The Transcription Factor PnWRKY15 Cooperating With Jasmonic Acid Signaling Positively Regulated The Resistance Of Panax Notoginseng To Root Rot

Panax notoginseng(Burk)F.H.Chen is a precious traditional Chinese medicine.However,the root rot caused by Fusarium solani and other fungi seriously affected medicinal value of P.notoginseng.WRKY transcription factors play an important role in plant defense response.A WRKY transcription factors PnWRKY15 responsived to jasmonic acid and F.solani was cloned from P.notoginseng and its function was studied in this study.Firstly,the subcellular localization of PnWRKY15 was analyzed;then PnWRKY15 was overexpressed in model plant tobacco(Nicotiana tabacum)to verify its resistance to F.solani and analyze the expression level of resistance-related genes;RNA sequencing and hormone content determination were carried out in PnWRKY15 transgenic tobacco.The promoter of root rot resistance gene PnOLP1 was cloned and analyzed.At the same time,the prokaryotic recombinant protein of PnWRKY15 was expressed and purified,and its binding to W-box on the promoter of PnOLP1 was analyzed through electrophoretic mobility shift assay.The trans-activation effect of PnWRKY15 on PnOLP1 promoter was verified by yeast one-hybrid assay.In addition,the PnOLP1 promoter was transferred into PnWRKY15 transgenic tobacco to further confirm the transcriptional regulation between PnWRKY15 and PnOLP1.The main results of this paper are as follows:1.The PnWRKY15 was cloned according to previous RNA sequencing data.The ORF frame is 420 bp and encodes a 15.87 k Da protein containing 139 amino acids and the protein isoelectric point is 7.51.A fusion expression vector of PnWRKY15-GFP was constructed,and the fluorescence localization was observed by laser confocal microscopy,which indicated that PnWRKY15 protein is localized in the nucleus.The expression levels of genes related to jasmonic acid biosynthesis pathway and disease resistance in PnWRKY15 transgenic tobacco implied that PnWRKY15 cooperated with jasmonic acid pathway to regulate disease-resistance-related gene expression.Moreover,RNAi assay results reconfirmed that PnWRKY15 is a positive regulator of P.notoginseng against F.solani infection.2.RNA sequencing was performed on PnWRKY15 transgenic tobacco and wild type tobacco.KEGG analysis showed that there were a large number of differentially expressed genes in plant hormone signal transduction pathway,plant-pathogen interaction pathway,phenylpropanoid biosynthesis pathway and photosynthesis pathway.Compared with wild-type tobacco,a series of genes related to plant hormone signal transduction pathway was up-regulated in PnWRKY15 transgenic tobacco.The expression of positive regulatory genes related hypersensitivity and defense response in PnWRKY15 transgenic tobacco increased,while the expression of negative regulatory genes decreased.These results suggested plant hormone signal transduction pathway and plant-pathogen interaction pathway are activated.Additionally,the activated phenylpropanoid biosynthesis pathway and the repressed photosynthesis pathway further confirmed that PnWRKY15 positively regulated disease-resistant defense response.Moreover,the content of jasmonic acid in PnWRKY15 transgenic tobacco was significantly higher than that in wild-type tobacco,indicating that PnWRKY15 in synergy with jasmonic acid signal pathway enhanced plant defense response.3.To identify the transcriptional regulation mechanism of PnWRKY15 on diseaseresistance-related genes in the P.notoginseng,the promoter of P.notoginseng osmotinlike protein gene PnOLP1 was cloned.Sequence analysis revealed that PPnOLP1 contained a W-box.The p BI121-PPnOLP1-GUS expression vector was transformed into wild-type tobacco,and hormones including methyl jasmonate,salicylic acid,abscisic acid,and gibberellin were found to be able to induce PPnOLP1 expression in the p BI121-PPnOLP1-GUS transgenic tobacco.A prokaryotic expression vector of PnWRKY15 was constructed,and His-PnWRKY15 recombinant protein was induced and purified for electrophoretic mobility shift assay,which showed that PnWRKY15 protein could specifically bind to the PPnOLP1 fragment containing W-box.To validate the trans-activating effect of PnWRKY15,yeast one hybrid assay was performed and revealed that PnWRKY15 has transcriptional activating effect on PnOLP1 promoter.PPnOLP1-GUS expression vector was constructed and transformed into PnWRKY15 transgenic tobacco,and the interaction betweeen PnWRKY15 and PPnOLP1 was analyzed by GUS activity.The results suggested that the PnWRKY15 transcription factor positively regulates the expression of PnOLP1 by transcriptionally activating PPnOLP1 through specific binding to the W-box.In summary,PnWRKY15 synergized with the jasmonic acid signal pathway to regulated the expression level of the root rot resistance gene PnOLP1 and enhanced the resistance of P.notoginseng to the root rot pathogen F.solani.This paper reveals the function of P.notoginseng WRKY transcription factor 15 and its regulation mechanisms in P.notoginseng growth-defense balance,which can help to further understand the transcriptional regulatory mechanism of molecular interaction between P.notoginseng and root rot pathogen.