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

Abstract

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

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

Identification of the Transcription Factor WRKY75 of CmPR4A in Citron C-05 and Its Function Analysis in Resistance to Citrus Canker Disease

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