Preliminary Functional Identification Of CsWRKY27 In Cucumber Disease Resistance And Screening Of Its Interacting Factors

Cucumber（Cucumis sativus L.）belongs to Cucumis,an annual crop of Cucurbitaceae.However,various diseases pose a serious threat to cucumber yield and quality.Although fungicides can slow down the impact of diseases on production,the abuse of fungicides has led to the continuous evolution of various bacteria,resulting in their increasing drug resistance;Moreover,the abuse of fungicides will not only pollute the soil,but also cause pesticide residues on fruit,which is a serious threat to human health.Therefore,breeding disease resistant varieties is the most economical,safe and effective way,but the conventional traditional breeding takes a long time,cumbersome steps and heavy workload.With the completion of the whole genome sequencing of cucumber,it is possible to clarify the resistance and susceptibility mechanism of cucumber to pathogens at the molecular level,which will be conducive to the genetic breeding of cucumber disease resistance.In this paper,CsWRKY27 was mined because of its differential expression in the transcriptomes done previously in our lab.The function and disease resistance mechanism of the gene were studied to enrich the theoretical basis of cucumber disease resistance mechanism.The main results are as follows:1.By analyzing the transcriptomes between resistant and susceptible cucumber lines in our research group,a total of six WRKYs differentially expressed were identified.Semi quantitative analysis further confirmed that the expression of these six WRKY genes were induced by downy mildew pathogen infection.The protein interaction network of differentially expressed genes identified in the transcriptomes showed that CsWRKY27,as a hub gene,could interact with two WRKY genes,LRR-RK and regulators of salicylic acid synthesis,respectively.The phylogenetic tree analysis showed that the homologous genes of CsWRKY27 were WRKY70 and WRKY54 in Arabidopsis.In previous studies,it was found that WRKY70 and WRKY54 in Arabidopsis played important roles in responding to biotic stresses.According to the above results,CsWRKY27 was chosen as the target gene to be further studied.2.Subcellular localization analysis showed that CsWRKY27 was localized in the nucleus.Through the infection of downy mildew to the resistant inbred line IL51 and the susceptible IL53,it was found that WRKY27 showed an upward trend in IL53 with the extension of infection time,but a downward trend in resistant variety IL51.In the detection of WRKY27 expression in different organs,it was found that there were significant differences in roots,stems,leaves,flowers and fruits,and the expression in leaves was the highest.3.The cucumber cotyledons were inoculated with 35S:WRKY27 for transient expression,then were infected by Botrytis cinerea and downy mildew,respectively.It was found that the overexpression of CsWRKY27 enhanced the resistance of the cucumber cotyledons to Botrytis cinerea and the sensitivity to downy mildew.The ion leakage rate also were consistant with their phenotypes.Me JA could increase the resistance of cucumber leaves to Botrytis cinereal,and SA could not.At 24 H after transient overexpression of CsWRKY27,the cucumber cotyledons were inoculated with Botrytis cinerea,and then the concentrations of SA and JA were detected.The results showed that overexpression of CsWRKY27 could not cause the change of SA content,but could significantly increase SA content after inoculation with Botrytis cinerea,which indicated that CsWRKY27 could enhance the accumulation of SA caused by Botrytis cinerea infection,.The content of JA is similar to that of SA.4.Using yeast one hybrid,we screened the upstream genes of CsWRKY27,and obtained a total of 384 genes,including 18 transcription factors.We selected 10 of the 18 genes for confirmation.Besides one,the nine transcription factors could bind to the promoter sequence of WRKY27,including zinc finger protein,MYB,b HLH,ERF and sigmafactor.5.Using yeast two hybrid,we screened the the interaction proteins of CsWRKY27,and obtained a total of 55 genes,including 29 genes located in nucleus,16 genes located in non nucleus,and the remaining 10 genes that where their subcellular localization are unknown.We selected seven genes related to resistance and located in nucleus as candidate genes.Of which,four proteins were confirmed to interact with CsWRKY27,and then their interactions were also verified by Bimolecular Fluorescence Complementation（Bi FC）.These data laid a foundation for revealing the regulatory network of CsWRKY27 in the cucumber disease resistance.