Screening And Functional Analysis Of Rho3 Interacting Proteins In Magnaporthe Oryzae

Rice blast fungus, Magnaporthe oryzae, is an important model for the study of host plant and pathogenic fungal interactions. Rho-family proteins, which act as molecular switches in eukaryotic signal transduction, can exert diverse biological effects through a variety of downstream effector proteins, and have been proved to be closely related to cell differentiation, polarized hyphal growth and plant infection process of M. oryzae.Our previous study showed MoRho3 play an important role in the growth, development and pathogenicity of M. oryzae, therefore screening out MoRho3 interaction proteins and making clear of their biological functions are conductive to exploring the regulation mechanisms of growth, development and pathogenicity in M. oryzae. In this study, after repeated verifications, seven MoRho3 interacting proteins （MGG₀0505, MGG₀3041, MGG₀4584, MGG₀6263, MGG₀9445, MGG₀9978 and MGG₁3563） have been obtained through screening the yeast two-hybrid cDNA library of hyphal stage of M. oryzae using the constitutively active form of MoRho3. Besides, four potential MoRho3 interacting proteins were identified by bioinformatics, and two of them encode the different subunits of protein kinase CK II, and interestingly, the follow-up yeast two-hybrid experiment exhibited that all the three subunits of CK Ⅱ could interact with MoRho3 respectively.In order to study the function of these MoRho3 interacting proteins, related gene knockout mutants were constructed through homologous recombination. Phenotype analysis results showed that, compared to the wild type strain, MGG₀0505 deletion mutants exihibited dramatical defects in polarized hyphal growth and conidial septum morphogenesis, while the pathogenicity on susceptible rice cultivar CO39 showed normal. The growth, development, cell differentiation and pathogenicity of MGG₀3041 and MGG₀6263 deletion mutants exhibited as normal as the wild-type strain.Taken together, it appeared more likely that MoRho3 regulated polarized hyphal growth and conidial septum morphogenesis of rice blast fungus through MGG₀0505.