Barbara Valent

Direct interaction of resistance gene and avirulence gene products confers rice blast resistance.

Rice expressing the Pi‐ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR‐Pita in a gene‐for‐gene relationship. Pi‐ta encodes a putative cytoplasmic receptor with a centrally localized nucleotide‐binding site and leucine‐rich domain (LRD) at the C‐terminus. AVR‐Pita is predicted to encode a metalloprotease with an N‐terminal secretory signal and pro‐protein sequences. AVR‐Pita176 lacks the secretory and pro‐protein sequences. We report here that transient expression of AVR‐Pita176 inside plant cells results in a Pi‐ta‐dependent resistance response. AVR‐Pita176 protein is shown to bind specifically to the LRD of the Pi‐ta protein, both in the yeast two‐hybrid system and in an in vitro binding assay. Single amino acid substitutions in the Pi‐ta LRD or in the AVR‐Pita176 protease motif that result in loss of resistance in the plant also disrupt the physical interaction, both in yeast and in vitro. These data suggest that the AVR‐Pita176 protein binds directly to the Pi‐ta LRD region inside the plant cell to initiate a Pi‐ta‐mediated defense response.

tA Single Amino Acid Difference Distinguishes Resistant and Susceptible Alleles of the Rice Blast Resistance Gene Pi-ta

The rice blast resistance (R) gene Pi-ta mediates gene-for-gene resistance against strains of the fungus Magnaporthe grisea that express avirulent alleles of AVR-Pita. Using a map-based cloning strategy, we cloned Pi-ta, which is linked to the centromere of chromosome 12. Pi-ta encodes a predicted 928–amino acid cytoplasmic receptor with a centrally localized nucleotide binding site. A single-copy gene, Pi-ta shows low constitutive expression in both resistant and susceptible rice. Susceptible rice varieties contain pi-ta– alleles encoding predicted proteins that share a single amino acid difference relative to the Pi-ta resistance protein: serine instead of alanine at position 918. Transient expression in rice cells of a Pi-ta+ R gene together with AVR-Pita+ induces a resistance response. No resistance response is induced in transient assays that use a naturally occurring pi-ta– allele differing only by the serine at position 918. Rice varieties reported to have the linked Pi-ta2 gene contain Pi-ta plus at least one other R gene, potentially explaining the broadened resistance spectrum of Pi-ta2 relative to Pi-ta. Molecular cloning of the AVR-Pita and Pi-ta genes will aid in deployment of R genes for effective genetic control of rice blast disease.

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.

Improved Vectors for Selecting Resistance to Hygromycin

Resistance to hygromycin B is an important dominant selectable marker in fungal transformation. Our goal was to improve vectors for hygromycin selection by making the gene more compact, by eliminating sites for commonly used restriction enzymes, and by subcloning the modified gene into convenient vectors. These improvements were made by modifying pCSN43 (Staben et al. 1989 Fungal Genetics Newsl. 36:79-81) through three rounds of megaprimer mutagenesis (Aiyar and Leis, 1993 Biotechniques 14:366-368 ), a technique based on polymerase chain reaction amplification. Plasmid pCSN43 has a 2.4 kb SalI fragment containing the bacterial hph gene (Gritz and Davies, 1983 Gene 25:179-188), encoding hygromycin B phosphotransferase, under control of the Aspergillus nidulans trpC promoter and terminator (Mullaney et al. 1985 MGG 199:37-45) (Fig. 1a).

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.

Roles for Rice Membrane Dynamics and Plasmodesmata during Biotrophic Invasion by the Blast Fungus

Rice blast disease is caused by the hemibiotrophic fungus Magnaporthe oryzae, which invades living plant cells using intracellular invasive hyphae (IH) that grow from one cell to the next. The cellular and molecular processes by which this occurs are not understood. We applied live-cell imaging to characterize the spatial and temporal development of IH and plant responses inside successively invaded rice (Oryza sativa) cells. Loading experiments with the endocytotic tracker FM4-64 showed dynamic plant membranes around IH. IH were sealed in a plant membrane, termed the extra-invasive hyphal membrane (EIHM), which showed multiple connections to peripheral rice cell membranes. The IH switched between pseudohyphal and filamentous growth. Successive cell invasions were biotrophic, although each invaded cell appeared to have lost viability when the fungus moved into adjacent cells. EIHM formed distinct membrane caps at the tips of IH that initially grew in neighboring cells. Time-lapse imaging showed IH scanning plant cell walls before crossing, and transmission electron microscopy showed IH preferentially contacting or crossing cell walls at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Analysis of biotrophic blast invasion will significantly contribute to our understanding of normal plant processes and allow the characterization of secreted fungal effectors that affect these processes.

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 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.

Translocation of Magnaporthe oryzae Effectors into Rice Cells and Their Subsequent Cell-to-Cell Movement

The authors imaged fungal transformants secreting fluorescent effector fusion proteins in first-invaded rice cells. Two effectors that accumulated in biotrophic interfacial complexes were translocated into the invaded cells cytoplasm. Depending on rice cell type and effector size, the translocated effectors moved into adjoining uninvaded rice cells, potentially preparing them for fungal entry. Knowledge remains limited about how fungal pathogens that colonize living plant cells translocate effector proteins inside host cells to regulate cellular processes and neutralize defense responses. To cause the globally important rice blast disease, specialized invasive hyphae (IH) invade successive living rice (Oryza sativa) cells while enclosed in host-derived extrainvasive hyphal membrane. Using live-cell imaging, we identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH. In each newly entered rice cell, effectors were first secreted into BICs at the tips of the initially filamentous hyphae in the cell. These tip BICs were left behind beside the first-differentiated bulbous IH cells as the fungus continued to colonize the host cell. Fluorescence recovery after photobleaching experiments showed that the effector protein PWL2 (for prevents pathogenicity toward weeping lovegrass [Eragrostis curvula]) continued to accumulate in BICs after IH were growing elsewhere. PWL2 and BAS1 (for biotrophy-associated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion. We report robust assays for elucidating the molecular mechanisms that underpin effector secretion into BICs, translocation to the rice cytoplasm, and cell-to-cell movement in rice.