Functional analysis of pathogenicity genes in a genomics world.

Genome-wide mutational and expression analyses have been performed in yeast and provide a model for large-scale analysis of gene function in filamentous fungi. The recent completion of the Neurospora crassa genome offers a resource for comparative analysis with plant pathogenic filamentous fungi. These advances have important implications for molecular genetic studies of pathogenicity genes.

A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta.

Genetic mapping showed that the rice blast avirulence gene AVR-Pita is tightly linked to a telomere on chromosome 3 in the plant pathogenic fungus Magnaporthe grisea. AVR-Pita corresponds in gene-for-gene fashion to the disease resistance (R) gene Pi-ta. Analysis of spontaneous avr-pita(-) mutants indicated that the gene is located in a telomeric 6.5-kb BglII restriction fragment. Cloning and DNA sequencing led to the identification of a candidate gene with features typical of metalloproteases. This gene is located entirely within the most distal 1.5 kb of the chromosome. When introduced into virulent rice pathogens, the cloned gene specifically confers avirulence toward rice cultivars that contain Pi-ta. Frequent spontaneous loss of AVR-Pita appears to be the result of its telomeric location. Diverse mutations in AVR-Pita, including point mutations, insertions, and deletions, permit the fungus to avoid triggering resistance responses mediated by Pi-ta. A point mutation in the protease consensus sequence abolishes the AVR-Pita avirulence function.

Magnaporthe grisea pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues.

Mutagenesis of Magnaporthe grisea strain 4091-5-8 led to the identification of PTH11, a pathogenicity gene predicted to encode a novel transmembrane protein. We localized a Pth11-green fluorescent protein fusion to the cell membrane and vacuoles. pth11 mutants of strain 4091-5-8 are nonpathogenic due to a defect in appressorium differentiation. This defect is reminiscent of wild-type strains on poorly inductive surfaces; conidia germinate and undergo early differentiation events, but appressorium maturation is impaired. Functional appressoria are formed by pth11 mutants at 10 to 15% of wild-type frequencies, suggesting that the protein encoded by PTH11 (Pth11p) is not required for appressorium morphogenesis but is involved in host surface recognition. We assayed Pth11p function in multiple M. grisea strains. These experiments indicated that Pth11p can activate appressorium differentiation in response to inductive surface cues and repress differentiation on poorly inductive surfaces and that multiple signaling pathways mediate differentiation. PTH11 genes from diverged M. grisea strains complemented the 4091-5-8 pth11 mutant, indicating functional conservation. Exogenous activation of cellular signaling suppressed pth11 defects. These findings suggest that Pth11p functions at the cell cortex as an upstream effector of appressorium differentiation in response to surface cues.

Magnaporthe grisea pathogenicity genes obtained through insertional mutagenesis.

We have initiated a mutational analysis of pathogenicity in the rice blast fungus, Magnaporthe grisea, in which hygromycin-resistant transformants, most generated by restriction enzyme-mediated integration (REMI), were screened for the ability to infect plants. A rapid primary infection assay facilitated screening of 5,538 transformants. Twenty-seven mutants were obtained that showed a reproducible pathogenicity defect, and 18 of these contained mutations that cosegregated with the hygromycin resistance marker. Analysis of eight mutants has resulted in the cloning of seven PTH genes that play a role in pathogenicity on barley, weeping lovegrass, and rice. Two independent mutants identified the same gene, PTH2, suggesting nonrandom insertion of the transforming DNA. These first 7 cloned PTH genes are described.

The PWL host specificity gene family in the blast fungus Magnaporthe grisea.

The PWL2 gene, isolated from a Magnaporthe grisea rice pathogen, prevents this fungus from infecting a second host grass, weeping lovegrass. We have investigated the distribution of sequences homologous to PWL2 in M. grisea strains isolated from diverse grass species. Multiple PWL2 homologs with varying degrees of sequence homology were identified. The presence of PWL2 homologs does not correlate with an avirulent phenotype on weeping lovegrass in many cases: some strains were fully pathogenic on weeping lovegrass although they carry multiple PWL2 homologs. Three weakly hybridizing PWL2 homologs were cloned and characterized. One of these, the PWL1 gene previously identified by genetic analysis, functioned to prevent infection of weeping lovegrass. Cloned PWL3 and PWL4 genes were nonfunctional, although PWL4 became functional if its expression was driven by either the PWL1 or the PWL2 promoter. The PWL1, PWL2, and PWL3/PWL4 genes map to different genomic locations. The amino acid sequences of the predicted PWL1, PWL3, and PWL4 proteins have 75, 51, and 57% identity, respectively, to the PWL2 protein. Our studies indicate that PWL genes are members of a dynamic, rapidly evolving gene family.

Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus.

Genetic analysis of host specificity in the rice blast fungus (Magnaporthe grisea) identified a single gene, PWL2 (for Pathogenicity toward Weeping Lovegrass), that exerts a major effect on the ability of this fungus to infect weeping lovegrass (Eragrostis curvula). The allele of the PWL2 gene conferring nonpathogenicity was genetically unstable, with the frequent appearance of spontaneous pathogenic mutants. PWL2 was cloned based on its map position. Large deletions detected in pathogenic mutants guided the gene cloning efforts. Transformants harboring the cloned PWL2 gene lost pathogenicity toward weeping lovegrass but remained fully pathogenic toward other host plants. Thus, the PWL2 host species specificity gene has properties analogous to classical avirulence genes, which function to prevent infection of certain cultivars of a particular host species. The PWL2 gene encodes a glycine-rich, hydrophilic protein (16 kD) with a putative secretion signal sequence. The pathogenic allele segregating in the mapping population, pwl2-2, differed from PWL2 by a single base pair substitution that resulted in a loss of function. The PWL2 locus is highly polymorphic among rice pathogens from diverse geographic locations.

Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea.

A gene from Magnaporthe grisea was cloned using a cDNA clone of the Colletotrichum gloeosporioides cutinase gene as a heterologous probe; the nucleotide sequence of a 2 kb DNA segment containing the gene has been determined. DNA hybridization analysis shows that the M. grisea genome contains only one copy of this gene. The predicted polypeptide contains 228 amino acids and is homologous to the three previously characterized cutinases, showing 74% amino acid similarity to the cutinase of C. gloeosporioides. Comparison with previously determined cutinase sequences suggests that the gene contains two introns, 115 and 147 bp in length. The gene is expressed when cutin is the sole carbon source but not when the carbon source is cutin and glucose together or glucose alone. Levels of intracellular and extracellular cutinase activity increase in response to growth in the presence of cutin. The activity level is higher in a transformant containing multiple copies of the cloned gene than in the parent strain. Non-denaturing polyacrylamide gels stained for esterase activity show a single major band among intracellular and extracellular proteins from cutin-grown cultures that is not present among intracellular and extracellular proteins prepared from glucose-grown or carbon-starved cultures. This band stains more intensely in extracts from the multicopy transformant than in extracts from the parent strain. We conclude that the cloned DNA contains a M. grisea gene for cutinase, which we have named CUT1.

Disruption of a Magnaporthe grisea cutinase gene.

Using a one-step strategy to disrupt CUT1, a gene for cutinase, cut1- mutants were generated in two strains of Magnaporthe grisea. One strain, pathogenic on weeping lovegrass and barley and containing the arg3-12 mutation, was transformed with a disruption vector in which the Aspergillus nidulans ArgB+ gene was inserted into CUT1. Prototrophic transformants were screened by Southern hybridization, and 3 of 53 tested contained a disrupted CUT1 gene (cut1::ArgB+). A second strain, pathogenic on rice, was transformed with a disruption vector in which a gene for hyg B resistance was inserted into CUT1. Two of the 57 transformants screened by Southern hybridization contained a disrupted CUT1 gene (cut1::Hyg). CUT1 mRNA was not detectable in transformants that contained a disrupted gene. Transformants with a disrupted CUT1 gene failed to produce a cutin-inducible esterase that is normally detected by activity staining on non-denaturing polyacrylamide gels. Enzyme activity, measured either with tritiated cutin or with p-nitrophenyl butyrate as a substrate, was reduced but not eliminated in strains with a disrupted CUT1 gene. The infection efficiency of the cut1- disruption transformants was equal to that of the parent strains on all three host plants. Lesions produced by these mutants had an appearance and a sporulation rate similar to those produced by the parent strains. We conclude that the M. grisea CUT1 gene is not required for pathogenicity.

Synthesis of the phytoalexin pisatin by a methyltransferase from pea.

Previous labeling studies in vivo suggest that the terminal step of (+)pisatin biosynthesis in Pisum sativum L. is methylation of the phenol (+)6a-hydroxymaackiain (HMK). We have found that extracts from pea seedlings perform this reaction, using S-adenosylmethionine as the methyl donor. The enzyme activity was induced by microbial infection or treatment with CuCl(2), which elicit pisatin synthesis, though some activity was also present in healthy tissues. It has been reported that CuCl(2)-treated pea tissue provided with (-)HMK or (-)maackiain can synthesize (-)pisatin. Our extract showed no methyltransferase activity dependent on either of these substrates. Methylation of (+)maackiain was detectable, but much slower than that of (+)HMK.