Screening And Functional Study Of E3 Ubiquitin Ligase Subunit MoGrr1 Binding Protein In Rice Blast Fungus

Rice blast disease,one of the most devastating fungal diseases in rice,has caused serious losses in rice cultivation and production.The causal agent of rice blast disease is the ascomycetes fungus Magnaporthe oryzae.Till now,the control of rice blast disease mainly relied on the selection and breeding of disease-resistant varieties.However,the pathogenic 1fungus M.oryzae are prone to mutation,making it difficult to be controlled effectively.In 2002,the Rice Genome Research Project obtained the full rice genome sequence,providing an opportunity to study interactions between the rice and M.oryzae at the genomic level,besides,it also provides new ideas for the control of the rice blast disease.The ubiquitination pathway is widely involved in a series of important physiological processes such as cell division,development,and immunity in eukaryotes.Previously,the study in our laboratory revealed the preliminary roles of the E3 ubiquitin ligase subunit Mo Grr1 in regulating growth and development of M.oryzae.To further understand the function and molecular mechanism of Mo Grr1 encoding gene Mogrr1,CoImmunoprecipitation(Co-IP)and liquid chromatography-mass spectrometry(LC-MS)were used to identify the interacting proteins,and Mo PPG1,Mo PPH21,Mo PP2 B,Mo SIT4,Mo RTS1 and other 136 proteins that may interact with Mo Grr1 were obtained.Western blot and yeast two-hybrid were further employed for in-vitro interaction test on Mo PPG1 to identify the direct interaction between them.Meanwhile,domain deletion assays which are validated by yeast two-hybrid,clarified that Mo PPG1 interacted with Mo Grr1 by combining with the F-box domain of Mo Grr1 in M.oryzae.To further identify the relation between Mo Grr1 and Mo PPG1 in biological function,the knock-out mutant of Mo PPG1 encoding gene Moppg1(MGG＿01690)was obtained using homologous recombination.The phenotypes of Moppg1 mutants were similar to Mogrr1 mutants,such as the slower mycelial growth,abnormal spore morphology and the remarkable reduction in sporulation.Besides,Moppg1 also involved in controlling conidium germination and appressorium development of M.oryzae and had a regulatory effect on the process of intracellular glycogen metabolism.In addition,the spore of Moppg1 mutants,similar to Mogrr1 mutants,could scarcely infect the host,and therefore their pathogenicity on barley and rice plants were significantly reduced.Meanwhile,the mutants became less tolerant to hydrogen peroxide but sensitive to other cytoderm stress factors like CFW and CR,indicating that its cell wall integration might have changed.To sum up,this research preliminarily obtained 136 proteins that may interacted with Mo Grr1 through the combining use of Co-IP and LC-MS.Western blot and yeast two-hybrid were further used to verify the interactions between Mo PPG1 and Mo Grr1.Moreover,the biological function of the gene was examined based on the construction of Moppg1 mutants.Meanwhile,the relationship between Moppg1 mutants and Mogrr1 mutants will provide a new basis for further investigation of Mo Grr1 regulating the development and pathogenesis of M.oryzae.