Functional Analysis Of Retromer Complex In Magnaporthe Oryzae And Fusarium Graminearum

In eukaryotes,retromer is an endosome-localized complex involved in protein trafficking.However,the role of such membrane trafficking events in pathogenic fungal development and pathogenicity still remains unclear.Different fungal pathogen uses different ways to infect their host.In order to understand the biological function of retromer complex,we here applied two different plant pathogens(Magnaporthe oryzae and Fusarium graminearum)to study due to their distinct infection ways.The filamentous ascomycetous fungus M.oryzae causes rice blast disease,one of the most severe fungal diseases of rice resulting in significant yield loss throughout the world.M.oryzae invades the host plants using key infection structures,called appressoria that are elaborated at the tips of the germ tubes.In this study,we found that MoVps35,the major component of the retromer is essential for appressorium-mediated host penetration in M.oryzae.Loss of MoVps35 function blocked glycogen breakdown and lipid droplet turnover,delayed nuclear degeneration in conidia,and reduced turgor development in appressoria.Interestingly,the ?Movps35 mutant was found to be impaired in the biogenesis of punctate autophagosomes(RFP-Atg8)in developing appressoria and significantly reduced the endoproteolytic cleavage and lipidation of MoAtg8 upon nitrogen starvation.The aforementioned defects in ?Movps35 were reminiscent of the atg8-deletion in M.oryzae.Remarkably,MoVps35 plays a recognition capability with cleaved MoAtg8 by visualizing the co-localization between MoVps35-GFP and RFP-MoAtg8 proteins and Co-IP assay.By using time-lapse videomicroscopy,we found MoVps35-GFP was present on highly mobile punctate structures within/around the vacuolar membrane.These results suggest that MoVps35 is likely required to retrieve the cleaved MoAtg8 via the vacuolar membrane sorting for autophagosome formation.Finally,deletion of two other retromer components MoVps26 and MoVps29 result in similar autophagy defects as the ?Movps35 mutant.Therefore,our data suggest that MoVps35/retromer plays a key role in regulating the autophagy process in appressorium-mediated initiation of blast disease in rice.F.gramimarum is the major causal agent of Fusarium head blight(FHB)on wheat,barley,which causes yield loss and affects the quality of grains.The mycotoxins,such as deoxynivalenol(DON)and its derivatives produced by the fungus in infected grains,pose a serious threat to human and animal health.By using time-lapse videomicroscopy,we revealed that the retromer core component FgVps35-GFP can be found in the cytoplasm as fast-moving vesicles.Co-localization assay identified that more than 60%fluorescence dots of FgVps35-GFP are associated with vacuolar membranes,while no more than 10%fluorescence co-localize with TGN marker FgKex2.These results suggest that FgVps35 functions at the donor membrane,that is the vacuolar membrane,to TGN trafficking pathway.To establish the physiological relevance of trafficking ability in FgVps35,we disturbed the trafficking by treating fungi with microtubule-destabilising agent nocodazole,the result showed mobility of punctate were significantly compromised and exhibits a strong inhibition against germination as well as radial growth.In addition,knockout of FgVPS35 caused significant reduction in growth,conidiation and virulence as well as blocked the ascospore formation.Using yeast two hybrid assays,we clarified the interactions among FgVps35,FgVps26,FgVps29,FgVps17 and FgVps5 that are homologous to components of the yeast retromer complex.Deletion any one of these genes resulted in similar defects as ?Fgvps35 mutant,including growth,sexual and asexual development and pathogenesis.Our data indicate that retromer is a well-conserved complex and mediates intracellular trafficking which would be required for F.graminearum development and virulence to the host.