Functional Analysis Of Four Key Genes In The TCHK Signaling Pathway In Pyrenophora Graminea

Barley leaf stripe is a seed-borne pathogen caused by Pyrenophora graminea and mainly distributed in Europe,the United States,Australia,Canada,India,and China’s northwest,northeast and other main production areas of barley.The incidence rate of the disease in the serious year is over 60%,and the yield reduction is more than 73%.At present,the most economical and effective prevention measures in production are to select disease-resistant varieties,establish disease-free reserved fields and sow disease-free seeds.However,resistant cultivars generally lost their resistance owing to the emergence of new virulent pathotypes.Therefore,it is imperative to understand the genetic basis of virulence and the inheritance of pathogenicity.Which can provide theoretical guidance for resistance breeding of barley leaf stripe.The two-component histidine-kinase(TCHK)signaling pathway is involved in a variety of life activities of fungi,such as regulation of pathogenicity,growth and development,antimicrobial sensitivity and osmotic stress response.The elements of the TCHK signaling pathway have never been characterized in P.graminea.In this study,we analysed 4 key genes in the TCHK signaling pathway in Pyrenophora graminea,and the main results were as follows:1.Cloned four important genes in TCHK signal pathway in Pyrenophora graminea: the HPK(Histidine protein kinase)encoding gene pgsln,RR(Response regulator protein)encoding gene pgssk1,MAPKK kinase encoding gene pgssk2 and MAPK kinase encoding gene pgpbs.The protoplast regeneration and transformation system of Pyrenophora graminea were established.To obtain the mutants of Pyrenophora graminea,we used the pSilent-1 vector and pBluescript Ⅱ KS(-)vector to constrct mutants.The interference vectors of pgssk1,pgssk2 and pgpbs were constructed and knockout vector pCM-hyg-PGSLN of pgsln was constructed by RNAi technology.2.In this study,we investigated functions of the four important TCHK components: pgsln,pgpbs,pgssk1 and pgssk2 in P.graminea.First,Mutant strains △pgsln and △pgpbs grew slowly and formed seriously distorted hyphal and swelling tips in P.graminea.,it indicated that pgsln and pgpbs took part in vegetative growth of mycelium.Second,the mutants of △pgsln,△pgssk1,△pgssk2 and △pgpbs showed increased sensitive to osmotic stress(sorbit or glycerol),the mutants △pgpbs and △pgssk2 showed increased sensitive to ion stress(Na),mutant △pgsln showed increased sensitive to ion stress(Co)compared to wild type train.Which showed pgsln、pgpbs、pgssk1and pgssk2 played key role in osmotic stress of mycelium.Third,the mutants of △pgsln and △pgpbs showed increased tolerance to fungicide stress of DCFs(Dicarboximides fungicide),the mutants of △pgsln showed increased tolerance to carbendazim and fludioxonil stress,the mutants of △pgssk2 showed increased tolerance to prochloraz stress compared with wild type strains,thus deduced pgsln and pgpbs might be as the target of DCFs,pgsln might be as the target of carbendazim and fludioxonil,and pgssk2 might be as the target of DMI.Fourth,the mutant △pgsln showed increased resistant to Congo red and SDS,increased sensitive to CFW,decreased content of chitin and increased release rate of protoplast compared with wild type strains.The mutants △pgpbs showed increased tolerance to CFW,increased content of chitin and decreased release rate of mycelial protoplast compared with wild type strains,and the chitin was dispersed in the lateral edge of cell wall in mutants △pgpbs.The mutants △pgssk1 showed increased sensitive to CR,SDS,CFW,decreased chitin content and increased protoplast release ratecompared to wild type strains.It showed that pgsln,pgpbs and pgssk1 took part in the regulation of cell integrity.3.Transcriptomic analysis of the wild strain QWC and Δ pgpbs1 mutant strains.There were 890 different expression genes in Δpgpbs compared with the wild strain,242 upregulated genes and 648 down-regulated genes.Among these,242 up-regulated genes and 648 down-regulated genes were detected.PGPBS regulates the pathogenicity of barley stripe disease by affecting the metabolism,transport,chitinase metabolism and the transcription of pathogenic genes of P.graminea.4.The preliminary localization of pgsln,pgssk1,pgssk2 and pgpbs in TCHK signal pathway.Fluorescence quantitative results showed that the expressions of pgpbs in mutants △pgsln,△pgssk1 and △pgssk2 were lower than that in WT,different stresses can stimulate the increase of pgpbs expression in wild WT,while the changes of pgpbs expression in mutant strains is not significant.Therefore,it was speculated that mutations of pgsln,pgssk1 and pgssk2 genes in TCHK pathway effected the expression of pgpbs.We decided that the genes were located at the up stream of pgpbs in TCHK signaling pathway.It was preliminarily speculated that the location relationship of the four genes was: PGSLN(HPK)→pgssk1(RR)→pgssk2(mapkkk)→ PGPBS(mapkk)or PGSLN(HPK)→pgssk2(mapkkk)→pgssk1(RR)→ PGPBS(mapkk).