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We examined the genomes of 100 isolates of Magnaporthe oryzae (Pyricularia oryzae), the causal agent of rice blast disease. We grouped current field populations of M. oryzae into three major globally distributed groups. A genetically diverse group, clade 1, which may represent a group of closely related lineages, contains isolates of both mating types. Two well-separated clades, clades 2 and 3, appear to have arisen as clonal lineages distinct from the genetically diverse clade. Examination of genes involved in mating pathways identified clade-specific diversification of several genes with orthologs involved in mating behavior in other fungi. All isolates within each clonal lineage are of the same mating type. Clade 2 is distinguished by a unique deletion allele of a gene encoding a small cysteine-rich protein that we determined to be a virulence factor. Clade 3 isolates have a small deletion within the MFA2 pheromone precursor gene, and this allele is shared with an unusual group of isolates we placed within clade 1 that contain AVR1-CO39 alleles. These markers could be used for rapid screening of isolates and suggest specific events in evolution that shaped these populations. Our findings are consistent with the view that M. oryzae populations in Asia generate diversity through recombination and may have served as the source of the clades 2 and 3 isolates that comprise a large fraction of the global population.

One major threat to global food security that requires immediate attention, is the increasing incidence of host shift and host expansion in growing number of pathogenic fungi and emergence of new pathogens. The threat is more alarming because, yield quality and quantity improvement efforts are encouraging the cultivation of uniform plants with low genetic diversity that are increasingly susceptible to emerging pathogens. However, the influence of host genome differentiation on pathogen genome differentiation and its contribution to emergence and adaptability is still obscure. Here, we compared genome sequence of 6 isolates of Magnaporthe species obtained from three different host plants. We demonstrated the evolutionary relationship between Magnaporthe species and the influence of host differentiation on pathogens. Phylogenetic analysis showed that evolution of pathogen directly corresponds with host divergence, suggesting that host-pathogen interaction has led to co-evolution. Furthermore, we identified an asymmetric selection pressure on Magnaporthe species. Oryza sativa-infecting isolates showed higher directional selection from host and subsequently tends to lower the genetic diversity in its genome. We concluded that, frequent gene loss or gain, new transposon acquisition and sequence divergence are host adaptability mechanisms for Magnaporthe species, and this coevolution processes is greatly driven by directional selection from host plants.

Sec4/Rab8 is one of the well-studied members of the Rab GTPase family, previous studies have shown that Sec4/Rab8 crucially promotes the pathogenesis of phytopathogens, but the upstream regulators of Rab8 are still unknown. Here, we have identified two Sec2 homologues FgSec2A and FgSec2B in devastating fungal pathogen Fusarium graminearum and investigated their functions and interactions with FgRab8 by live-cell imaging, genetic and functional analyses. Yeast two-hybrid assay shows that FgSec2A specifically interacts with FgRab8DN(N123I) and itself. Importantly, FgSec2A is required for growth, conidiation, DON production and virulence of F. graminearum. Live-cell imaging shows that FgSec2A and FgSec2B are both localized to the tip region of hyphae and conidia. Both N-terminal region and Sec2 domain of FgSec2A are essential for its function, but not for localization, whereas the C-terminal region is important for its polarized localization. Furthermore, constitutively active FgRab8CA(Q69L) partially rescues the defects of ΔFgsec2A. Consistently, FgSec2A is required for the polarized localization of FgRab8. Finally, FgSec2A and FgSec2B show partial functions, but FgSec2A does not interact and co-localize with FgSec2B. Taken together, these results indicate that FgSec2A acts as a FgRab8 guanine nucleotide exchange factor and is necessary for polarized growth, DON production and pathogenicity in F. graminearum.

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The oxidative degradation of lipids through lipid peroxidation processes results in the generation of free fatty acid radicals. These free radicals including reactive oxygen species (ROS) serve as a substrate for generating reactive aldehydes. The accumulation of free fatty acid radicals, ROS, and reactive aldehydes in cell compartments beyond physiological threshold levels tends to exert a damaging effect on proximal membranes and distal tissues. Living organisms deploy a wide array of efficient enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and aldehyde dehydrogenases (ALDHs) for scavenging reactive molecules and intermediates produced from membrane lipid peroxidation events. Although the contributions of SOD, CAT, and POD to the pathogenesis of microbial plant pathogens are well known, the influence of ALDH genes on the morphological and infectious development of plant pathogenic microbes is not well understood. In this study, we deployed RNA interference (RNAi) techniques and successfully silenced two putative family-four aldehyde dehydrogenase genes potassium-activated aldehyde dehydrogenase (MoKDCDH) and delta-1-pyrrorine-5-carboxylate dehydrogenase (MoP5CDH) in the rice blast pathogen Magnaporthe oryzae. The results obtained from the phenotypic analysis of individual knock-down strains showed that the RNAi-mediated inactivation of MoKDCDH and MoP5CDH triggered a significant reduction in conidiogenesis and vegetative growth of ΔMokdcdh and ΔMop5cdh strains. We further observed that downregulating the expression of MoKDCDH and MoP5CDH severely compromised the pathogenesis of the rice blast fungus. Also, the disruption of MoKDCDH and MoP5CDH M. oryzae undermined membrane integrity and rendered the mutant strains highly sensitive to membrane stress inducing osmolytes. However, the MoKDCDH and MoP5CDH knock-down strains generated in this study displayed unaltered cell wall integrity and thus suggested that family-four ALDHs play a dispensable role in enforcing cell wall-directed stress tolerance in M. oryzae. From these results, we deduced that family-four ALDHs play a conserved role in fostering membrane integrity in M. oryzae possibly by scavenging reactive aldehydes, fatty acid radicals, and other alcohol derivatives. The observation that downregulating the expression activities of MoKDCDH had a lethal effect on potential mutants further emphasized the need for comprehensive and holistic evaluation of the numerous ALDHs amassed by the rice blast fungus for their possible engagement as suitable targets as antiblast agents.

Fusarium graminearum is a fungal pathogen that causes Fusarium head blight (FHB) in wheat and barley. Autophagy is a highly conserved vacuolar degradation pathway essential for cellular homeostasis in which Atg9 serves as a multispanning membrane protein important for generating membranes for the formation of phagophore assembly site. However, the mechanism of autophagy or autophagosome formation in phytopathogens awaits further clarifications. In this study, we identified and characterized the Atg9 homolog (FgAtg9) in F. graminearum by live cell imaging, biochemical and genetic analyses. We find that GFP-FgAtg9 localizes to late endosomes and trans-Golgi network under both nutrient-rich and nitrogen starvation conditions and also show its dynamic actin-dependent trafficking in the cell. Further targeted gene deletion of FgATG9 demonstrates that it is important for growth, aerial hyphae development, and pathogenicity in F. graminearum. Furthermore, the deletion mutant (ΔFgatg9) shows severe defects in autophagy and lipid metabolism in response to carbon starvation. Interestingly, small GTPase FgRab7 is found to be required for the dynamic trafficking of FgAtg9, and co-immunoprecipitation (Co-IP) assays show that FgAtg9 associates with FgRab7 in vivo. Finally, heterologous complementation assay shows that Atg9 is functionally conserved in F. graminearum and Magnaporthe oryzae. Taken together, we conclude that FgAtg9 is essential for autophagy-dependent development and pathogenicity of F. graminearum, which may be regulated by the small GTPase FgRab7.

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