Heat Shock Protein FpLhs1 Is Impotarnt For Conidiation And Pathogenicity Of Fusarium Pseudograminearum

Wheat Crown Rot is a soil-borne disease caused by a variety of pathogens,posing a serious threat to wheat production and food security worldwide.The pathogens mainly include Fusarium pseudograminearum,Fusarium graminearum,and Fusarium culmorum.Particularly,F.pseudograminearum caused FCR has become a serious issue in the Huanghuai wheat growing area of China.However,most research only concern its biological morphology or genetic diversity,and few reports on its pathogenic mechanism.Hsp70s(heat shock protein 70s)are a class of molecular chaperones that are highly conserved and ubiquitous in organisms ranging from microorganisms to plants and humans.Hsp70s play key roles in cellular development and protecting living organisms from environmental stresses such as heat,drought,salinity,acidity,and cold.However,their functions in F.pseudograminearum are largely unknown.In order to provide a theoretical basis for revealing the molecular mechanism of F.pseudograminearum.In our study,the ER lumenal Hsp70 protein FpLhslwas selected for further study.According to the known Hsp70 protein,a total of 14 FpHsp70 genes were identified in F.pseudograminearum,which were designated as Hsp70-1～14,including 3 in the mitochondria,7 in the cytoplasm,2 in the ER,1 in the nucleus,and 1 in the plastid.By analyzing the genetic structure of FpHsp70s,we found that the mitochondria-localized FpHsp70s have 1-4 introns,and the cytoplasm-localized FpHsp70s have 1-8 introns.Other localized FpHsp70s have no obvious rules.In addition,the protein motifs of the FpHsp70 have no regularity in the same subfamily,either.The transcriptome data showed that the expression level of FpHsp70s in the hyphae of F.pseudograminearum was different.Except for FpHsp70-3 and FpHsp70-6,all other FpHsp70s were induced to express in the infection stage,among which FpHsp70-12 The expression was highest in both hyphae and infection stages,while FpHsp70-4 induced the highest fold in the infection stage,reaching 1383 to 5196 times.Two endoplasmic reticulum-localized Hsp70 proteins,FpLhs1 and FpKar2,were induced to express 2 to 5 fold during the infection phase.The results of expression analysis showed that most of FpHsp70 may be involved in the pathogenic process of F.pseudograminearum.Studies have shown that secreted proteins are very important in the pathogenesis of plant pathogenic fungi,and endoplasmic reticulum is a key site for protein secretion,so we selected two endoplasmic reticulum-localized FpHsp70 proteins for further functional analysis.To explore potential effects of these two proteins of FpLhsl and FpKar2 in F.pseudograminearum,the target gene replacement construct was generated by the split marker approach and transformed into the wild type strain WZ-8A using polyethylene glycol(PEG)-mediated protoplast stable transformation method.In this study,two putative △fplhsl mutants,T3 and T10 were further confirmed by PCR and re-sequencing analysis.The FpLhsl-GFP fusion construct under the control of its native promoter was transformed for genetic complementation.The putative complementation was examined by G418 resistance screening,PCR and GFP fluorescence.In addition,we also try to get the Afpkar2 mutant,but multiple tests were failed.The FpLhsl protein localization showed that FpLhsl was localized on the endoplasmic reticulum consistent with the predicted results.Comparing with wild-type and complement strains strains,the biological phenotype of △fplhsl mutant showed that the growth rate decreased,but the colony and mycelial morphology did not change significantly;The yield of conidia was significantly reduced,and the conidia were significantly shorter,with the rounded ends;the number of inner membranes was reduced,and the rate of conidial germination was significantly reduced.Wheat hypocotyls,barley leaves and potted inoculation experiments showed that the pathogenicity of△fplhsl mutant was significantly reduced compared with wild-type and complement strains,and the infecting and spreading ability was significantly blocked.To find out the possible mechanism of FpLhsl in the pathogenesis of F.pseudograminearum,we test the tolerance of △fplhsl mutant in Congo red,copper sulfate,hydrogen peroxide,SDS and manganese chloride.There was no significant change in the growth of the △fplhsl mutant compared to the wild type and the complement strain.Sequencing the secretory proteomes of wild-type strains and △fplhsl mutants showed that 60 proteins were significantly reduced in the secretory proteomes of △fplhsl mutants.Six of them were selected for real-time quantitative fluorescence PCR analysis.Compared with wild-type strains,their expression in △fplhsl mutants did not change significantly,suggesting that the deletion of FpLhsl affected the secretion of some proteins.All of the results indicate that endoplasmic reticulum-localized heat shock protein FpLhs1 is involved in the growth,conidiation and pathogenesis of F.pseudograminearum,and affects the secretion of pathogenic proteins.This study will provide a theoretical basis for the analysis of the molecular mechanism of F.pseudograminearum.