A Study On Striatin-like Pcg7 In Asexual Development And Pathogenesis Of Pyricularia Oryzae

Striatin family proteins are widely distributed.Striatin,which involved in signal transduction,RNA synthesis and cytoskeleton assembly in mammals,is an important component of the STRIP AK complex.However,reports on its role in plant pathogenic fungi are few.Rice blast caused by Pyricularia oryzae is one of the most serious rice diseases in the world.Wide and deep research on pathogenesis of Pyricularia oryzae will be helpful to establish new effective control theory and technology.This study aimed to explore the function and mechanism of stratin in the growth and pathogenesis of the rice blast fungus Pyricularia oryzae by means of molecular techniques.The main results and conclusions are obtained.First of all,one mutant with slow mycelial growth was obtained and the fact that the T-DNA insertion destroys the striatin encoding gene was confirmed through genomic Southern blot and gene complementation assay.Furthermore,phenotype test on this gene knockout mutant showed some results.The striatin gene knockout mutant not only has obvious defects in vegetative mycelium growth,but also produce very little deformity conidia,especially the loss of pathogenicity to the host plant.Based on this,it was named PCG7.RT-qPCR showed that high expression of PCG7 in the appressorium and the expansion of primary infected hyphae.Secondly,in order to clarify the mechanism of Pcg7 involved in the mycelium growth and pathogenicity of rice blast fungus Pyricularia oryzae,the interacting proteins were separated and identified.Using pull down and mass spectrometry,a total of 73 proteins that were coprecipitated with Pcg7 were identified.Similar components of STRIP 1/2,SLMAP,and PP2AA in STRIP AK complex were found confirming that Pcg7 is a component of the STRIP AK complex.In coprecipitation proteins with Pcg7,two proteins Mst7 and Pmk1 which were important components in Pmk1-MAPK signaling pathway controlling the pathogenicity of Pyricularia oryzae were found.Yeast two hybrid experiments were carried out to determine the direct interaction between Pcg7/Mst7 and Pcg7/Pmk1.Furthermore,the point mutation vector keeping Mst7 phosphorylation was respectively transferred into the wild type P131 and the PCG7 deletion mutant SK3 to analyze the phosphorylation state of Pmkl.The phosphorylation of Pmk1 in SK3 transformants was enhanced than that of P131 transformants.This result suggests that Pcg7 may mediate the modification of Pmk1 by protein phosphatase.In this respect,the proteins coprecipitated with Pcg7 were further analyzed founding that two phosphatase proteins respectively encoded by MGG₁0195 and MGG₀3154 were contained.Pcg7 was proved directly interacted with phosphatase MGG₁0195 and MGG₀3154 by yeast two hybrid.This result indicates that Pcg7 is likely to mediate the interaction between protein phosphatase and Pmk1,so that the phosphorylation of Pmk1 in PCG7 knockout SK3 is enhanced.In order to verify whether Pcg7 mediates the interaction between protein phosphatase MGG 10195 or MGG 03154 and Pmk1 in vivo,MGG 10195.6 and MGG 03154.6 knockout vector also been constructed and one deletion mutant 3154K01 of MGG₀3154.6 was obtained.Phenotypic analysis of 3154K01 showed that compared with the wild type P131,3154K01 had obvious defects in mycelial growth and conidialsporulation.Experiment is currently carried out to analyze the effect of MGG₀3154 on the dephosphorylation of Pmkl.Thirdly,small G proteins and other proteins were also obtained as the interaction proteins of Pcg7 by yeast two two hybrid experiments.In addition,the interaction domain between Pcg7 itself and between Pcg7 and other proteins are analyzed.The pGBKT7 vectors carried different segments of Pcg7 were constructed and were used in yeast two two hybrid experiments.The results showed that,Pcg 7 maybe interacted with itself through the N-terminal 1-130 amino acids to form dimers and through the N-terminal 150-180 amino acids to interacted with the protein kinase?protein phosphatases and small G proteins.In summary,this study identified a new Pyricularia oryzae pathogenic gene PCG7,of which the encoding product is striatin protein.This study also found Pcg7 may regulate the phosphorylation of two pathogenic necessary proteins Mst7 and Pmkl thus participate in the pathogenesis of rice blast fungus.Pcg7 was also found interacted with small G proteins and other proteins.This study provides an important new information for the further study of the signal pathway in the growth,development and pathogenesis of rice blast fungus.