CsWRKY33 Regulates Citrus Resistance To Canker Through ROS Synthesis Mediated By CsRBOH5

Citrus canker（Citrus bacterial canker,CBC）is the most harmful and widespread citrus bacterial disease at present,which is caused by Citrus xanthomonas citrus subspecies（Xanthomonas citri subsp.Citri,Xcc）.Because of the difficulty and low efficiency of traditional cross breeding and mutation breeding of citrus,screening high quality resistant and susceptible genes for molecular breeding is one of the most efficient methods to prevention and treatment canker.Previous studies found that overexpression of Cs LYK6 could enhance the resistance of citrus to canker.Analysis of phosphorylated proteome sequencing and transcriptome sequencing of transgenic plants in overexpression of Cs LYK6showed that the transcriptional levels of CsRBOH5 and CsWRKY33 were significantly up-regulated caused by Cs LYK6 overexpression.The interaction between CsRBOH5 and CsWRKY33 may be exsist,but the mechanism is not clear.Therefore,after Xcc induction,the expression and localization of CsRBOH5 and CsWRKY33 were dectected.The interaction between CsRBOH5 and CsWRKY33 was analyzed by electrophoretic mobility shift assay and dual-luciferase reporter.Functional verification of CsWRKY33 and CsRBOH5 was carried out by citrus genetic transformation,and the mechanism of their regulation of citrus canker resistance was analyzed combined with physiological and biochemical results.The main results are as follows:（1）CsRBOH5 is a resistance gene to citrus canker.After Xcc induction,the expression of CsRBOH5 in susceptible variety Wanjincheng was generally lower than resistant variety kumquat.The response to Xcc in Wanjincheng was not obvious,while the expression of CsRBOH5 in kumquat increased significantly within 24 hours after infection.H₂O₂ and O₂^·-increased significantly in the Wild Wanjincheng by overexpressing CsRBOH5.These results indicate that CsRBOH5 is the key factor to ROS production.It is speculated that CsRBOH5 plays a role in the resistance of Xcc.In this experiment,6 overexpressing CsRBOH5 plants and 5 interfering CsRBOH5 plants were obtained by agrobacterium-mediated genetic transformation.There was no significant difference in phenotype between transgenic plants and wild types.The resistance of transgenic plants to Xcc was evaluated by acupuncture and injection methods.The lesion area,disease index and disease degree of overexpressed transgenic plants were lower than those wild plants,while interferingtransgenic plants were higher than-WT.This shows that CsRBOH5 is positively correlated with citrus canker resistance and CsRBOH5 is the resistance gene of citrus canker.（2）CsWRKY33 binds to CsRBOH5 promoter and activates its expression.The previous Y1H results were further verified by EMSA,and the recombinant plasmids（p ET32a-CsWRKY33）were constructed to express and purify the recombinant protein.According to CsRBOH5 promoter sequence,WT,MT and cold probe with different multiples are designed for EMSA.CsWRKY33 protein specifically binds to WT probes.The LUC experiment also showed that CsWRKY33 activated the expression of CsRBOH5 promoter.（3）CsWRKY33 is a resistance gene to citrus canker.Subcellular localization shows that CsWRKY33 is located in the nucleus,which is consistent with the localization of transcription factor.After Xcc induction,the expression of CsWRKY33 in Wanjincheng presented downward trend.On the contrary,the expression of CsWRKY33 in kumquat showed a significant upward trend in the first 36 hours.Different expression characteristics in different varieties showed that the expression level of CsWRKY33 was positively correlated with citrus canker resistance.,The content of ROS and RBOH activity in leaves had been significantly increasedin CsWRKY33-overexpressed transgenic plants.This phenomenon showed that CsWRKY33 could promote the production of ROS.In this experiment,three transgenic plants with CsWRKY33-disturbed were obtained by genetic transformation mediated by Agrobacterium tumefaciens.The expression level of CsWRKY33 was significantly down-regulated to 62%,79%and 74%,respectively.After Xcc infection,the disease symptoms of transgenic plants with CsWRKY33-disturbed were aggravated.The disease spot area and disease index were significantly higher than those of WT,indicating that CsWRKY33 is a resistance gene to citrus canker.（4）CsWRKY33 regulates citrus resistance to canker through CsRBOH5-mediated ROS synthesis.The expression of CsRBOH5 was significantly down-regulated in three transgenic-with CsWRKY33-distrubed by RT-q PCR.This common expression trend again verified the positive regulatory effect of CsWRKY33 on CsRBOH5.When CsRBOH5 was transiently overexpressed in the leaves of transgenic plants with CsWRKY33-disturbed,the expression of CsRBOH5 was incresed and the RBOH enzyme activity,H₂O₂ content and O₂^·-content also significantly increased,indicating that CsWRKY33 positively regulates the expression of CsRBOH5 by combining with CsRBOH5 promoter andinduces the production of ROS.Above indicated CsRBOH5 enhances citrus resistance to Xcc as a key regulator in plant disease resistance.（5）CsWRKY33 interacts with a MYB transcription factor.The citrus yeast library was screened using CsWRKY33 promoter as bait.Sequencing showed that a MYB transcription factor had a strong interaction with CsWRKY33 promoter.Point-to-point gyration verification also confirms the interaction.between CsWRKY33 promoter and MYB.