Functional Analyses Of SNF1Pathway And The Autophagy-related Protein MoAtg14in Magnaporthe Oryzae

Magnaporthe oryzae, a heterothallic ascomycete fungus, is the causal agent of the rice blast disease, which causes substantial losses of cereal production worldwide. The genome sequence availability coupled with the experimental amenability of both M. oryzae and rice makes them an excellent model for the study of fungus-plant interaction. From conidial germination to invasive hyphae differentiation, the early infection stage of M. oryzae is in a nutrient-free environment, which highlights the importance of cellular energy homeostasis. SNF1pathway and autophagic process are well conserved in eukaryotic cells, both of which are crucial to the maintenance of energy balance. Previous studies in phytopathogenic fungi attached little attention to the energy sensor role of SNF1. And due to the high divergence, the function of Atg14, a key player in autophagy, is little known in organisms except yeast and mammals. In this study, based on the targeted-gene knockout strategy and phenotypic analyses, combined with the investigation of fluorescent signals, we identified and characterized three subunits of SNF1complex, two putative SNF1upstream kinases, as well as a novel autophagy-related protein MoAtgl4in the rice blast fungus, to contribute a better understanding of how SNF1pathway and MoAtg14facilitate the pathogenicity of M. oryzae. The main results acquired are listed below:1) SNF1pathway in M. oryzae retains the role of energy sensor via its participation in peroxisomal maintenance and lipid metabolism.Investigation of GFP-PTS1signals revealed that the SNF1pathway mutants developed enlarged peroxisomes in mycelia and the peroxisomal amount in conidia and appressoria was sharply decreased, indicating SNF1pathway is essential for peroxisomal maintenance in M. oryzae. Furthermore, the SNF1mutants performed inability to utilize non-fermentable carbon sources, and the lipid droplets mobilization was much retarded, both of which might result from the insufficient peroxisomal function in SNF1pathway mutants. 2) SNF1pathway is essential for normal appressorial morphogenesis, turgor generation and the pathogenicity of M. oryzae.During appressorial development, lipid reserves in conidia are rapidly transferred to appressoria where they are degraded and supplied as the sources of glycerol and other intermediates important to cellular differentiation. Due to the inability in lipid droplets mobilization and degradation, the interruption of SNF1pathway led to reduced appressorial size, increased appressorial wall porosity, decreased turgor pressure, ultimately the loss of pathogenicity.3) External glucose supplement relieved the defects of mutants impaired in SNF1function.Glucose metabolism can compensate acetyl-CoA and other intermediates via the glycolytic pathway and citric acid cycle independent of peroxisomal function. When supplemented with external glucose to conidial suspensions, the SNF1pathway mutants partially restored the appressorial morphology and virulence, and could form expansive infection hyphae.4) The SNF1complex integrity and upstream kinases are crucial to fungal various developments.Apart from the catalytic subunit MoSnfl previously reported, MoSip2and MoSnf4were additionally identified and confirmed as the β and γ subunits of SNF1complex respectively, based on the phenotypic evidences and their strong interaction with MoSnfl in this study. The null mutants△Mosip2and△Mosnf4performed multiple disorders as△Mosnfl, such as sparse aerial hyphae, poor conidiogenesis, defects in appressorial formation and virulence, suggesting the integral SNF1complex is critical to pathogenesis-ralated developments in M. oryzae. Furthermore, two putative upstream Snfl-activating kinases, MoSakl and MoTos3, were identified and characterized. They played unequal roles in SNF1activation with a clear preference to MoSakl, while MoTos3played a more auxiliary role. Mutant lacking both of them exhibited SNFl--like phenotypes and completely lost virulence, indicating they are crucial to MoSnfl activity.5) MoAtg14, the highly divergent homolog of the yeast autophagy protein Atg14, is vital to the autophagic process in M. oryzae.Via the position specific iterated and pattern hit-initiated BLAST tool, a novel autophagy-related protein MoAtg14was identified in M. oryzae protein database, which showed only21%sequence identity to its yeast counterpart. A conserved coiled-coil domain was predicted in the N-terminal region of MoAtg14protein, which mediated the interaction between MoAtg14and MoAtg6. The autophagic process was blocked in△Moatg14mutant, and the autophagic degradation of marker protein MoAtg8didn’t occur in the mutant. Therefore, the MoATG14deletion mutant exhibited typical△atg phenotypes, such as collapse in the middle of colony, poor conidiation and complete loss of virulence.6) The coiled-coil domain of the MoAtg14protein is essential for its function.In the mutant MoAtg14-AC or MoAtg14-AN, which expressed truncated MoAtg14lacking the C-terminal or N-terminal half, no obvious defect was observed in the colony morphology, conidiogenesis, pathogenicity and autophagic activity, indicating the C-terminal or N-terminal region of MoAtg14is dispensable for autophagy. While the MoAtg14-ACCD mutant lacking the coiled-coil motif of MoAtg14resembled the phenotypes of MoATGl4full deletion mutant, suggesting the MoAtg14functions in a manner dependent on its coiled-coil domain.Taken together, our results demonstrate that the integral SNF1pathway is required for fungal development and facilitates pathogenecity by its contribution to peroxisomal maintenance and lipid metabolism in M. oryzae, and highlight the significance of a novel coiled-coil domain protein MoAtg14in autophagic process and pathogenesis of M. oryzae.