Forrest G. Chumley

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.

A Mechanism for Surface Attachment in Spores of a Plant Pathogenic Fungus

Rice blast disease is caused by a fungus that attacks all above-ground parts of the rice plant. In a study of the means by which the fungus attaches to the hydrophobic rice leaf surface, it was found that spores(conidia) of the rice blast fungus Magnaporthe grisea have a mechanism for immediate and persistent attachment to various surfaces, including Teflon. This attachment occurs at the spore apex and is blocked by the addition of the lectin concanavalin A. Microscopy of hydrated conidia shows that a spore tip mucilage that binds concanavalin A is expelled specifically from the conidial apex before germ tube emergence. Ultrastructural analysis of dry conidia shows a large periplasmic deposit, presumably spore tip mucilage, at the apex. The results indicate a novel mechanism for the attachment of phytopathogenic fungal spores to a plant surface.

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.

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.

Disruption of a Magnaporthe grisea cutinase gene.

SummaryUsing 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.

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

SummaryA 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.

Genetic fusions that place the lactose genes under histidine operon control

Abstract The genes of the Salmonella histidine operon ( his ) have been placed on an F′ pro lac plasmid using genetic methods that rely on recombinational homology provided by Tn10 transposon insertions. The position and orientation of the transposed his genes permit subsequent deletion mutations to form operon fusions that put the lac genes under his operon control. Strains carrying such fusions show co-ordinate regulation of histidinol dehydrogenase and beta-galactosidase expression. While all of the operon fusions have an intact hisD gene, complementation testing and deletion mapping reveal that the genes downstream of hisD are deleted to varying extents. The beta-galactosidase produced by these operon fusions is itself a fused protein containing the amino terminus of one or another of the his enzymes. Two of the operon fusions having join-points in the hisB gene retain histidinol phosphate phosphatase activity and may produce a bifunctional protein having beta-galactosidase as well as the phosphatase activity. The methods that have been used to isolate these his-lac fusions should be applicable to other genetic systems.

Genes for Cultivar Specificity in the Rice Blast Fungus, Magnaporthe Grisea

Field isolates of the heterothallic Ascomycete, Magnaporthe grisea Barr (anamorph, Pyrlcularia oryzae Cav. or P. grisea), include pathogens of many grasses. Individual isolates, however, have a limited host range, parasitizing one or a few grass species (MACKILL and BONMAN 1986). Strains of the fungus that parasitize rice (Oryza sativa) are subdivided into races, depending on the rice cultivars they can successfully infect. The rice blast fungus shows a high degree of variability in the field; new races frequently appear with the ability to attack previously resistant rice cultivars (OU 1985).

Repeated DNA Sequences and the Analysis of Host Specificity in the Rice Blast Fungus

Field isolates of the heterothallic Ascomycete, Magnaporthe grisea Barr (anamorph, Pyricularia oryzae Cav. or P. grisea), include pathogens of many grasses, with rice (Oryza sativa) being the most important example from an economic perspective. Individual field isolates have a limited host range, parasitizing one or a few grass species (MACKILL and BONMAN 1986). We have recently shown that rice pathogens from around the world contain a family of repeated DNA sequences, MGR sequences, that appear to be absent from or present in low copy number in field isolates that infect grasses other than rice (HAMER et al. 1989a). We have hypothesized that the correlation between MGR sequence conservation and rice pathogenicity is due to genetic isolation and independent evolution of rice pathogens descended from a small ancestral population. As we will discuss, MGR sequences have become useful as tools for strain identification and as genetic markers for cloning host specificity genes of interest.

Strategies for Characterizing and Cloning Host Specificity Genes in Magnaporthe grisea, the Rice Blast Fungus

Although field isolates of the heterothallic Ascomycete, Magnaporthe grisea Barr (anamorph, Pyricularia oryzae Cav. or P. grisea), include pathogens of many grasses, individual isolates have a limited host range, parasitizing one or a few grass species (Mackill et al., 1986). Strains of the fungus that parasitize rice (Oryza sativa) are subdivided into races, depending on the rice cultivars they can successfully infect. The rice blast fungus shows a high degree of variability in the field; new races frequently appear with the ability to attack previously resistant rice cultivars (Ou, 1985).