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Reports that the rice blast fungus, which has become a model system for studying the aerial attack of fungi on plants, also invades roots, using a typical root-specific pathway. Occurrence of infection when airborne spores land on rice plants; Formation of large, highly pigmented runner hyphae on the root surface when these fungi were introduced to the roots.

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. [ABSTRACT FROM AUTHOR]

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome. [ABSTRACT FROM AUTHOR]

Wheat blast, caused by the Magnaporthe oryzae Triticum pathotype, is an economically important fungal disease of wheat. Wheat blast symptoms are similar to Fusarium head scab and can cause confusion in the field. Currently, no in-field diagnostic exists for M. oryzae Triticum. Loop-mediated isothermal amplification (LAMP) primers were designed to target the PoT2 and MoT3 loci, previously shown to be specific for M. oryzae and M. oryzae Triticum, respectively. Specificity was determined using 158 M. oryzae strains collected from infected wheat and other grasses and representing geographic and temporal variation. Negative controls included 50 Fusarium spp. isolates. Sensitivity was assessed using 10-fold serial dilutions of M. oryzae Triticum gDNA. PoT2- and MoT3-based assays showed high specificity for M. oryzae and M. oryzae Triticum, respectively, and sensitivity to approximately 5 pg of DNA per reaction. PoT2 and MoT3 assays were tested on M. oryzae Triticum-infected wheat seed and spikes and identified M. oryzae and M. oryzae Triticum, respectively, using a field DNA extraction kit and the portable Genie II system. The mitochondrial NADH-dehydrogenase (nad5) gene, an internal control for plant DNA, was multiplexed with PoT2 and MoT3 and showed results comparable with individual assays. These results show applicability for M. oryzae Triticum field surveillance, as well as identifying nonwheat species that may serve as a reservoir or source of inoculum for nearby wheat fields. [ABSTRACT FROM AUTHOR]

Phytopathogenic microorganisms, including the fungal pathogen Magnaporthe oryzae, secrete a myriad of effector proteins to facilitate infection. Utilizing the transient expression of candidate effectors in the leaves of the model plant Nicotiana benthamiana, we identified 11 suppressors of plant cell death (SPD) effectors from M. oryzae that were able to block the host cell death reaction induced by Nep1. Ten of these 11 were also able to suppress BAX-mediated plant cell death. Five of the 11 SPD genes have been identified previously as either essential for the pathogenicity of M. oryzae, secreted into the plant during disease development, or as suppressors or homologues of other characterized suppressors. In addition, of the remaining six, we showed that SPD8 (previously identified as BAS162) was localized to the rice cytoplasm in invaded and surrounding uninvaded cells during biotrophic invasion. Sequence analysis of the 11 SPD genes across 43 re-sequenced M. oryzae genomes revealed that SPD2, SPD4 and SPD7 have nucleotide polymorphisms amongst the isolates. SPD4 exhibited the highest level of nucleotide diversity of any currently known effector from M. oryzae in addition to the presence/absence polymorphisms, suggesting that this gene is potentially undergoing selection to avoid recognition by the host. Taken together, we have identified a series of effectors, some of which were previously unknown or whose function was unknown, that probably act at different stages of the infection process and contribute to the virulence of M. oryzae. [ABSTRACT FROM AUTHOR]

Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation. [ABSTRACT FROM AUTHOR]

Biotrophic invasive hyphae (IH) of the blast fungus Magnaporthe oryzae secrete effectors to alter host defenses and cellular processes as they successively invade living rice (Oryza sativa) cells. However, few blast effectors have been identified. Indeed, understanding fungal and rice genes contributing to biotrophic invasion has been difficult because so few plant cells have encountered IH at the earliest infection stages. We developed a robust procedure for isolating infected-rice sheath RNAs in which ∼20% of the RNA originated from IH in first-invaded cells. We analyzed these IH RNAs relative to control mycelial RNAs using M. oryzae oligoarrays. With a 10-fold differential expression threshold, we identified known effector PWL2 and 58 candidate effectors. Four of these candidates were confirmed to be fungal biotrophy- associated secreted (BAS) proteins. Fluorescently labeled BAS proteins were secreted into rice cells in distinct patterns in compatible, but not in incompatible, interactions. BAS1 and BAS2 proteins preferentially accumulated in biotrophic interfacial complexes along with known avirulence effectors, BAS3 showed additional localization near cell wall crossing points, and BAS4 uniformly outlined growing IH. Analysis of the same infected-tissue RNAs with rice oligoarrays identified putative effector-induced rice susceptibility genes, which are highly enriched for sensor-transduction components rather than typically identified defense response genes. [ABSTRACT FROM AUTHOR]

In planta secretion of fungal pathogen proteins, including effectors destined for the plant cell cytoplasm, is critical for disease progression. However, little is known about the endoplasmic reticulum (ER) secretion mechanisms used by these pathogens. To determine if normal ER function is crucial for fungal pathogenicity, Magnaporthe oryzae genes encoding proteins homologous to yeast Lhs1 p and Kar2p, members of the heat shock protein 70 family in Saccharomyces cerevisiae, were cloned and characterized. Like their yeast counterparts, both LHS1 and KAR2 proteins localized in the ER and functioned in an unfolded protein response (UPR) similar to the yeast UPR. Mutants produced by disruption of LHS1 were viable but showed a defect in the translocation of proteins across the ER membrane and reduced activities of extracellular enzymes. The Δlhs1 mutant was severely impaired not only in conidiation, but also in both penetration and biotrophic invasion in susceptible rice (Oryza sativa) plants. This mutant also had defects in the induction of the Pi-ta resistance gene--mediated hypersensitive response and in the accumulation of fluorescently-labeled secreted effector proteins in biotrophic interfacial complexes. Our results suggest that proper processing of secreted proteins, including effectors, by chaperones in the ER is requisite for successful disease development and for determining host-pathogen compatibility via the gene-for-gene interaction. [ABSTRACT FROM AUTHOR]

Molecular analyses of early disease events require infected plant tissue in which the pathogen is present in high quantities and interacts with the plant in a way found in the field. In this study, a quantitative polymerase chain reaction (Q-PCR) assay was developed to determine an "infection ratio" of fungal to plant cells in infected tissues. This assay was used to evaluate four inoculation methods (spray, mist, dip, and sheath) as well as use of whole plants or excised parts. Fluorescence stereomicroscopy was used to follow individual lesions developing from appressoria to macroscopic symptoms. Disease progression and outcomes were documented from 24 to 96 h postinoculation (hpi), as well as effectiveness of Pi-ta-mediated resistance. Even at 96 hpi, fungus proliferated well ahead of visible plant damage, especially in veins. Developing lesions sometimes were surrounded by greener areas in detached leaves. Spray inoculation was not sufficient for detecting fungal gene expression in planta before 96 h. Alternatively, a leaf sheath assay produced infected tissues containing 10 to 30% fungal DNA by 34 h. Used together, Q-PCR quantification and fluorescence stereomicroscopy will facilitate studies of early plant invasion because infection density and fungal growth stages are directly observed, not assumed from incubation time. [ABSTRACT FROM AUTHOR]

The resistance gene Pi-ta protects rice crops against the fungal pathogen Magnaporthe grisea expressing the avirulence gene AVR-Pita in a gene-for-gene manner. Pi-ta, originally introgressed into japonica rice from indica origin, was previously isolated by positional cloning. In this study, we report the nucleotide sequence of a 5,113-base pair region containing a japonica susceptibility pi-ta allele, which has overall 99.6% nucleotide identity to the indica Pi-ta allele conferring resistance. The intron region shows the levels of sequence diversity that typically differentiate genes from indica and japonica rices, but the other gene regions show less diversity. Sequences of the Pi-ta allele from resistant cultivars Katy and Drew from the southern United States are identical to the resistance Pi-ta sequence. Sequences from susceptible cultivars El Paso 144 and Cica 9 from Latin America define a third susceptibility haplotype. This brings the total number of Pi-ta haplotypes identified to four, including the resistance allele and three susceptibility alleles. The Pi-ta locus shows low levels of DNA polymorphism compared with other analyzed R genes. Understanding the natural diversity at the Pi-ta locus is important for designing specific markers for incorporation of this R gene into ricebreeding programs. [ABSTRACT FROM AUTHOR]

Rice expressing the Pi-ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR-Pita in a gene-for-gent 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 secretary signal and pro-protein sequences. AVR-Pita176 lacks the secretary and proprotein 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. [ABSTRACT FROM AUTHOR]