A Glycine-rich Poly(u)-binding Nuclear Protein MoGrp1 Regulates Asexual Development And Pathogenicity Of Magnaporthe Oryzae

Glycine rich RNA binding protein（GRP）is a type of protein widely existing in eukaryotes.It is an important protein involved in transcription and post-transcriptional regulation and affects many metabolic pathways.At present,the research on this type of protein is mainly concentrated on plants and animals.There are few studies on other species,and there is no research on filamentous fungi.Rice blast caused by rice blast fungus（Magnaporthe oryzae）is one of the most serious diseases in the world.The research on the pathogenesis of rice blast will help us to further understand the occurrence of disease and establish effective control theory and technology.In this study,the role and mechanism of GRP related genes in the growth and pathogenesis of Magnaporthe oryzae were studied by means of molecular biology,and the main results and conclusions were obtained.By using bioinformatics and other analysis,we identified a potential encoding GRP protein gene MGG₀7511,which named MoGRP1.A knockout mutation of this gene was obtained by knock-out methods.The specific phenotype of this mutation was as follows:MoGRP1 gene knockout not only slowed down the growth of mycelium obviously,but also defected in conidiation and conidia development,and its loss also decreased the formation rate of appressorium and seriously affected the pathogenic ability of the strain.After that,the gene complementation experiment confirmed that all the phenotypes were caused by the deletion of MoGRP1.The analysis of gene expression in the various stages of wild type showed that MoGRP1 was characterized by its composition and up to a certain amount of expression in the conidiation,which was about 4 times that of the mycelium stage.In order to clarify the mechanism of MoGRP1 in the growth and pathogenicity of Magnaporthe oryzae,we purified the protein MoGrp1 and identified the interaction protein.Using the intermolecular interaction method pull-down and mass spectrometric analysis,we verified two proteins strongly binding to MoGrpl,a RNA precursor splicing factor encoded by the MGG₀6457 gene,and we named MoRSE1;the other was a protein of the MGG₀3556 gene encoded H/ACA nucleoprotein complex,and we named MoGAR1.By co-immunoprecipitation to confirm this result,the authors suggest that MoGrpl may be involved in the post transcriptional process of precursor RNA with MoRsel and MoGarl.The RNA binding experiments show that MoGrpl can effectively combine poly（U）RNA and not bind to DNA,suggesting that it may have binding activity in the U-rich area of the precursor mRNA.After that,we observed that MoGrpl protein could be localized in the nucleus.The structure of glycine enriched domain is also important to MoGrp1.The deletion was complementary to ΔMogrp1 mutation by using characteristic sequence arginine glycine glycine（Arg-Gly-Gly,RGG）.The deletion of the amino acids after 264th does not affect the normal function of the protein;the loss of amino acids after the 190th shows a defect similar to that of the knockout body,but it can partially complement the phenotypic defect of the knockout body;the loss of the 181 amino acid and its subsequent peptide leads to the complete inability to restore the phenotype.It is confirmed that the 182-190 peptide is the most important in MoGRP1,and is the key domain for MoGrpl to perform all functions.In addition,we also observed the subcellular localization of the missing complement,and found that the nuclear localization were unstable with loss after the 190 amino acids.After deleted after 181 amino acids,MoGrpl were completely lost nuclear localization.To sum up,a new pathogenic gene MoGRPl of rice blast fungus was identified in this study,and its encoding product was a GRP protein.It was found that MoGrp1 could combine with poly（U）RNA,suggesting that it transcriptional regulation of certain pathogenic essential proteins through the splicing modification of pre-mRNA,thus participating in the pathogenicity process of rice blast fungus.In addition,it was also found that MoGRPl could interact with MoGar1 and MoRse1.The functional domain of the protein must be determined.This study provides important information for revealing the important transcriptional regulation process of rice blast fungus growth,development and pathogenesis in the further.