.