The pH plays a key role in the growth and colonization of plant pathogens as well as the onset and progression of the symptoms they cause within the host. Plants may quickly alter their apoplastic pH (pHapo) to protect themselves against infection. However, pathogens can also alter the pH of their ambient environment to promote their own growth. Citrus canker is a serious plant disease caused by Xanthomonas citri subsp. citri (Xcc). This Gram-negative aerobic rod is usually cultured in Luria–Bertani (LB) medium at pH 7. However, little is known about the changes in pH both in this medium as Xcc grows and in the leaf apoplast in response to Xcc infection and colonization. Moreover, the differences in leaf apoplast pH between Xcc-resistant and Xcc-susceptible citrus genotypes are also unknown. Here, Xcc grew well in liquid LB medium at initial pH 6–8 and the pathogen altered the medium pH to 6.8 ± 0.4. Xcc growth declined at pH 5 and was zero at pH 3, 4, 9, and 10. In susceptible sweet orange infected with Xcc inoculum, canker symptoms were inhibited at pH 3, 4, and 10 but did not differ in the range of pH 5–9. As expected, canker symptoms were absent at all inoculum pH in the resistant Citron C-05. For both genotypes, Xcc only grew well in the leaves exposed to pH 5–8 inoculums. At four days post-inoculation (4 dpi), the foliar pHapo of resistant Citron C-05 had rapidly declined from 5.6 to 4.4. At 2 dpi, the pHapo of susceptible sweet orange had rapidly increased from 5.6 to 6.7, Xcc grew quickly, and canker symptoms appeared. Plasma membrane (PM) H+-ATPase activation with fusicoccin (FC) acidified the apoplast and upregulated the pathogenesis-related genes (PRs) in the sweet orange leaves. Hence, Xcc colonization and canker development were inhibited. The results of this study revealed that apoplastic acidification is implicated in the resistance of Citron C-05 to Xcc infection and provided insight into the association between pHapo regulation and resistance to bacterial pathogen invasion in plants.
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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.
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.
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.
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.
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.
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