Identification And Functional Analysis Of Pathogencity-Related Genes GCN5 And MoNMD5 In Magnaporthe Oryzae

Rice(Oryza sativa L.)is the most widely consumed food crop.Rice blast,caused by the filamentous ascomycete fungus Magnaporthe oryzae,is one of the most destructive diseases in rice and severely affects crop stability and sustainability worldwide.Therfore,to better control the rice-blast,we need to understand the molecular basis of M.oryzae pathogenicity in depth,which would provide a theoretical basis to develop a novel disease control strategy.In eukaryotes,autophagy is a highly conserved catabolic process for vacuolar degradation of proteins,membranes and organelles,which is important for life activity.Previous study reported that light can induce expression of the GCN5 in M.oryzae.In current study,to investigate the function of GCN5,we generated the GCN5 deletion(gcn5(35))mutants and GCN5 overexpressed(GCN5OX)strains.Based on epifluorescence microscopy,western blotting as well as biological function of GCN5,we found that autophagy is induced during dark or when cultured on rich(CM)medium condition in the gcn5(35)mutants.We also observed that condiation is increased while pathogenicity is decreased in the gcn5(35)mutants.However,induction of autophagy is repressed in light or nitrogen starvation condition,and conidiation is decreased the GCN5 OX strains.Pathogenicity is similar with WT in the GCN5 OX strains.These results suggest that Gcn5 negatively regulates autophagy and M.oryzae conidiation.We compared the acetylated proteins differentially present in gcn5(35)vs GCN5 OX mutant by LC-MS/MS.We identified differentially acetylated protein Atg7 that is autophagy-related.Further study indicated that acetylation of Atg7 at K338 might be specifically catalyzed by Gcn5 in the dark or rich medium,repressing induction of autophagy.Taken together,we propose that light triggered translocation of Gcn5 from cytosol to nucleus,and meanwhile de-repressed autophagy by removing the Gcn5-catalyzed acetylation on Atg7,to promote conidiation(asexual reproduction)in the rice-blast fungus.Additionally,to explore the mechanism of transducing environmental signals or macromolecules to nucleus,we identified an ortholog of β-importin Nmd5 in M.oryzae,which is responsible for transducing signals or macromolecular to nuclear.We found that deletion of Mo NMD5 results in decreased growth rate,less pigmentation and defects in formation of appressuorium-like structure,and loss of pathogenicity.We identified potential Mo Nmd5-interacting proteins by LC-MS/MS,and chose Mo Cks1,the cyclin dependent kinase subunit as a downstream component of MAPK(mitogen-activated protein kinase)cascade,for further verification.We generated strains expressing Mo Cks1-GFP in WT and monmd5(35)background.Our result showed that Mo Cks1-GFP localized in both cytosol and nuclei in the WT,while it localized in cytosol.We performed a yeast-two-hybrid assay and co-immunoprecipitation to verify the direct interaction of Mo Nmd5 with Mo Cks1.In summary,we found that acetylation of Atg7 might be specifically catalyzed by Gcn5 in the dark,repressing induction of autophagy.Light induces autophagy by triggering nuclear translocation of Gcn5 protein and thus releases Atg7 acetylation to promote M.oryzae conidiation.Additionally,Mo Nmd5 mediates nuclear import of Mo Cks1 to regulate development process and pathogenicity in M.oryzae.