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

doi:10.3864/j.issn.0578-1752.2023.21.013

摘要/Abstract

摘要：

【背景】柑橘溃疡病（Citrus canker）是由黄单胞杆菌柑橘致病变种（Xanthomonas citri subsp. citri，Xcc）引起的危害严重的柑橘病害之一，目前尚无根治方法，而现有栽培品种中很少有对柑橘溃疡病存在显著抗性的品种，因此，抗病品种的选育对根治该病害尤其重要，而抗性基因的发掘又非常利于抗病育种。【目的】以枸橼C-05的抗溃疡病相关基因CmPR4A为诱饵，筛选其上游转录因子，探究转录因子参与调控枸橼C-05抗溃疡病的功能作用，为选育柑橘抗病品种提供基因信息。【方法】基于前期冰糖橙和枸橼C-05叶片接种Xcc的转录组测序结果，结合实时荧光定量PCR分析PR4A在抗病和感病种质中的表达差异，使用PlantCARE对枸橼C-05（抗病）和冰糖橙（感病）PR4A启动子序列进行差异分析，利用酵母单杂交筛选PR4A上游转录因子，并使用酵母回转验证、双荧光素酶验证CmPR4A和候选转录因子的互作关系。在8种抗病和感病柑橘种质中，人工接种Xcc后，于0、2、4、6和8 d时取注射点附近的叶片，对转录因子的表达水平进行分析，验证其与抗病的关系。通过农杆菌介导瞬时转化法，将带有35S启动子的转录因子载体在枸橼C-05和冰糖橙叶片中瞬时过表达，使用qRT-PCR对转录因子和PR4A表达水平进行分析，并在瞬时过表达24 h后接种Xcc，进行Xcc菌定量和症状观察。【结果】接种Xcc的枸橼C-05和冰糖橙的转录组分析及定量PCR表达结果显示，在接种4、6和8 d时，抗病种质枸橼C-05中的PR4A表达量显著高于感病的冰糖橙。枸橼C-05和冰糖橙的PR4A启动子在-236 bp处存在顺式作用元件W-box的差异，以此为依据进行CmPR4A启动子短截并构建诱饵载体。依据自激活结果，在200 ng·mL^-1的金担子素（AbA）浓度下，以proCmPR4A-2为诱饵，在枸橼C-05受Xcc诱导下的酵母文库中进行酵母单杂交筛选，表明CmWRKY75可以与proCmPR4A-2互作，双荧光素酶报告系统也证实二者的互作关系，且CmWRKY75正调控CmPR4A的表达。在8种柑橘种质叶片接种Xcc后进行WRKY75的表达模式分析表明，WRKY75表达量在抗病种质枸橼C-05、美国枸橼AV、矮果香橼中显著上调，在感病种质冰糖橙、沙田柚及柠檬、南川香橼和丹娜香橼中只出现微量上调。在枸橼C-05和冰糖橙叶片中瞬时过表达WRKY75，发现PR4A的表达量在接种4 d时显著上调且能增强叶片对Xcc的抗性。【结论】CmWRKY75可以结合到CmPR4A启动子的W-box上，并正调控CmPR4A的表达，增强叶片对Xcc的抗性。同时WRKY75的表达受Xcc诱导，在抗病种质中呈显著上调表达，与PR4A在抗病感种质中的表达变化趋势一致，可能是上游调控因子WRKY75在不同抗病和感病柑橘种质中表达差异导致，从而使其在枸橼C-05抗溃疡病过程中起作用。

关键词: 柑橘溃疡病, 枸橼C-05, PR4A, 转录因子, WRKY75

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.

Key words: citrus canker disease, Citron C-05, PR4A, transcription factor, WRKY75

颜培涵, 罗健铭, 郝晨星, 孙紫青, 叶蓉春, 李益, 刘恋, 盛玲, 马先锋, 邓子牛. 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.

YAN PeiHan, LUO JianMing, HAO ChenXing, SUN ZiQing, YE RongChun, LI Yi, LIU Lian, SHENG Ling, MA XianFeng, DENG ZiNiu. Identification of the Transcription Factor WRKY75 of CmPR4A in Citron C-05 and Its Function Analysis in Resistance to Citrus Canker Disease[J]. Scientia Agricultura Sinica, 2023, 56(21): 4304-4317.

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用途 Use	引物名称 Primer name	引物序列 Primer sequence (5′-3′)
酵母单杂交 Yeast one hybrid	proCmPR4A-P1-AbAi-F-Sac I	CTTGAATTCGAGCTCGGGTTGCTCAAGAGCCAGATG
	proCmPR4A-P2-AbAi -F-Sac I	CTTGAATTCGAGCTCCCCTTAGCAGCCCCCGC
	proCmPR4A-AbAi -R-Xho I	AGCACATGCCTCGAGATTTTTCTCTTAATTTTTTTTAGCTTAACTTACC
	CmWRKY75-pGADT7-F-EcoR I	GGAGGCCAGTGAATTCATGGAAAATTGCCAAATGTTTTTC
	CmWRKY75-pGADT7-R-Bam HI	CGAGCTCGATGGATCCTCAAAATGGAGTGTAAATTTGCAACT
双荧光素酶 Dual-luciferase	proCmPR4A-pGreen-F-Hind III	GACGGTATCGATAAGCTTCCCTTAGCAGCCCCCGC
双荧光素酶 Dual-luciferase	proCmPR4A-pGreen-R-Bam HI	AGAACTAGTGGATCCATTTTTCTCTTAATTTTTTTTAGCTTAACTTACC
瞬时过表达 Over-expression	CmWRKY75-P1300-F-Sac I	ACGGGGGACGAGCTCATGGAAAATTGCCAAATGTTTTTC
瞬时过表达 Over-expression	CmWRKY75-P1300-R-Kpn I	GCTCACCATGGTACCTCAAAATGGAGTGTAAATTTGCAACT
实时荧光定量PCR Quantitative Real-time PCR	Actin-qPCR -F	CATCCCTCAGCACCTTCC
	Actin-qPCR -R	CCAACCTTAGCACTTCTCC
	PR4A-qPCR-F	TATGGCTGGACCGCTTTCTG
	PR4A-qPCR-R	TCTAAGCCTCCATTGGCACA
	WRKY75-qPCR-F	TGCTTTCCAAACAAGAAGCCAA
	WRKY75-qPCR-R	CAACGGTAGTAGCTTCTGGGG

基因名称 Gene name	基因ID Gene ID	转录因子家族 Transcription factor family	功能 Function
HDG1	Cme072170	HD-ZIP	茉莉酸代谢；角质层发育；花器官特性的维持 Jasmonate metabolism; Development of cuticle; Maintenance of floral organ characteristics
MADA4	Cme017590	ZF-HD	细胞分化、防御反应、调节代谢 Cell differentiation, defense response, regulation of metabolism
RMA3	Cme174580	ZF-HD	解旋酶和E3泛素连接酶活性 Activity of helicase and E3 ubiquitin ligase
WRKY33	Cme018250	WRKY family	SUMO化修饰调控植物免疫反应 SUMO modification regulates plant immune response
bHLH122	Cme111260	bHLH	表皮发育；光周期；调节气孔运动 Epidermal development; Photoperiod; Regulate stomatal movement
TGA10	Cme058810	bZIP	调节植物信号分子水杨酸和茉莉酸甲酯 Regulate plant signal molecules salicylic acid and methyl jasmonate
bZIP 60	Cme155220	bZIP	调控种子萌发；增强耐热性 Regulating seed germination; Enhanced heat resistance
WRKY75	Cme125500	WRKY	参与叶片与花瓣的衰老与脱落；参与抗病反应 Participate in the senescence and shedding of leaves and petals; Participate in disease resistance response