Abstract:

【Objective】Non-expressor of pathogenesis-related genes 1 (NPR1) is an important transcription factor in salicylic acid (SA)-mediated signal transduction of systemic acquired resistance and plays a key role in regulating plant disease resistance. The objective of this study is to evaluate the resistance of CiNPR4, a NPR1-like gene from Huanglongbing-tolerant ‘Jackson’ grapefruit (Citrus paradisi), to citrus canker and preliminarily explore the resistance mechanism of CiNPR4 transgenic plants to citrus canker.【Method】The fully mature leaves from Huanglongbing-resistant CiNPR4 transgenic citrus plants were selected to challenge the Xanthomonas citri subsp. citri (Xcc) pathogen via in vitro pin-prick inoculation. Then, the CiNPR4 transgenic plants with enhanced resistance to citrus canker were inoculated with Xcc in vitro by the pin-prick method and then counted the number of Xcc at 0, 1, 3, 5, 7 and 9 d after inoculation. The leaves of CiNPR4 transgenic plants with enhanced resistance to citrus canker were inoculated with Xcc by injection and collected at 0, 3 and 5 d after inoculation. The contents of SA and jasmonic acid (JA) in the leaves were measured. Meanwhile, the expression level of defense-related genes CsPR1 and CsPDF1.2 mediated by SA and JA, respectively, was analyzed by quantitative real-time PCR. According to the interaction network of CiNPR4 protein and TGA transcription factor, it is predicted that Ciclev10005080m and Ciclev10001081m can interact with CiNPR4, respectively, these two genes were analyzed by blastx on the website https://www.citrusgenomedb.org/ to predict the candidate proteins that interact with CiNPR4 in the sweet orange genome. Then, the cDNA sequences of CiNPR4 and candidate protein genes were cloned, and inserted into the pGBKT7 and pGADT7 plasmid to construct the bait and prey vectors by homologous recombination method, respectively. After that, the bait and prey plasmids were co-transformed into yeast strain Y2HGold for point-to-point yeast two-hybridization analysis.【Result】The disease resistance evaluation in vitro showed that overexpression of CiNPR4 alleviated the symptoms of citrus canker on transgenic citrus plants. Xcc grew slowly in the leaves of CiNPR4 transgenic plants with enhanced resistance to citrus canker during the entire observation period, and the number of Xcc in the transgenic plants was significantly lower than that of wild-type (WT) plants at 9 d after inoculation. The SA contents among the transgenic citrus lines and WT plants were not significantly different at 0 d. However, with the prolongation of Xcc induction time, the SA content in CiNPR4 transgenic plants significantly increased. The JA content in WT plants was significantly higher than that in CiNPR4 transgenic plants at 0 d after inoculation, after that, the JA content in CiNPR4 transgenic plants gradually increased and was significantly higher than that in WT plants at 5 d after Xcc induction. However, the SA and JA contents in WT plants were not significantly changed during the entire induction period. The expression level of CsPR1 was significantly increased in the transgenic plants with increased canker resistance than that in WT plants after Xcc induction for 3 d, and the expression level of CsPDF1.2 in the above-mentioned transgenic plants increased at 5 d after inoculation. However, WT plants exhibited no significantly different expression of CsPR1 after Xcc induction, but had an increased expression level of CsPDF1.2 at 5 d after inoculation, which was significantly higher in WT plants than that in the CiNPR4 plants. The interaction between CiNPR4 and CsTGA2 was confirmed by the yeast two-hybrid analysis.【Conclusion】These results indicated that CiNPR4 interacts with CsTGA2 to regulate the resistance of transgenic plants to citrus canker by promoting SA- and inhibiting JA-mediated defense response.

Key words: CiNPR4, citrus canker, salicylic acid, jasmonic acid, TGA transcription factor