Talks: Plant and Animal Fungal Pathogenesis

Cloning of the Candida albicans genes encoding virulence factors is the essential first step in the molecular genetic dissection of their role in pathogenesis

John E. Edwards, Jr. and Brad Spellberg. Harbor-UCLA Medical Center

Adherence and extracellular phospholipase production have been identified as putative virulence factors. To determine the role of these two factors in candidal pathogenicity we have cloned two genes from C. albicans that encode an adhesin and an extracellular phospholipase respectively. Saccharomyces cerevisiae was transformed with genomic library from C. albicans and the transformed cells were screened for adherent and phospholipase producing clones, respectively. Adherent clones of the transformed S. cerevisiae were selected by repetitive passages over human umbilical vein endothelial cells. One clone was identified that exhibited 50-fold greater adherence to endothelial cells compared to control organisms transformed with an empty plasmid. This clone was found to contain candidal gene with a 1737 bp open reading frame. The predicted amino acid sequence had significant homology with both transcription factors and structural genes. Immunoprecipitation of an epitope- tagged fusion protein from this gene identified a 65-67 kd protein. Experiments are in progress to determine whether the gene product localizes to the nucleus or to the cell surface in S. cerevisiae. Also mutants of C. albicans in which both alleles of the adherence gene have been deleted are being constructed. Clones expressing phospholipase were identified on egg yolk agar medium. The phospholipase gene was localized to a 2.5 kb XhoI-PstI fragment. Sequencing of this fragment revealed a single open reading frame 1055 bp in length. The putative protein encoded by the open frame is 352 amino acids in length with a predicted molecular weight of 37,904 Daltons. The predicted protein contains an hydrophobic amino terminus consistent with the secreted nature of the phospholipase. Five N-glycosylation consensus sites are present at the amino acid positions 114, 202, 300, 305, and 334. No amino acid sequence similarity was found with other proteins, including other phospholipases. However, the predicted protein contains the sequence (97)GYSGG(101) which matches the G-X-S-X-G motif characteristic of extracellular lipase from C. cylindracea. This same motif has a significant homology with a lipid-binding site of lipases from human, pig, dog, rat, and mouse. The cloned phospholipase gene was confirmed to be a structural gene and not a regulatory one by gene dosage.

Genetic control of race specificity in Cochliobolus heterostrophus

B.G. Turgeon and O.C. Yoder. Dept. of Plant Pathology, Cornell University, Ithaca, NY 14853 USA

Pathogen populations are inherently unstable, frequently giving rise to races with altered virulence or host specificity. New forms of the distantly related corn pathogens, C. heterostrophus and Mycosphaerella zeae-maydis appeared in 1970, distinguished by their specificity to corn containing Texas male sterile (T) cytoplasm. In both cases high virulence is conditioned by production of similar polyketide toxins which bind to a protein unique to T cytoplasm corn. Thus, race change is marked by production of a distinctive secondary metabolite: T-toxin in C. heterostrophus and PM- toxin in M. zeae-maydis. Our goal is to investigate race changes by comparing the genetic mechanisms underlying toxin production by these fungi. Although it is known that new pathogenic races can result from nucleotide substitutions, complex genome rearrangements can also accompany a change in race. We know that T-toxin production by C. heterostrophus is associated with a reciprocal translocation, a large insertion, and repeated, AT-rich DNA, features not yet detected in M. zeae-maydis. Moreover, at least two unlinked loci are required for T-toxin production by C. heterostrophus; one encodes a polyketide synthase which has no close homolog in M. zeae-maydis, or in nontoxin producing strains of C. heterostrophus. Induced Tox- mutants of both fungi have drastically reduced virulence, indicating that both toxins play prominent roles in pathogenesis.

The search for virulence associated genes in Cryptococcus neoformans

K.J. Kwon-Chung and Yun C. Chang. Clinical Mycology Section, Laboratory of Clinical Investigation, NIAID, NIH, Bethesda, MD. 20892

The search for virulence associated genes in Cryptococcus neoformans began soon after the discovery of the heterothallic life-cycle of the fungus. Since heterothallism provided the means of recombinational analysis, the first trait of C. neoformans that we have focused on was diphenol oxidase activity. The rationale for this approach was that C. neoformans is the only undisputed pathogen within the genus Cryptococcus and the only species among more than 20 members of the genus that expresses diphenol oxidase activity in addition to good growth at 37 C. The Mel+ and Mel- siblings of opposite mating types were crossed and the progeny of Mel+ and Mel- were tested in a mouse model. As we expected, the Mel+ progeny killed mice much faster than Mel- isolates. The enzyme diphenol oxidase was purified and the analysis of the amino acid sequence of the enzyme indicated that it is a laccase. The gene CNLAC1 encoding this laccase in C. neoformans was recently cloned and characterized in our laboratory. Work is underway to molecularly prove that the CNLAC1 gene is associated with virulence in C. neoformans. The second factor we have focused was mating type. Although laboratory analysis shows alpha and a mating types to be alleles, and alpha and a progeny grows equally well, most clinical isolates are of alpha type. We suspected that alpha type may be more virulent for mice. An isogenic set with identical karyotype, DNA finger-print pattern, Mel phenotype and growth characteristics at both 30 and 37 C were constructed and their progeny of alpha and a type were tested for virulence in mice. As expected, the alpha type parent as well as the alpha type progeny killed more mice in a shorter period of time than did a type strains. The gene has been cloned by J. Edman and is being characterized in his laboratory. Although every member of the genus Cryptococcus produces a large extracellular polysaccharide capsule, there is evidence that the capsule of C. neoformans is clearly associated with virulence. We have focused on capsule formation and cloned 2 (CAP59 and CAP64) of the 6 loci proposed to be associated with capsule formation according to classical mutational analysis. Although the biochemical function of these gene products is unknown, both CAP59 and CAP64 genes are found to be essential for the virulence in mice. Work is underway to identify all the genes involved in capsule formation.

Avirulence genes and pathogenicity genes of the tomato pathogen Cladosporium fulvum

Pierre J.G.M. De Wit, M.H.A.J. Joosten, P.J.M.J. Vossen, A.J. Cozijnsen, G. Hon e, M. Kooman- Gersmann, R. Laug, R. Vogelsang, H.W.J. van den Broek* and J.J.M. Vervoort**, Departments of Phytopathology, Genetics* and Biochemistry**, Agricultural University, Wageningen, The Netherlands.

The interaction between C. fulvum and tomato has a gene-for-gene basis. Here we report on the race-specific elicitors encoded by the avirulence genes Avr9 and Avr4. The AVR9 elicitor consists of 28 amino acids and induces a hypersensitive response (HR) in Cf9 tomato genotypes. (1)H NMR studies revealed that the AVR9 elicitor is a compact sandwich-like molecule consisting of 3 antiparallel beta-sheets. By in vitro mutagenesis we have produced mutant peptides with the same, higher or lower HR-inducing activities than the wild type AVR9 elicitor. The (125)I-AVR9 peptide binds to membranes of different tomato genotypes. A positive correlation exists between affinity of (125)I-AVR9 to membranes and HR-inducing activity. However, it is yet unclear whether the cloned Cf9 resistance gene encodes a receptor for the AVR9 elicitor. The mature AVR4 elicitor is a 106 amino acid peptide. All strains avirulent on Cf4 genotypes contain an identical Avr4 gene, while virulent strains contain alleles with single basepair changes in the coding region, or a frameshift mutation (in one case). Peptides encoded by virulent Avr4 alleles appear to be very unstable. Disruption of the putative pathogenicity genes ecp1 and ecp2 affect the sporulation of C. fulvum on susceptible tomato plants.

Saponin detoxification by plant pathogenic fungi

Anne Osbourn, Paul Bowyer, Greg Bryan, Patricia Lunness, Belinda Clarke and Michael Daniels, Sainsbury Laboratory, Norwich, UK.

Saponins occur in many plant species, and because of their antifungal properties they have been implicated as pre-formed determinants of resistance to fungal attack. Some fungi produce enzymes which remove sugars from saponins, to give molecules which are less fungitoxic. Mutants of the cereal-infecting fungus Gaeumannomyces graminis var. avenae which do not produce the saponin glucosyl hydrolase avenacinase can no longer infect the saponin-containing host oats (but are still fully pathogenic to wheat, which does not contain saponins) (see accompanying poster by Bowyer et al). Southern blots using avenacinase cDNA as a probe revealed cross-hybridising DNA sequences in a number of other phytopathogenic fungi, suggesting that enzymes related to avenacinase may be widespread. We have demonstrated for one of these fungi (the tomato pathogen, Septoria lycopersici), that the cross-hybridising DNA in this fungus also encodes a saponin detoxifying enzyme (in this case tomatinase). While avenacinase and tomatinase are clearly related, the relative activities of these enzymes towards avenacin and tomatine reflect the host specificity of the fungi from which they originate. Structure/function analysis of these two highly conserved saponin glucosyl hydrolases should allow us to identify the regions of the enzymes which are important for activity and for substrate specificity, and to design inhibitors of enzyme action which may have significance for crop protection strategies. The occurrence of DNA sequences which hybridise to avenacinase cDNA in genomic DNA of other phytopathogenic fungi suggests that saponin-saponinase combinations may be more important in determining host range than has previously been appreciated.

Genetic improvement and the molecular basis of fungal pathogenesis

Raymond J. St. Leger, Boyce Thompson Institute, Ithaca, NY 14853-1801 USA.

Entomopathogenic fungi represent an untapped reservoir of pesticidal genes for the production of advanced engineered pestcides; an important consideration given that the lack of "useful" pesticidal genes for transfer has been a major constraint in the implementation of biotechnology in crop protection We are assembling a bank of pathogenicity related genes from Metarhizium anisopliae and Beauveria bassiana which could be used to transform other fungi, bacteria, or viruses to create novel combinations of insect specificity, or to produce transgenic plants with improved resistance to insect pests.To perform these studies, specific vectors are being constructed which facilitate strain construction to enhance virulence using constitutive and regulatory promoter regions for expression of homologous and heterologous genes. The potential for this approach has been demonstrated by transferring the gene for the Prl protease from M. anisopliae to Aschersonia aleyrodis, which consequently became a pathogen of late instar whitefly. We have developed a direct strategy for engineering enhanced virulence in M. anisopliae by constitutive expression of some of the many, normally inducible anti-insect proteins. Our initial candidates for this approach have been genes encoding cuticle-degrading enzymes and toxins, since the active agents are encoded by single genes and have been shown to be active in vitro against insects. Constitutive expression of Prl was obtained by transforming M. anisopliae with cDNA for Prl behind the Neurospora crassa cross pathway control promoter. Transgenic strains continued to produce Prl in the haemocoel of Manduca sexta caterpillars following penetration of the cuticle causing extensive melanization in the body cavity and cessation of feeding 30-40 hr earlier than controls infected with wild type. These studies provide the first conclusive demonstration of the utility of heterologous gene expression in molecular analysis of the insect-fungus interaction, and for strain construction of improved mycoinsecticides.

Characterisation of the pathogenicity gene MPG1 from the rice blast fungus Magnaporthe grisea

Nicholas J. Talbot, Michael Kershaw, Nicholas Tongue1, John E. Hamer2, Onno de Vries, Joseph. G.H. Wessels3. 1University of Exeter, Exeter, EX4 4QG, 2UK, Purdue University, West Lafayette, IN 47907, 3University of Groningen, Haren, The Netherlands.

Magnaporthe grisea infects its host by producing a specialized cell known as an appressorium. This cell works by adhering tightly to the leaf surface and generating high internal turgor which is translated into the mechanical force necessary to break the underlying plant cuticle. Recently, we identified a gene known as MPG1 which appears to play an important role in the elaboration of appressoria. The gene was identified as a fungal transcript produced abundantly in planta. Temporal analysis revealed that MPG1 was highly expressed as soon as 18h after inoculation of rice seedlings and was also expressed during disease symptom expression 72-96h later. A directed gene replacement showed that MPG1 is required for efficient appressorial development and mpg1-mutants therefore showed a reduced pathogenicity phenotype. MPG1 appears to encode a hydrophobin-like protein with homology to the Sc3, Sc1 and Sc4 genes from Schizophyllum commune, the rodA gene from Aspergillus nidulans and the eas gene from N. crassa. Consistent with this, M. grisea mpg1 mutants show an 'easily wettable' phenotype showing that cell surface hydrophobicity of aerial hyphae is reduced. Hydrophobins are unusual proteins which are known to be produced during aerial growth of fungi. Hydrophobins appear to undergo self-assembly into high molecular weight amphipathic complexes when they reach interfaces between liquids and air. Such physical characteristics would predict a number of potential roles for MPG1p in appressorial development and pathogenesis. It is, for example, conceivable that MPG1p acts either as a structural component of the appressorium, or as an adhesion protein. Its secretion and potential incorporation into cell wall complexes may therefore be a rate limiting step in the transduction of the inductive signals required for appressorial morphogenesis. In order to test these hypotheses a number of strategies have been adopted to purify and characterise MPG1p and to determine its precise role in the pathogenesis of M. grisea. Progress in these areas will be discussed.

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