Establishment Of Immune Activation Reporting System And Its Application In Screening Of Immune Related Secreted Peptides In Arabidopsis Thaliana

Using elicitors to trigger plant immune responses to resist pathogens is a new idea for plant disease control,which is of great significance for the sustainable development of agriculture.Screening and identification of novel elicitors requires the establishment of an efficient,sensitive and simple plant immune activation reporting system.Therefore,it’s urgent to dig out the immune activation reporter genes with high response to immune elicitor signals and construct an immune activation reporting system based on these gene expression elements..In this study,the reporter genes with high response to different elicitor signals were screened from several immune-related genes in Arabidopsis,and the luciferase(LUC)transient expression vectors driven by the promoters were constructed.Based on the transient protoplast expression system of Arabidopsis thaliana mesophyll cells,the efficient transient reporting systems for immune activation were screened and established after treatment with different elicitors.Using the reporting system,the Arabidopsis immune-related secreted peptides were preliminarily screened and identified from the up-regulated genes of Arabidopsis transcripts treated by PAMP(pathogen-associated molecular pattern).The main results were as follows:(1)Arabidopsis col-0 wild type seedlings were treated with PAMP(FLG22)and DAMP(damage-associated molecular pattern)(PEP1),elicitors respectively.The expression levels of immune-related genes FRK1,PR2,CYP71B15,NHL10,PDF1.2,CYP71A13,WRKY33 and WRKY29 were detected by q RT-PCR.The results showed that WRKY33 was up-regulated most,indicating that it could respond to PTI elicitors efficiently.(2)The promoter sequences of the immune-related genes WRKY33,WRKY29,FRK1,PR2,PDF1.2 were cloned and the promoters driven LUC transient expression vectors(Promoter::LUC)were constructed and transformed into Arabidopsis protoplasts for transient expression.After treatment with different types and concentrations of PTI(PAMP-triggered immunity)elicitors(FLG22,ELF18,PEP1),the relative expression of LUC was detected.The results showed that p WRKY33 had very significant activation response to PTI elicitors at high concentration(1 μmol/L)and low concentration(100nmol/L),indicating that the p WRKY33::LUC reporter system is a plant immune activation transient reporting system that can respond to PTI elicitors efficiently and sensitively.(3)The effectors Avr Rpt2 and Avr Pphb,which were known to activate ETI,were cloned from Pseudomonas syringae DC3000,and their transient expression vectors were constructed.After transiently co-expression with the above Promoter::LUC vectors in Arabidopsis protoplasts respectively,the relative expression of LUC was detected.The results showed that the up-regulation response of p PR2 was the highest and extremely significant,and the up-regulation of p WRKY33 was also extremely significant,indicating that the p PR2::LUC reporter system can be used as a efficient plant ETI(Effector-triggered immunity)activation transient reporting system for the screening of effector-type elicitors,while p WRKY33::LUC reporting system is an efficient,broad-spectrum plant immune activation transient reporting system for screening a variety of plant immune elicitors;(4)From the up-regulated genes of col-0 wild type Arabidopsis transcriptome treated by FLG22,20 secreted peptide precursor genes were predicted by bioinformatics method according to the common structural characteristics of secreted peptide precursor genes.Eighteen of them were cloned and their transient expression vectors(HBT95:: Secreted peptide)were constructed.Using p WRKY33::LUC immune activation transient reporter system,the HBT95::Secreted peptide and p WRKY33::LUC were co-expressed in Arabidopsis protoplasts,then the relative expression levels of LUC were detected.The results showed that the expression of AT2G28460(PIP1)(positive control),AT1G02900,AT1G22890,AT1G68765,AT5G62150,AT1G13590,AT2G20070,AT1G51915,and AT2G33130 could significantly activate the p WRKY33::LUC transient reporting system.Among them,AT2G28460(PIP1),AT1G51915,and AT2G33130 had very significant activation effects,indicating that the secreted peptides encoded by the above genes might participate in the Arabidopsis immune response;(5)The plant over-expression vectors of AT1G51915 and AT2G33130 were constructed and transformed into Arabidopsis col-0 wild type plants.After resistance screening and PCR identification,T2 generation over-expressing transgenic plants were obtained,which provided material basis for the subsequent identification of the functions of these secreted peptide genes.