Involvement Of NPR1 In H₂S Mediated Systematic Acquired Resistance Of Cucumber

During long-term evolution,plants have developed salicylic acid（SA）mediated systemic acquired resistance（SAR）to resist pathogen invasion.SA signal induces interaction between NPR1 and transcription factors such as WRKY and TGA to regulate the expression of Pathogenesis Related Protein（PRP）,which is its main mode of action.There are many researches on the involvement of gas signaling molecule H₂S in plant responses to abiotic stresses,but there are few related researches on response and resistance to biological stresses.Previous researches showed that SA processes and activates endogenous H₂S signal in Arabidopsis thaliana.H₂S pretreatment activated the expression of Pathogenesis Related Protein genes（PR1,PR4）and inhibited the growth of Botrytis cinerea in cucumber leaves,but the molecular mechanism was unclear.This research used cucumber 9930 to investigate the role of NPR1 transcription factor in the production of systemic acquired resistance of H₂S signal involved.By cloning Cs NPR1 transcription factor,the physiological and molecular mechanisms of the Cs NPR1transcription factor in the H₂S signal participating in the protein expression related to cucumber pathogenesis and enhancing the resistance to gray mold were explored,so as to provide help for oriented analysis and breeding of cucumber in the disease resistance process.The main experimental results are as follows:1.After cucumber was fumigated with H₂S and inoculated with Botrytis cinerea,the changes of disease resistance related indexes of cucumber in 72 h were analyzed.The results showed that H₂S treatment could significantly reduce the content of H₂O₂and MDA in cucumber leaves after inoculation,which could alleviate the oxidative damage caused by bacterial invasion.H₂S treatment can increase the soluble sugar content of the whole plant,maintain the energy balance of the plant to a certain extent,and enhance the ability of the plant to resist pathogenic bacteria.H₂S treatment can activate the expression of NPR1,indicating that NPR1 responded to the plant disease resistance process induced by H₂S.2.Constructing prokaryotic and eukaryotic expression vectors of NPR1;The dissocation degree of NPR1 oligomer was significantly increased when the recombinant protein was treated with H₂S,indicating that H₂S could affect the polymerization of NPR1oligomer and monomer.The transient expression system of cucumber leaves transformed with NPR1 eukaryotic expression vector showed that the expression levels of disease resistance genes PR1 and PR4 in cucumber increased,indicating that NPR1 could promote the expression of downstream disease resistance genes.3.The yeast two-hybrid system showed that there was an interaction between NPR1and WRKY46,and the interaction between them could not be enhanced by exogenous application of H₂S.The expression vector of WRKY46 was constructed and the recombinant WRKY46 protein was obtained,which provided a basis for further research on the mechanism of NPR1 and WRKY46 in regulating the expression of PR1 and PR4.4.In order to detect the bacteriostatic effect of PR1 and PR4,eukaryotic expression vectors of PR1 and PR4 were constructed and transformed into Arabidopsis thaliana.At present,PR4 gene overexpressing plants were screened out and identified at DNA level,which can provide plant materials for subsequent experiments.Combined with the previous researches,it is shown that H₂S affects the polymerization state of NPR1,a key transcription activator in the SAR signal transduction pathway,which changes the redox state of Cys of NPR1,transforming it from an inactive oligomer to an active monomer,thereby promoting the overexpression of downstream disease resistance genes PR1 and PR4,and enhancing the disease resistance of cucumber plants.However,the roles of NPR1 and WRKY transcription factors in the regulation of PR1 and PR4 expression need to be further studied.This research provides experimental basis and theoretical support for revealing the mechanism of systemic acquired resistance of cucumber and the breeding selection of cucumber varieties with disease resistance.