Functional Analysis Of Regulator Of G-Protein Signaling MoRgs1 Binding Protein MoLia1 And MoCmc1 In Magnaporthe Oryzae

Rice is an important food crop in global food production.Rice blast caused by(Magnaporthe oryzae)infection causes 10% to 30% reduction in rice production every year.In order to meet the growing dietary needs of the global population,rice production must be doubled in the next 40 years.Therefore,the in-depth study of the pathogenic mechanism in the rice blast fungus provides important theoretical guidance value for winning the food defense war.As a eukaryotic organism,M.oryzae uses its G protein signal pathway to recognize the surface signal of the host plant rice leaf.And G protein regulatory factors(regulators of Gprotein signaling,RGS)by accelerating the hydrolysis of heterotrimeric G protein subunit GTPase and exercising its(GTPase-activating protein,GAP)activity,negatively regulate the G protein signaling pathway.Among the rice blast fungus,eight RGS proteins were identified in the early stage of the laboratory,namely MoRgs1-MoRgs8.MoRgs1 is located on the late endosomes and interacts with the Gα subunit of the heterotrimeric G protein.Studies have shown that MoRgs1 negatively regulates the G protein-c AMP signal pathway,which in turn affects the formation and pathogenicity of the functional appressorium of M.oryzae.However,people’s understanding of the intracellular regulatory mechanism of MoRgs1 in M.oryzae is still not perfect.In the early stage of the laboratory,affinity chromatography and yeast two-hybrid(Y2H)were used to screen two candidate binding proteins for MoRgs1,namely deoxycoprotein hydroxylase MoLia1 and 3-carboxymuconate Cyclase MoCmc1.This article uses yeast two hybrid(Y2H),bimolecular fluorescence complementation technology(BIFC),and co-immunoprecipitation(Co-IP)technology to verify MoRgs1 and MoLia1 and MoCmc1.The results show that MoRgs1 interacts with MoLia1 and MoCmc1,indicating that MoLia1 and MoCmc1 are binding proteins of MoRgs1.For MoLia1,firstly,the phylogenetic tree method was used to analyze the protein homology,and it was found that the rice blast fungus MoLia1 was a homologous protein of the yeast Sc Lia1.Next,construct knockout mutant of ΔMolia1 gene,confirm that the knockout mutant is a single copy by Southern Blot,and construct a complement strain.The biological phenotypes of the wild-type strains,the knockout mutants and the complement strains were observed and analyzed.It was found that the pathogenicity of ΔMolia1 strain was significantly reduced compared with the wild-type and complementary strains.Its growth rate slows down;The spore production decreased significantly and the appressorium formation was blocked.The c AMP content in ΔMolia1 decreased.Further biochemical analysis showed that MoLia1 could inhibit the GAP activity of MoRgs1.For MoCmc1,first create knockout mutant ΔMocmc1,and use Southern Blot analysis to confirm that a single-copy mutant strain is obtained,and to construct a complement strain.Phenotypic analysis found that compared with the wild-type and complementary strains of M.oryzae,the virulence of the ΔMocmc1 strain was decreased;the sporulation rate was significantly reduced;the appressorium formation rate was decreased,and the intracellular c AMP level was increased;In addition,the staining analysis of the glycogen lipids in M.oryzae found that MoCmc1 regulates the transfer of glycogen within appressorium,but does not affect lipid degradation;Further observation of the subcellular localization of MoCmc1 revealed that the co-localization of MoCmc1 and Actin indicated that it is located on the cytoskeleton.In ΔMorgs1,the localization of MoCmc1 is abnormal.In summary,deoxycoprotein hydroxylase MoLia1 inhibits the GAP activity of MoRgs1,affects the intracellular c AMP content in M.oryzae,thereby regulating the formation of functional appressorium,and ultimately regulating the pathogenicity in M.oryzae.In addition,3-carboxymuconate cyclase MoCmc1 interacts with MoRgs1 and participates in the G protein signaling pathway.MoRgs1 regulates the subcellular localization of MoCmc1 and affects its biological functions,leading to a decrease in the rate of intracellular glycogen transfer and an increase in c AMP content of the rice blast fungus,which ultimately affects the formation and pathogenicity of the fungus functional appressorium.This article reveals that MoLia1 and MoCmc1 are involved in the G protein-c AMP signal transduction mechanism of M.oryzae through binding to MoRgs1,and the molecular mechanism of regulating the pathogenicity of M.oryzae.It enriches the interaction network between pathogenic microorganisms and hosts,and provides a clearer theoretical basis for the research and development of target drugs and biological control technology.