Justice Norvienyeku

Directional Selection from Host Plants Is a Major Force Driving Host Specificity in Magnaporthe Species

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.

Two different subcellular-localized Acetoacetyl-CoA acetyltransferases differentiate diverse functions in Magnaporthe oryzae

The mevalonate pathway is an efficient biosynthesis pathway that yields isoprenoids for promoting different crucial cellular functions, including ergosterol synthesis and growth regulation. Acetoacetyl-CoA acetyltransferase (EC2.3.1.9) is the first major catalytic enzyme constituting the mevalonate pathway and catalyzes the transformation of Acetoacetyl-CoA from two molecules of acetyl-CoA enroute ergosterol production in fungi. We identified two homologous genes encoding Acetoacetyl-CoA acetyltransferase (MoAcat1 and MoAcat2) in Magnaporthe oryzae, the rice blast fungus. Phylogenetic analysis indicates these two genes have different evolutionary history. We subsequently, conducted targeted gene deletion using homologous recombination technology to ascertain the unique roles of the two MoAcat homologues during the fungal morphogenesis and pathogenesis. The findings from our investigations showed that the activity of MoAcat1 promoted virulence in the rice blast fungus as such, the ΔMoacat1 mutants generated exhibited defect in virulence, whilst ΔMoacat1 mutants did not portray growth defects. ΔMoacat2 mutants on the other hand were characterized by reduction in growth and virulence. Furthermore, MoAcat1 and MoAcat2 showed different expression patterns and subcellular localizations in M. oryzae. From our investigations we came to the conclusion that, different subcellular localization contributes to the diverse functions of MoAcat1 and MoAcat2, which helps the successful establishment of blast disease by promoting efficient development of cell morphology and effective colonization of host tissue.

WD40-repeat protein MoCreC is essential for carbon repression and is involved in conidiation, growth and pathogenicity of Magnaporthe oryzae

Carbon catabolite repression (CCR) is a common regulatory mechanism used by microorganisms to prioritize use of a preferred carbon source (usually glucose). The CreC WD40-repeat protein is a major component of the CCR pathway in Aspergillus nidulans. To clarify the function of the CreC ortholog from Magnaporthe oryzae in regulating gene expression important for pathogenesis, MoCreC was identified and genetically characterized. The vegetative growth rate of the MoCreC deletion mutant on various carbon sources was reduced. The MoCreC mutant produced fewer conidia and with about 60% of conidia having septation defects. Appressorium formation was impaired in the MoCreC mutant. Although some appressoria of the mutant could penetrate the leaf surface successfully, the efficiency of penetration and invasive growth of infection hyphae was reduced, resulting in attenuated virulence toward host plants. The CCR was defective as the mutant was more sensitive to allyl alcohol in the presence of glucose, and 2-deoxyglucose was unable to fully repress utilization of secondary carbon sources. qRT-PCR results indicated that the genes encoding cell wall degradation enzymes, such as β-glucosidase, feruloyl esterase and exoglucanase, were upregulated in MoCreC mutant. Taken together, we conclude that MoCreC is a major regulator of CCR and plays significant roles in regulating growth, conidiation, and pathogenicity of M. oryzae.

FgNoxR, a regulatory subunit of NADPH oxidases, is required for female fertility and pathogenicity in Fusarium graminearum.

Fusarium graminearum is a filamentous fungal pathogen that causes wheat Fusarium head blight. In this study, we identified FgNoxR, a regulatory subunit of NADPH oxidases (Nox) in F. graminearum, and found that it plays an important role in the pathogenicity of F. graminearum. FgNoxR is localized on punctate structures throughout the cytoplasm in aerial hyphae while these structures tend to accumulate at or near the plasma membrane, septa and hyphal tips in germinated conidia. Deletion of the FgNOXR gene results in reduced conidiation and germination. Importantly, sexual development is totally abolished in the FgNOXR deletion mutant. In addition, the disease lesion of FgNOXR deletion mutant is limited to the inoculated spikelets of wheat heads. Finally, FgNoxR interacts with FgRac1 and FgNoxA, and all three proteins are required for female fertility. Taken together, our data indicate that FgNoxR contributes to conidiation, sexual reproduction and pathogenesis in F. graminearum.

Small GTPase Rab7-mediated FgAtg9 trafficking is essential for autophagy-dependent development and pathogenicity in Fusarium graminearum

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.

A HOPS Protein, MoVps41, Is Crucially Important for Vacuolar Morphogenesis, Vegetative Growth, Reproduction and Virulence in Magnaporthe oryzae

The homotypic fusion and protein sorting protein complex (HOPS) is the first known tether complex identified in the endocytic system that plays a key role in promoting homotypic vacuolar fusion, vacuolar biogenesis and trafficking in a wide range of organisms, including plant and fungi. However, the exact influence of the HOPS complex on growth, reproduction and pathogenicity of the economically destructive rice blast fungus has not been investigated. In this study, we identified M. oryzae vacuolar protein sorting 41 (MoVps41) an accessory subunit of HOPS complex and used targeted gene deletion approach to evaluate its contribution to growth, reproduction and infectious life cycle of the rice blast fungus. Corresponding results obtained from this study showed that MoVps41 is required for optimum vegetative development of M. oryzae and observed that MoVps41 deletion mutant displayed defective vegetative growth. Our investigation further showed that MoVps41 deletion triggered vacuolar fragmentation, compromised membrane integrity and pathogenesis of the ΔMovps41 mutant. Our studies also showed for the first time that MoVps41 plays an essential role in the regulation of sexual and asexual reproduction of M. oryzae. In summary, our study provides insight into how MoVps41 mediated vacuolar fusion and biogenesis influences reproduction, pathogenesis, and vacuolar integrity in M. oryzae and also underscores the need to holistically investigate the HOPS complex in rice blast pathogen.

Comparative genomic analysis revealed rapid differentiation in the pathogenicity-related gene repertoires between Pyricularia oryzae and Pyricularia penniseti isolated from a Pennisetum grass

Backgrounds Pyricularia is a multispecies complex that could infect and cause severe blast disease on diverse hosts, including rice, wheat and many other grasses. Although the genome size of this fungal complex is small [~40 Mbp for Pyricularia oryzae (syn. Magnaporthe oryzae), and ~45 Mbp for P. grisea], the genome plasticity allows the fungus to jump and adapt to new hosts. Therefore, deciphering the genome basis of individual species could facilitate the evolutionary and genetic study of this fungus. However, except for the P. oryzae subgroup, many other species isolated from diverse hosts, such as the Pennisetum grasses, remain largely uncovered genetically. Results Here, we report the genome sequence of a pyriform-shaped fungal strain P. penniseti P1609 isolated from a Pennisetum grass (JUJUNCAO) using PacBio SMRT sequencing technology. We performed a phylogenomic analysis of 28 Magnaporthales species and 5 non-Magnaporthales species and addressed P1609 into a Pyricularia subclade that is distant from P. oryzae. Comparative genomic analysis revealed that the pathogenicity-related gene repertoires were fairly different between P1609 and the P. oryzae strain 70-15, including the cloned avirulence genes, other putative secreted proteins, as well as some other predicted Pathogen-Host Interaction (PHI) genes. Genomic sequence comparison also identified many genomic rearrangements. Conclusion Taken together, our results suggested that the genomic sequence of the P. penniseti P1609 could be a useful resource for the genetic study of the Pennisetum-infecting Pyricularia species.

Population genomic analysis of the rice blast fungus reveals specific events associated with expansion of three main clades

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.

Family-Four Aldehyde Dehydrogenases Play an Indispensable Role in the Pathogenesis of Magnaporthe oryzae

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.

FgSec2A, a guanine nucleotide exchange factor of FgRab8, is important for polarized growth, pathogenicity and deoxynivalenol production in Fusarium graminearum : Sec2A functions as a Rab8 GEF in F. graminearum

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.