Function Analysis Of MoArf Family And The Associated Proteins MoGga1 And MoGlo3 In The Development And Pathogenicity Of Magnapothe Oryzae

Rice is the most important food crop all of the world,more than half of the world’s population lives in rice.The planting area of rice is about 30 million hectares in china,which is a quarter of the agricultural area.However,plant diseases affect the production of rice severely,causing 20 billion kilograms of grain losses every year for us in the current controlling levels.Among all of the diseases,rice blast is the most serious,resulting in 3 billion kilograms of grain losses every year in china.Rice blast,caused by Magnaporthe oryzae,occurs wherever rice is grown,with the losses enoughing to feed more than 60 million people each year.Currently,resistant cultivars and chemical pesticides are the major controls for rice blast.However,the variation of races make the resistant cultivars lose the resistance and the pathogen acquire the ability of anti-fungicide.Long-time controlling of the disease is a major worldwide challenge,therefore,elucidating the new pathogenic mechanisms of fungi will be contribute to the breeding of resistant cultivars and the exploration of new targets for efficient fungicides.Eukaryotic cells conclude many membrane-related organelles and the vesicle transport between the various organelles is a prerequisite for its normal metabolism.Inhibiting the vesicle transport in Fusarium graminearum is a good example of controlling the fusarium head blight.However,there are none related measures for rice blast.Our previous studies suggested the role of cellular transport in growth and development of pathogenic in M.oryzae,but the mechanism was unclear.In this study,we systematically characterized all of Arf GTPase family proteins,regulating vesicle traffic in other organisms,and the associated proteins MoGgal and MoGlo3 on the developmental and pathogenicity in M.oryzae,and major results are showed below:Here,we have systematically characterized all six MoArf and MoArl(Arf-like)proteins(MoArf1,MoArf6,MoArl1,MoArl3,MoArl8,and MoCin4)and determined their shared and specific functions.We have shown that MoArf6,MoArl 1,and MoCin4 are important for growth and sexual reproduction.In addition,MoArf6 and MoCin4 regulate conidia formation whereas MoArl3 regulates vegetative growth.Meanwhile,through FM4-64 staining,we found that MoArf6,MoArll,MoArl3 and MoCin4 play important roles in endocytosis;Our results also suggested that MoArfl could have an essential function,while MoArl8 have no apparent function;Importantly,Mo Arl1 and MoCin4 are involved in pathogenicity through the regulation of host penetration and invasive hyphae growth.Moreover,we showed that MoCin4 is localized through the cytoplasm,while MoArll exhibit Golgi-cytoplasm cycling for the GTP and GDP binding forms and that the cycling is required for the full function of MoArl1.To investigate the function of MoArll in Golgi,we identified MoGgal as an interacting protein of GTP-binding MoArll through co-immunoprecipitation(co-IP)assay and Mass Spectrometry analysis in the second chapter.We tested the interaction between the other five MoArf/MoArl with MoGgal,the results showed that the GTP-binding MoArfl also interacted with MoGgal.We further acquired AMoggal mutant and found that,like ΔMoarl1,ΔMogga1 also showed delayed endocytosis and decreased pathogenicity,meanwhile,AMoggal showed decreased conidiation and abnormal conidia.We further demonstrated that the Golgi-localized MoArll and MoArfl are required for the Golgi localization and function of MoGga1 through their interactions.When we mutated the 208th isoleucine to asparagine in MoGga1,it did not interact with MoArll and MoArfl anymore,causing descread pathogenicity for the pathogen.We have previously showed that the small G protein MoArf and the adaptor protein MoGgal involve in multiple biological process in M oryzae.However,it is unknown for the roles of their GEFs or GAPs.We identified MoGlo3 as an ArfGAP protein that is homologous to ScGlo3p of the budding yeast Saccharomyces cerevisiae,and we found that MoGlo3 partially complements the function of yeast ScGlo3p.We further found MoGlo3 is localized to the Golgi,and that the localization is dependent on the conserved BoCCS domain.Through phenotype analysis of the AMoglo3 mutant,we concluded that MoGlo3 is involved in vegetative growth,conidial production,the response to endoplasmic reticulum(ER)stress and scavenging of the reactive oxygen species.The combined effects result in failed appressorium function and decreased pathogenicity in ΔMoglo3 mutant.Finally,we provided evidence showing that the domains including the GAP,BoCCS and GRM are all important for normal MoGlo3 functions.In summary,we characterized that the MoArf family members and their related proteins MoGgal and MoGlo3 play critical roles in the development and pathogenicity in M oryzae.Among them,MoArl1,MoCin4,MoGga1,and MoGlo3 are involved in both endocytosis and pathogenicity,indicating the relationships between these two processes,but further studies are needed.At least,we imply a new insight for controlling the rice blast.