Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (21): 4304-4317.doi: 10.3864/j.issn.0578-1752.2023.21.013

• HORTICULTURE • Previous Articles     Next Articles

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

YAN PeiHan(), LUO JianMing, HAO ChenXing, SUN ZiQing, YE RongChun, LI Yi, LIU Lian, SHENG Ling, MA XianFeng, DENG ZiNiu()   

  1. College of Horticulture, Hunan Agricultural University/Engineering Research Center for Horticultural Crop Germplasm Creation and New Variety Breeding, Ministry of Education/National Center for Citrus Improvement (Changsha), Changsha 410128
  • Received:2023-04-06 Accepted:2023-06-09 Online:2023-11-01 Published:2023-11-06
  • Contact: DENG ZiNiu

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

Table 1

Primers used in this study"

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
proCmPR4A-pGreen-R-Bam HI AGAACTAGTGGATCCATTTTTCTCTTAATTTTTTTTAGCTTAACTTACC
瞬时过表达
Over-expression
CmWRKY75-P1300-F-Sac I ACGGGGGACGAGCTCATGGAAAATTGCCAAATGTTTTTC
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

Fig. 1

PR4A expression level comparison between resistant and susceptible citrus genotypes in leaves after Xcc inoculation A: Expression levels of PR4A in the RNA-seq of Citron C-05 and Bingtang Sweet orange; B: qRT-PCR detection of PR4A in Citron C-05 and Bingtang Sweet orange after inoculation with Xcc. * indicate significant difference (P<0.05). The same as below"

Fig. 2

Construction and self-activation verification of CmPR4A bait vector A: Schematic diagram of the short fragment of the CmPR4A promoter of Citron C-05; B: Cloning of the short fragment of the CmPR4A promoter; C: Self-activation validation of the proCmPR4A-1-pAbAi (0--516 bp) bait vector; D: Self-activation validation of the proCmPR4A-2-pAbAi (0--317 bp) bait vector"

Table 2

Screening results of CmPR4A upstream transcription factors in Xcc-induced Citron C-05 yeast library"

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

Fig. 3

Rotary verification in Yeast one-hybrid The positive control involves the interaction between p53-AbAi and pGADT7-53; The negative control involves the interaction between p53-AbAi and pGADT7-T"

Fig. 4

Verification of interaction between CmWRKY75 and proCmPR4A A: Yeast one-hybrid assay was used to verify the interaction between proCmPR4A-2 and CmWRKY75; B: Dual-luciferase reporter assay was employed to validate the interaction between proCmPR4A and CmWRKY75"

Fig. 5

Heatmap analysis of WRKY transcription factor family expression in citrus induced by Xcc and verification of its interaction with CmPR4A A: Expression heatmaps of WRKY family genes under Xcc infection in Bingtang Sweet orange and Citron C-05; B: Validation of the interaction between proCmPR4A-2-pAbAi and 9 differentially expressed WRKY family genes by yeast one hybrid. The red arrow indicates the differentially expressed WRKY transcription factors between Citron C-05 and Bingtang Sweet orange induced by Xcc"

Fig. 6

WRKY75 expression pattern of different citrus genotypes leaves after Xcc inoculation"

Fig. 7

Expression of WRKY75 and PR4A after transient expression of CmWRKY75 in Citron C-05 and Bingtang Sweet orange"

Fig. 8

The growth of Xcc and symptom observation after transient expression of WRKY75 in Citron C-05 and Bingtang Sweet orange A, B: Citron C-05; C, D: Bingtang Sweet orange"

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