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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]

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]

Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae , indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and A vr P iz-t I nteracting P rotein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP -triggered immunity in plants. [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]

To cause rice blast disease, the fungus Magnaporthe oryzae produces biotrophic invasive hyphae that secrete effectors at the host–pathogen interface. Effectors facilitate disease development, but some (avirulence effectors) also trigger the host''s resistance gene-mediated hypersensitive response and block disease. The number of cloned M. oryzae avirulence effector genes has recently doubled, largely based on resequencing with a Japanese field isolate and association of avirulence activity with presence/absence polymorphisms in novel genes for secreted proteins. Effectors secreted by hyphae in rice cells accumulate in biotrophic interfacial complexes, and this property correlates with their translocation across plasma membrane into the rice cytoplasm. Interestingly, the translocated effectors moved into surrounding uninvaded cells, suggesting that effectors prepare host cells before the fungus enters them. [Copyright &y& Elsevier]

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. [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]

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]

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]

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.

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]

Blast disease destroys up to 30% of the rice crop annually and threatens global food security. The blast fungus Magnaporthe oryzae invades plant tissue with hyphae that proliferate and grow from cell to cell, often through pit fields, where plasmodesmata cluster. We showed that chemical genetic inhibition of a single fungal mitogen-activated protein (MAP) kinase, Pmk1, prevents M. oryzae from infecting adjacent plant cells, leaving the fungus trapped within a single plant cell. Pmk1 regulates expression of secreted fungal effector proteins implicated in suppression of host immune defenses, preventing reactive oxygen species generation and excessive callose deposition at plasmodesmata. Furthermore, Pmk1 controls the hyphal constriction required for fungal growth from one rice cell to the neighboring cell, enabling host tissue colonization and blast disease [ABSTRACT FROM AUTHOR]

Wheat blast first emerged in Brazil in the mid-1980s and has recently caused heavy crop losses in Asia. Here we show how this devastating pathogen evolved in Brazil. Genetic analysis of host species determinants in the blast fungus resulted in the cloning of avirulence genes PWT3 and PWT4, whose gene products elicit defense in wheat cultivars containing the corresponding resistance genes Rwt3 and Rwt4. Studies on avirulence and resistance gene distributions, together with historical data on wheat cultivation in Brazil, suggest that wheat blast emerged due to widespread deployment of rwt3 wheat (susceptible to Lolium isolates), followed by the loss of function of PWT3. This implies that the rwt3 wheat served as a springboard for the host jump to common wheat. [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]