Regulator Of G-protein Signaling MoRgs1 Regulates Appressorium Formation And Pathogenicity In Rice Blast Fungus Magnaporthe Oryzae

As one of the most important crops in China,the cultivated area of rice accounts for about one-fourth of the total cultivated land and the annual rice yield accounts for about one-half of the total grain output.Rice blast fungus Magnaporthe oryzae is a devastating fungal disease in rice production,threatening global food production security and causing food loss which could feed sixty million people annually.At present,the control measures of rice blast mainly focus on the breeding of high-quality varieties with high yield and resistance to rice blast,supplemented by scientific cultivation measures and integrated control strategies of phytochemistry protection.However,because rice blast is prone to genetic variation in the field and new pathogenic races are produced,the resistance of the selected resistant varieties will gradually decrease or even completely disappear after three to five years of planting and the control effect of cultivation measures is limited.The chemical control uses the kind of medicaments to be quite simple that mainly is the tricyclazole,the long-term application also can cause the pathogen to produce the medicine resistance similarly.Therefore,further study on the molecular mechanism of pathogenicity in Magnaporthe oryzae will help to explore new fungicide targets and provide the possibility for better control of the disease.Like other eukaryotes,rice blast fungus recognizes and transmits extracellular signals through a G-protein coupled receptors（GPCRs）cascade,regulators of G-protein signaling（RGS）negatively regulate this process.Previous research found that there are 8 RGS genes in M.oryzae,named from MoRGS1 to MoRGS8.Four genes MoRGS1,MoRGS3,MoRGS4and MoRGS7 not only regulate the intracellular c AMP level but also play an important role in the pathogenicity of M.oryzae.After further exploration,people found that although both MoRgs7 and MoRgs8 had seven transmembrane domains,only MoRgs7 could undergo endocytic internalization and function at the late endosomes,the presence or absence of transmembrane domain is important for the subcellular localization and biological function of MoRgs7 and its involvement in the internalization process is regulated by MoCrn1.In addition,the interaction between MoRgs7 and MoMag A^G187S is also regulated by MoMip11.Unlike MoRgs7,the N-terminal sequence of MoRgs1 has no transmembrane domain,but it has two DEP domains that also regulate the correct subcellular localization of proteins.MoRgs1 is also localized in the late endosomes,but until now the underlying molecular mechanism of the protein negatively regulating the G-protein signaling pathway is still unclear.In Saccharomyces cerevisiae,Sst2 that is homologous to MoRgs1 could be phosphorylated by Fus3 MAPK cascade kinase pathway in response to the accumulation of pheromones.Another research about phosphoproteome analysis on M.oryzae showed that there might be phosphorylated modification of MoRgs1.Therefore,the potential roles of MoRgs1 phosphorylation and functions of its associated binding proteins of M.oryzae were studied in this paper.Identification of MoRgs1-binding proteins from M.oryzaeFirst,Mn²⁺-phos-tag methods were used to confirm the phosphorylation of MoRgs1.Therefore,the protein co-immunoprecipitation technique and yeast two-hybrid system were performed to screen and identify the binding proteins and potential kinases of MoRgs1.By analyzing the mass spectrum results,47 candidate binding proteins of MoRgs1 were identified.In addition,38 binding proteins were isolated from the rice blast fungus c DNA library by yeast two-hybrid system.Bioinformatics analysis found that the candidate proteins were involved in lipid transport and metabolism,cell wall or membrane biosynthesis,signal transduction,intracellular cytoskeleton and vesicles transport.Luckily we identified that MoCkb2 might be the binding protein of MoRgs1 that functions as a regulatory subunit of Casein kinase 2（CK2）.The interaction between MoRgs1 with one catalytic subunit MoCka and two regulatory subunit MoCkb1 and MoCkb2 of casein kinase2（CK2）were performed by yeast two-hybrid,co-immunoprecipitation and bimolecular fluorescence complementary assays,respectively.It turned out that MoCka and MoCkb2combine as MoCk2 complex that directly interact with MoRgs1.Phosphorylation of MoRgs1 regulates appressorium formation and pathogenicity in M.oryzaeIn order to explore the roles of MoCk2 subunits in MoRgs1 functioning.We conducted phosphorylation assays in vivo and in vitro by Mn²⁺-phos-tag methods and FDIT assays,respectively.The results showed that MoCka coordinated MoCkb2 as a MoCk2 complex to phosphorylate MoRgs1,but the role of MoCkb1 in the phosphorylation of MoRgs1 is unclear.Then,five phosphorylation sites S396,S399,S585,S696 and S700 regulated by MoCk2 complex were identified by differential phosphorylation mass spectrometry.In this paper,whole five sites were subjected to unphosphorylated and phosphomimetic mutations,that is,all of them were site-directed mutagenesis into Alanine and L-Aspartic Acid,respectively.The phenotypic analysis showed that the phosphorylation of these five sites was involved in the regulation of growth,conidiation,biomass,appressorium penetration and pathogenicity of M.oryzae.In addition,MoRgs1 phosphorylation regulates the intracellular c AMP level and plays an important role in the formation of appressorium at the hydrophobic interface.In addition,by scanning the results of MoRgs1 binding proteins,an endoplasmic reticulum membrane protein complex subunit protein MoEmc2 was performed for further study.Here,we found that MoEmc2 is directly involved in the phosphorylation of MoRgs1 mediated by CK2.First,MoEmc2 interacts with MoRgs1 and the three subunits of CK2 protein kinase MoCka,MoCkb1 and MoCkb2,respectively.Besides,the interaction between MoCk2 subunits MoCka,MoCkb1 and MoCkb2 with MoRgs1 were regulated by MoEmc2.Second,MoEmc2 regulates the subcellular localization of the CK2 protein kinase subunits MoCka,MoCkb1 and MoCkb2 at the early stage of appressorium formation（3h）.Then,phosphorylation analysis in vitro showed that the level of MoRgs1 phosphorylation was decreased in the MoEMC2 mutant.The analysis of MoRgs1 phosphorylation mass spectrometry showed that five phosphorylation sites activated by MoCk2 of MoRgs1 were also unphosphorylated in MoEMC2 depletion mutant.In conclusion,MoEmc2 is required for MoCk2 and MoRgs1 interactions and phosphorylation modifications,suggesting that MoEmc2 serves as a scaffold for MoCk2and MoRgs1 interactions to regulate MoCk2 phosphorylation of MoRgs1,and that phosphorylation equilibrium of MoRgs1 is necessary for the appressorium formation and pathogenicity of M.oryzae.MoEmc2 is involved in G-protein signaling to regulate appressorium formation and pathogenicity in M.oryzaeIn organisms,the endoplasmic reticulum membrane protein complex（EMC）is involved in the biogenesis of new polypeptide chains and helps proteins with transmembrane domain（TM）enter the endoplasmic reticulum to form the topological structures.In addition,MoEmc2 was found to be directly involved in the regulation of the G-protein signaling.MoEmc2 was confirmed to interact with Gαs（MoMag A）,Gαi(MoMag B^G183S),Gαi(MoMag B^Q204L),Gβ（MoMgb1）and Gγ（MoMgg1）by yeast two-hybrid system.MoEmc2 is also involved in the subcellular localization of MoMag A at the early stage（3h）in appressorium formation,and the interaction between MoEmc2 and MoMag A in M.oryzae.It was found that the normal function of MoMag A was important to inhibit the decrease of c AMP level,the deficits of appressorium formation and the pathogenicity of MoEMC2 depletion mutant.To sum up,on the one hand the endoplasmic reticulum complex（EMC）protein subunit MoEmc2 regulates the phosphorylation of MoRgs1,on the other hand it regulates the subcellular localization and function of G-protein alpha subunit Gαs to transport signal to c AMP pathway,finally regulates the growth,differentiation and pathogenicity of M.oryzae.In summary,we found that the phosphorylation of MoRgs1 is dependent on MoCk2,and the subunit of endoplasmic reticulum complex MoEmc2 acts as an adaptor for the phosphorylation.At the same time,MoEmc2 regulates the subcellular localization and function of MoMag A,participates in the G-protein/c AMP signaling,and regulates the Pmk1 MAPK kinase cascade,and ultimately affects the growth and differentiation and pathogenicity of M.oryzae.This paper revealed the important role of the endoplasmic reticulum complex in intracellular signal transduction of organisms,which has enriched the understanding of G-protein dependent signal transduction and MoRgs1 negatively regulating G-protein signaling.