Functional Characterization Of NDR Family Protein Kinase MoCbkl And Regulator Of G-protein Signaling MoRgs7 In Development And Pathogenicity Of Magnaporthe Oryzae

Rice blast caused by Magnaporthe oryzae is one of the most serious diseases on rice,which will pose a great threat to food production at home in the world.Meanwhile,due to the genetic variation of rice blast fungus,it will cause the bacteria to become resistant to fungicides and lose resistance to resistant varieties.With the completion of sequencing of the genome-wide sequence of M.oryzae,the discovery and identification of genes related to pathogenicity of the pathogenic fungus have become more mature.Study on the molecular mechanism of pathogenicity of rice blast can not only understand the pathogenesis of rice blast fungus more comprehensively,but also provide theoretical support for the breeding of resistant varieties and the screening of targets for chemical control.After the sequencing of the whole genome sequence of M.oryzae,the research on the molecular mechanism of pathogenic bacteria is more and more extensive and in-depth.In this paper,the mechanism of regulation of mitosis-associated NDR family protein kinase MoCbkl and regulator of G-protein signaling MoRgs7 in rice blast fungus development and recognition of host signals was studied.Results are as follows:Cbkl is a terminal kinase in the RAM(regulation of Ace2 functionand cell morphogenesis)signaling network,The RAM signaling network is very conserved in many eukaryotic cells and regulates membrane hydrolysis after mitosis to facilitate the separation of the two daughter cells.Cbkl is a protein kinase of the AGC/NDR family that activates the transcriptional Ace2 which regulates the disruption of the membrane.In this paper,the ΔMocbk1 knockout mutant was obtained in M.oryzae,and it "was found that theΔMocbk1 had obvious defects in growth,sporulation,appressorium formation and pathogenicity.Furthermore,the reason for the decrease in the infectivity of the △Mocbk1 was analyzed.It was found that when the △Mocbk1 formed the appressorium,the transfer and degradation of glycogen lipids were defective,resulting in a decrease in the turgor pressure of the appressorium,and the invading nails could not normally invade the host cells,eventually leading to a reduction in virulence.The yeast homologous proteins MoKicl and MoAce2 were searched for rice blast fungus,and the genes encoded by their homologous proteins were knocked out,and the pathogenicity of ΔMokic1 and ΔMoace2 was found to be unchanged.Then,co-immunoprecipitation techniques were further used to search for proteins that may interact with MoCbk1.The next step is to reveal the effects of these interaction proteins on the growth and pathogenesis of the pathogen.The appressorium of M.oryzae are a special infection structure produced by M.oryzae invading host plants.The formation and function of appressorium are dependent on signal transduction pathways,including G-protein coupled receptors(G protein coupled receptors,GPCRs)/Gprotein-mediated cAMP signaling.It was previously found in our laboratory that regulator of G-protein signaling MoRgs7 regulates the formation of pathogen appressorium and plays an important role in pathogen infection of the host,and its RGS and 7 transmembrane(7-TM)functional domains play an important role in MoRgs7 function.But how does MoRgs7 perceives surface cues and activates the signals needed for disease is still unclear.We demonstrated the interaction of MoRgs7-MoMagA by Co-IP experiments,and confirmed that MoRgs7 and MoMagA act through the endocytic pathway involved in actin protein by exogenous addition of inhibitors.Furthermore,we found through fluorescence bleaching recovery technology that MoRgs7 and MoMagA are rarely transmitted from the intracellular system to the plasma membrane in order to the response of the hydrophilic interface.In contrast,in the hydrophobic interface,the hydrophobic interface induces localization of MoRgs7 from the germ tube to the plasma membrane in a manner that interacts with MoRgs7.By disturbing the hydrophobic interface,We found that the appressorium formation rate of rice blast fungus decreased significantly.The results showed that the interaction between MoRgs7 and hydrophobic interface plays an important role in the identification of hydrophobic interface signals by rice blast,thereby regulating the formation of appressorium and pathogenicity of M.oryzae.In summary,we found that MoCbkl plays a very important role in the growth,development and pathogenesis of M.oryzae.On the other hand,the regulator of G-protein signaling MoRgs7,through the interaction with the hydrophobic interface,transmits the extracellular signal to the intracellular through endocytosis,so as to participate in the sensing of hydrophobic interface signal by M oryzae,and affect the appressorium formation and pathogenicity of M.oryzae.