中国农业科学2023，Vol.56Issue(21)：4304-4317,14.DOI:10.3864/j.issn.0578-1752.2023.21.013
枸橼C-05 CmPR4A上游转录因子CmWRKY75的筛选及其抗溃疡病功能分析
Identification of the Transcription Factor WRKY75 of CmPR4A in Citron C-05 and Its Function Analysis in Resistance to Citrus Canker Disease
摘要
Abstract
[Background]Citrus canker is one of the serious citrus diseases caused by Xanthomonas citri subsp.citri(Xcc).There is currently no radical cure method for it,and few of the existing cultivars have sufficient resistance to citrus canker.Therefore,the breeding for resistant varieties is crucial for the radical cure of the disease,and the identification of resistant genes is beneficial to disease-resistant cultivar breeding.[Objective]The aim of this study was to use the resistance related gene CmPR4A to screen its upstream transcription factors,and to explore the role of transcription factors in resistance to Xcc,which could provide genetic information for the breeding of citrus disease resistant varieties.[Method]Based on the transcriptome results of Citron C-05(resistant)and Bingtang Sweet orange(susceptible)after inoculated with Xcc,and combined with the results of qRT-PCR,PR4A was differentially expressed in resistant and susceptible genotypes.Differential analysis on PR4A promoter sequence of Citron C-05 and Bingtang Sweet orange was performed using PlantCARE.Yeast one hybrid was used to screen the upstream transcription factors of PR4A.Further interaction between CmPR4A and candidate transcription factors was verified by yeast gyration test and dual-Luciferase.The expression of candidate transcription factors was detected among 8 resistant and susceptible citrus genotypes after inoculation with Xcc at 0,2,4,6,and 8 days to verify their relationship with disease resistance.By transient overexpression of candidate transcription factors in Citron C-05 and Bingtang sweet orange leaves,the expression of transcription factors and PR4A were analyzed using qRT-PCR.Xcc bacterial quantification and symptom observation were executed in transgenic leaves after 24 h inoculated with Xcc.[Result]The expression of PR4A was significantly higher in resistant Citron C-05 than that in the susceptible Bingtang sweet orange at 4,6,and 8 dpi after inoculated with Xcc.There was a difference in the cis acting element W-box in PR4A promoter between Citron C-05 and Bingtang sweet orange at-236 bp location.Therefore,the CmPR4A promoter was truncated and the bait vector was constructed.Yeast one hybrid screening was conducted using Citron C-05 yeast library induced by Xcc,resulting in CmWRKY75 could interact with proCmPR4A-2.Further dual Luciferase reporting system also confirmed that the interaction between CmWRKY75 and CmPR4A,and CmWRKY75 was positive regulating the expression of CmPR4A.Additionally,the expression of WRKY75 was significantly upregulated in resistant genotypes Citron C-05,American citron and Aiguo citron after inoculation with Xcc,while it was only slight upregulation in susceptible genotypes Bingtang Sweet orange,Shatian Yu pummelo,lemon,Nanchuan and Danna citron.Transient overexpression WRKY75 in Citron C-05 and Bingtang Sweet orange leaves revealed a significant upregulation expression of PR4A at 4 dpi of Xcc and enhanced leaf resistance to Xcc.[Conclusion]CmWRKY75 could bind to the W-box in CmPR4A promoter and positively regulate the expression of CmPR4A,resulting in enhancing leaf resistance to Xcc.Moreover,the expression of WRKY75 was induced by Xcc and showed significant upregulation in disease-resistant genotypes,which was consistent with the expression pattern of PR4A.These results indicated that the differential expression of WRKY75 in different disease-resistant and susceptible citrus genotypes influenced the expression of PR4A,which made it play a role in the resistance of Citron C-05 to canker disease.关键词
柑橘溃疡病/枸橼C-05/PR4A/转录因子/WRKY75Key words
citrus canker disease/Citron C-05/PR4A/transcription factor/WRKY75引用本文复制引用
颜培涵,邓子牛,罗健铭,郝晨星,孙紫青,叶蓉春,李益,刘恋,盛玲,马先锋..枸橼C-05 CmPR4A上游转录因子CmWRKY75的筛选及其抗溃疡病功能分析[J].中国农业科学,2023,56(21):4304-4317,14.基金项目
国家重点研发计划(2022YFD1200503)、湖南省研究生科研创新项目(CX20210671) （2022YFD1200503）
