Study On The Function Of VdPRMT1 Of Verticillium Dahliae Protein Arginine Methyltransferase

As a kind of soil-borne plant pathogenic fungus,Verticillium dahliae has strong pathogenicity and causes severe vascular disease called Verticillium wilt after infecting plants.Protein arginine methyltransferase(PRMT)plays a critical role in the cellular activities of eukaryotic cells,involving multiple biological processes such as pre-m RNA splicing,gene expression regulation,and DNA repair.The current research on arginine methyltransferases has generally focused on humans,mammals,plants and fungal model organism Saccharomyces cerevisiae.However,little has been reported on V.dahliae.This study focuses on the biological function of the protein arginine methyltransferase VdPRMT1 of V.dahliae.The main results are as follows:1.Four kinds of protein arginine methyltransferases from V.dahliae were obtained by Blast P.Their amino acid sequences were aligned,all of them had the conserved domain of methyltransferase(MT)including Motif I,Post I,Motif II and Motif III and THW loop of the PRMT family.The phylogenetic tree shows that V.dahliae and other filamentous fungi such as Neurospora crassa and Fusarium graminearum contain four kinds of PRMT and the kinship are relatively close.Among them,VdPRMT1 is homologous to S.cerevisiae HMT1.The related research in S.cerevisiae show that HMT1 is the main PRMT.The related research in plant pathogenic fungus Magnaporthe oryzae find that HMT1 homologous protein Mo HMT1 plays an important role in pathogenicity,so VdPRMT1 is selected for follow-up studies.2.The VdPRMT1 gene of V.dahliae was knocked out by the principle of DNA homologous recombination using polyethylene glycol(PEG)-mediated protoplast transformation.The growth of the deletion mutants on PDA,different carbon sources and various stress medium were significantly affected,the colony growth rate and the spore production were remarkably reduced.The pathogenicity to cotton seedlings and the fungal biomass in root tissue were also dramatically decreased.The GFP-tagged VdPRMT1 fusion plasmid was transformed into the VdPRMT1 deletion mutant strain by Agrobacterium tumefaciens-mediated transformation(ATMT)and the VdPRMT1 complementary strain was obtained.All the phenotypes of the complementary strain were restored to wild-type levels.It indicates that VdPRMT1 plays a key role in the growth,development and pathogenicity of V.dahliae.3.The subcellular localization of VdPRMT1 protein in V.dahliae was observed by Laser scanning confocal microscope.The distribution in the nucleus and cytoplasm of the hyphae were relatively uniform.However,there were more granular distributions in spores,which are similar to the autophagosome structure of M.oryzae spores.It is speculated that VdPRMT1 protein may be involved in the process of autophagy.4.The expression pattern of VdPRMT1 in different infection stages of V.dahliae were determined by RT-q PCR,and its expression level was greatly increased in the early stage of infection.The VdPRMT1 gene was transiently silenced by host-induced gene silencing(HIGS)in cotton seedlings.After V.dahliae inoculation,the disease index and the biomass of V.dahliae in root tissue was markedly lower than that of control,which confirmed the correlation between the VdPRMT1 gene and the pathogenicity of V.dahliae.5.The cDNA library of V.dahliae was screened in a yeast two-hybrid approach,and then verified by yeast two-hybrid assays and bimolecular fluorescence complementation(Bi FC),indicating that there is an interaction between VdPRMT1 protein and VdLuc7 protein in S.cerevisiae and V.dahliae.According to the literature,the substrate of PRMT usually has RG/RGG domains,and the arginine residue in this domain is the preferred site for PRMT methylation modification,while VdLuc7 contains seven RG/RGG domains.Therefore,it is suggested that VdLuc7 is the substrate of VdPRMT1.In this study,the biological function of VdPRMT1 of V.dahliae was preliminarily explored.It would lay a foundation for in-depth analysis of VdPRMT1 role in the pathogenic mechanism of the pathogen,and provide candidate target genes for the effective control of Verticillium wilt in cotton.