The Functional Study Of Proline-rich Protein PnPRPL1 Involved In The Defense Response Of Panax Notoginseng To Root Rot

Panax notoginseng(Burk.)F.H.Chen is a precious perennial medicinal herb.However,the root rot caused by pathogens such as Fusarium solani seriously affected the yield and quality of P.notoginseng.The use of pesticides to control root rot cannot completely solve the problem of P.notoginseng disease.Further research on the defense mechanism of root rot disease can help cultivate excellent resistant varieties and promote the healthy development of the P.notoginseng industry.In this study,a prolinerich protein gene(Pn PRPL1)responding to F.solani infection was mined based on previous transcription sequencing data of P.notoginseng,and the Pn PRPL1 was cloned by RT-PCR.The expression pattern of Pn PRPL1 after hormone treatments and F.solani infection was analyzed by q RT-PCR;The Pn PRPL1 recombinant protein was expressed in Escherichia coli to study its antifungal activity;The function of Pn PRPL1 in the defense response to F.solani was studied through RNAi in P.notoginseng and overexpression in tobacco.The positive regulatory transcription factors of P.notoginseng root rot,WRKYs,were obtained in previous studies,and this study further explored the molecular mechanism of WRKY on regulating Pn PRPL1.The research results of this paper are as follows:1.The c DNA sequence of the proline-rich protein gene Pn PRPL1 from P.notoginseng is 766 bp in length with an open reading frame of 444 bp,encoding a protein with 147 amino acids.The results of q RT-PCR showed that Pn PRPL1 responded to the treatment of methyl jasmonate(Me JA),ethephon(ETH),gibberellin,abscisic acid and salicylic acid(SA).The expression level of Pn PRPL1 was upregulated after F.solani infection.2.Pn PRPL1-GFP fusion protein was expressed in onion epidermis,and the subcellular localization analysis indicated Pn PRPL1 was a cell wall localization protein.The Pn PRPL1 recombinant protein was expressed in E.coli Rosetta(DE3),and the Pn PRPL1 recombinant protein significantly inhibited the growth of F.solani,Fusarium oxysporum,Curvularia trifolii,and Fusarium graminearum.The expression level of Pn PRPL1 in P.notoginseng leaves was reduced by RNAi,that caused the increased susceptibility to F.solani.The overexpression of Pn PRPL1 in tobacco enhanced the resistance to F.solani.Compared to wild type tobacco,the transgenic tobacco overexpressing Pn PRPL1 showed down-regulation of gene expression levels of reactive oxygen species(ROS)producing enzymes and up-regulation of gene expression levels of ROS cleaning enzymes,higher activities of peroxidase,superoxide dismutase,catalase and phenylalanine ammonia-lyase,and lower content of ROS as well as increased accumulation of callose.3.The promoter sequence of Pn PRPL1(PPn PRPL1)was cloned,which contains the core sequence of W-box element.The PPn PRPL1-GUS plant expression vector was constructed and transformed into tobacco,as a result,the activity of PPn PRPL1 in transgenic tobacco significantly increased after the infection with F.solani and F.oxysporum,as well as treatment with Me JA,ETH,sodium chloride,aluminum chloride and damage.The transcription factors Pn WRKY27/15/5/12/9 was transiently coexpressed with Pn PRPL1 promoter in tobacco,and it was found that the interaction between PPn PRPL1 and Pn WRKY27 was most visible.Therefore,Pn WRKY27 recombinant protein was expressed and purified in prokaryotic cells.The result of EMSA showed that Pn WRKY27 recombinant protein specifically bound to the sequence of PPn PRPL1 containing W-box.Yeast one-hybrid experiment indicated that Pn WRKY27 had transcriptional activation on PPn PRPL1.In addition,the β-glucuronidase activity of PPn PRPL1-GUS and Pn WRKY27 co-expressed transgenic tobacco was significantly higher than the that of PPn PRPL1-GUS transgenic tobacco,indicating that Pn PRPL1 was positively regulated by Pn WRKY27 transcription factor.In conclusion,Pn PRPL1 responded to plant hormones and the infection of F.solani.Pn PRPL1 is a cell wall localization protein,and its prokaryotic expression protein had antifungal activity.Pn PRPL1 enhanced the tolerance of plants to F.solani by improving the balance of oxidative stress response and the accumulation of callose.Moreover,Pn PRPL1 was regulated by the transcription factor Pn WRKY27 and participated in the defense response to F.solani.