Signal Transduction Of PsnWRKY70 Gene On Stress Response In Populus Simmonii × Populus Nigra

WRKY proteins widely regulated the growth,development,senescence and stress response progresses of plant,and they constituded one of the largest plant stress responsive transcription factor(TF)families.Therefore,it is very significative to explore the functions and stress response mechanisms of WRKY TFs.According to our previous transcriptome sequencing results,20 Populus simonii × Populus nigra WRKY(PsnWRKY)genes which belonged to WRKY Group ?,? and ? subfamilies were selected as objects to conduct quantitative real-time PCR(qRT-PCR),and the results indicated that PsnWRKY70 gene not only can response to abiotic stress but also can response to biotic stress.In order to further explore the functions and response mechanisms of PsnWRKY70 gene/PsnWRKY70 TF(Group? member of P.simonii × P.nigra WRKY superfamily)under biotic and abiotic stresses,we performed a series of molecular biological and physiological experiments which included yeast one hybrid,yeast two hybrid,genetic transformation,stress resistance/tolerance analysis and transcriptome sequencing in the present study.First,the PsnWRKY70 promoter was cloned by genome walking method,the subsequent sequencing results and in silico sequence analysis suggested that lots of stress responsive cis-elements(such as W-box,GT1,MYBST1,MYCCONSENSUSAT,CURECORECR and ERELEE4 etc.)were enriched in the upstream of the encoding area of PsnWRKY70 gene,and these inforamtion implied the potential importance of PsnWRKY70 gene in the stress response signal transduction networks in P.simonii × P.nigra.To further seek the upstream regulation factors of PsnWRKY70 gene and the interactive proteins of PsnWRKY70 TF,a salt stress treated pGADT7-DEST cDNA library of P.simonii × P.nigra was constructed to perform yeast one hybrid and yeast two hybrid screenings.Yeast one hybrid screening and transient transformation verification in tobacco demonstrated that PsnWRKY70,PsnNAM,PsnMYB and PsnGT1 proteins regulated the expression of PsnWRKY70 gene by binding to the W-box or GT1 elements to response to salt stress under plant cellular environment.Yeast two hybrid screening and GST-Pull Down in vitro verification indicate that HP1(Hypothetical protein 1,contained a clpP domain),RRM(RNA recognition motif-containing protein)and Ulp1(Ulpl protease)proteins could directly bind to PsnWRKY70 TF to response to salt stress.After A.tumefaciens-mediated leaf disc transformation in P.simonii × P.nigra,8 PsnWRKY70-overexpressing(OEX)and 10 PsnWRKY70-repressing(REX)lines were finally achieved.Then NaCl(high salinity stress),PEG-6000(drought stress)and Alternaria alternata(Fr.)Keissl(leaf blight disease stress)treated transgenic and non-transgenic(NT)lines were subjected to perform stress resistance/tolerance analysis.According to phynotypic observation,plant height measurements,leaf injury index survey,photosynthesis parameters detection and malondialdehyde(MDA)content detection,OEX lines exhibited stronger leaf blight disease resistance than NT,while REX lines displayed stronger salt and drought tolerance than NT.Moreover,according to the stress responsive expression pattern analysis of PsnWRKY70 gene,on one hand,PsnWRKY70 TF probably acted as a positive regulation factor in leaf blight disease responsive progress,on the other hand,it possibly played negative regulation role in salt and drought response signal transduction networks.Then transcriptome sequencing was conducted on salt stress treated NT,OEX1 and REX1 lines.According to the sequencing data analysis,except for the normal salt stress responsive genes,there were 6 pathogen responsive genes among the differentially expressed genes(DEGs)from NT vs OEX1.The subsequently performed qRT-PCR verification and promoter sequence analysis suggested the 6 pathogen responsive DEGs are possible downstream target genes of PsnWRKY70 TF.Additionally,there were a lot of MAPK cascade members among all the DEGs from NT vs OEX1 and NT vs REX1,and in consideration of the previously published reports,we speculated that the corresponding proteins of these MAPK cascade DEGs probably response to salt stress through interacting with some stress responsive WRKY TFs(including PSnWRKY70)in P.simonii × P.nigra.Taken together,we selected Psn WRKY70 gene and its corresponding protein PsnWRKY70 TF as the objects,and orderly conducted biotic and abiotic stress responsive temporal expression pattern analysis,promoter functional research,gene functional research and stress response signal transduction pathway exploration in the present study.The results revealed the functions of PsnWRKY70 gene under high salinity,drought and leaf blight disease stresses,and summarized the stress response signal transduction pathway that PsnWRKY70 TF participated in.Moreover,the finally achieved results and conclusions in this study not only provide valuable clues for the completion of the huge and intricated stress response signal transduction netwoks but also provide alternative germplasm resources and theoretical support for stress resistance/tolerance breeding work in the near future in P.simonii x P.nigra.