Screening Of Retromer-Interacting Proteins And Analyses Of Their Biological Functions In Magnaporthe Oryzae

Retromer complex is a conserved,eukaryotic heteropentameric protein complex that sorts cargo proteins on endosomal membranes and mediates their retrograde transport to the trans-Golgi network(TGN)or plasma membrane,thereby preventing them from vacuolar/lysosomal degradation.Different retromer cargoes have been identified in many different species and this demonstrated the crucial roles of the retromer in regulating different physiological conditions.Although this protein complex has been established to regulate pathogenesis and autophagy in the model filamentous fungus,Magnaporthe oryzae,the actual cargoes it recycles in this species still remain to be investigated.This project aims to dissect the retromer-interacting proteins and establish their biological roles in the rice blast fungus.This will give an excellent clue to unveiling the retromer cargoes in M.oryzae.We used high-throughput immunoprecipitation and mass spectrometry techniques to pull-down the core retromer subunit,Vps35,and identify its interacting proteins.Live cell fluorescence microscopy was used to investigate their subcellular localizations and how their localizations alter in the absence of Vps35.Mutants lacking the Vps3 5-interacting proteins were generated,respectively,and used for phenotypic and pathogenesis analyses.We identified two putative Vps35-interacting proteins with high coverage:AAA ATPase family 3B(Tob3)and trehalose phosphatase(Tps2).MoTob3 localizes to the endosomes and was obviously mis-localized to the vacuoles in ΔMovps35 mutant.There was significant increase in virulence and conidiation of ΔMotob3 mutants as compared to the 70-15(wild-type,WT)strain.MoTps2 is expressed ubiquitously in the cytosol.ΔMotps2 mutants were unable to infect intact rice and barley leaves and conidiation was completely abolished.We therefore conclude that MoTob3 is a potential retromer-interacting protein which serves as a negative regulator of conidiation and pathogenicity in M.oryzae.MoTps2 is crucial for conidiation and host penetration necessary for the fungal pathogenesis.