Functional Analysis Of Two Endocytosis Regulating Proteins MoEnd3 And MoCrn In Pathogenicity Of Magnaporthe Oryzae

Rice is one of the most important food crops in the world.The rice blast fungus Magnaprothe oryzae causes the most serious disease of cultivated rice and is a significant challenge to global food security.This fungus cause a lose of 10 to 30%of rice production each year.Currently,chemical pesticides and resistant cultivars are always applied to control the rice blast.But the resistance of resistant cultivars are lost in a short time because that M.oryzae evolves rapidly to overcome the resistance.And the application of chemical pesticides lead to generate anti-fungicide variation of M.oryzae.To better control the rice blast,we urgently need to understand the molecular basis of pathogenicity of M.oryzae.However,the availability of the genome sequences of this pathogenic fungi accelerated the identification and characterization of pathogenesis related proteins.This provides new platform for identifying new fungicide targets and developing manage strategy.Endocytosis is playing a key role in eukaryotic cells,controlling such processes including cell growth and development,recognition of extracellular environment,transduction of signaling and the initiation of autophagic cell death.During endocytosis,endocytic vesicles pack cargos from plasma membrane,such as nutrition,receptors,membrane proteins,lipids and large molecular proteins.Those cargos are brought to endosomes,then are delivered to lysosomes for degradation,or retrograded back to plasma membrane for recycling.Endocytosis also governs the pathogenicity of pathogenic fungus,such as M.oryzae,by unknown mechanisms.Previously,we found that deletion of the genes which encode SNARE MoSec22 and MoVam7 causes defects in endocytosis,also affects growth,conidiation and pathogenicity.We also found that disruption of actin-regulating kinase gene MoARK1 causes the delay in endocytosis and significant decreases in conidiation and virulence.These results suggest that endocytosis is required for development and pathogenicity of M.oryzae.How endocytosis regulates pathogenicity remains unknown.To investigate the mechanism by which endocytosis regulating pathogenicity and the MoArk1 molecular basis,we here identified MoArk1-interacting proteins and studied the functions of endocytic protein MoEnd3(a homologue of Saccharomyces cerevisiae End3p)that is one member of the MoArk1-interacting proteins.Deletion of MoEND3 results in delay in appressorium formation and penetration.Further study shows that deletion of MoEND3 leads to defect in endocytosis of GPCR Pth11 and membrane sensor MoSho1,which transports Pth11 and MoShol from membrane into endosomes.It is known that Mst11-Mst7-Pmk1 MAPK pathway controls appressorium formation and penetration.The defect also causes that Pmk1 MAPK that functions downstream of Pth11 and MoSho1 cannot be normally phosphorylated,and that autophagy is inhibited.To activate Pmk1 phosphorylation,we expressed constitutive Mst7 in the ?Moend3 mutant.We found that expressing constitutive Mst7 significantly suppressed the defects of the ?Moend3 mutant.These results suggest that MoEnd3-mediated endocytosis of Pth11 and MoSho1 is important for pmk1 phosphorylation,autophagy,appressorium formation and penetration.Moreover,we demonstrated that MoEnd3 functions are regulated by MoArk1 though phosphorylation.Given that endocytosis is closely coupled with exocytosis,we found MoEnd3 has additional role in facilitating secretion of effectors Avr-Pia and AvrPiz-t to suppress host immunity.Additionally,to further investigate how MoRgs7 links to cAMP signaling,here we identified a MoRgs7-binding protein MoCrn,a homologue of coronin,and studied its functions.We found that MoCrn is an actin-binding protein and regulates actin assembly and endocytosis.The interaction between MoCrn and MoRgs7 allows MoCrn impacting MoRgs7 localization.Furthermore,MoCm interacts with other two proteins involved in cAMP signaling,Gas subunit MagA and adenylate cyclase activator MoCap1.Also,the normal localizations of MagA and MoCapl require MoCrn.In this way,MoCrn has a role in modulating cAMP pathway.Consistently,deletion of MoCRN gene results in defects in cAMP synthesis,degradation of glycogen and lipids,and turgor generation during appressorium development.Strikingly,exogenous cAMP addition and MagAG187S expression suppressed these defects of ?Mocrn.Finally,we show that MoCrn binding with actin is required for MoCrn interacting with MoRgs7?MagA and MoCap1.Mutations on actin-binding sites of MoCrn leads to a non-functional MoCrn.In summary,we found that endocytosis transport the membrane receptors or sensors to regulates Pmk1 MAPK pathway necessary for appressorium formation and penetration.Also,the endocytic protein MoEnd3 is required for secretion of some effectors which function to suppress rice defense.Additionally,MoCrn interacts with cAMP signaling components such as MoRgs7,MagA and MoCapl,which allows MoCrn to impact cAMP signaling,appressorium formation and penetration.