转CiNPR4基因柑橘抗溃疡病的机制解析

doi:10.3864/j.issn.0578-1752.2021.18.007

摘要/Abstract

摘要：

【目的】病程相关基因非表达子1（non-expressor of pathogenesis-related gene 1,NPR1）是水杨酸（salicylic acid,SA）介导的系统获得性抗性信号转导途径中一个重要的转录因子,在调节植物的抗病方面发挥着关键性的作用。研究耐黄龙病的‘Jackson’葡萄柚（Citrus paradisi）中的NPR1-like基因CiNPR4对柑橘溃疡病的抗性,解析过表达CiNPR4的转基因晚锦橙（C. sinensis）抗柑橘溃疡病的机理。【方法】选取黄龙病抗性增强的CiNPR4转基因柑橘,采取完全成熟的叶片利用针刺法进行柑橘溃疡病的离体抗性评价分析;在此基础上,对溃疡病抗性增强的CiNPR4转基因植株利用针刺法离体接种溃疡病菌（Xanthomonas citri subsp. citri,Xcc）,统计接种Xcc后0、1、3、5、7和9 d的细菌数量;利用注射法对溃疡病抗性增强的CiNPR4转基因植株叶片接种Xcc,接种后0、3和5 d收集接种部位的叶片,测定叶片中SA和茉莉酸（jasmonic acid,JA）含量;同时,收集接种Xcc后0、3和5 d接种部位的叶片,利用实时荧光定量 PCR分析SA和JA分别介导的防御反应相关基因CsPR1和CsPDF1.2在Xcc诱导下表达水平的变化;根据CiNPR4蛋白与TGA转录因子相互作用网络预测CiNPR4互作蛋白为Ciclev10005080m和Ciclev10001081m,以甜橙为参考基因组,将这两个基因在https://www.citrusgenomedb.org/网站上进行blastx分析,预测CiNPR4在甜橙基因组中互作的候选蛋白,克隆CiNPR4和候选蛋白基因的cDNA,利用同源重组法分别构建诱饵载体和猎物载体,并将诱饵质粒和猎物质粒共转入酵母菌株Y2HGold中,进行点对点酵母双杂交分析。【结果】CiNPR4的超量表达减轻了转基因柑橘叶片上溃疡病的症状,柑橘溃疡病抗性增强的CiNPR4转基因植株叶片上Xcc的生长比较缓慢,在接种Xcc后9 d,转基因植株Xcc数量显著低于野生型（wild type,WT）晚锦橙;Xcc诱导0 d,溃疡病抗性增强的CiNPR4转基因植株含有与WT植株无差异的SA含量,随着Xcc诱导时间的延长,CiNPR4转基因植株中SA的含量显著增加;而Xcc处理0 d时,WT植株含有显著高于CiNPR4转基因植株的JA含量,随后,JA含量在CiNPR4转基因植株中逐步上升,Xcc处理5 d时,达到显著高于WT植株的水平;而WT植株在整个处理期间SA和JA的含量基本保持不变;溃疡病抗性增强的转基因植株受Xcc诱导3 d时CsPR1的表达水平迅速上升,达到与WT植株差异显著的水平,而CsPDF1.2的表达水平在Xcc诱导5 d时上升;WT植株受Xcc诱导后CsPR1的表达水平没有发生显著变化,而CsPDF1.2的表达水平在Xcc诱导5 d时上升,且显著高于CiNPR4转基因植株中CsPDF1.2的表达水平;酵母双杂交分析证实,CiNPR4与甜橙基因组中的CsTGA2转录因子互作。【结论】CiNPR4与CsTGA2转录因子互作,通过促进SA而抑制JA介导的防御反应,调控转基因柑橘对溃疡病的抗性。

关键词: CiNPR4, 柑橘溃疡病, 水杨酸, 茉莉酸, TGA转录因子

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

张婧芸,刘语诺,王兆昊,彭爱红,陈善春,何永睿. 转CiNPR4基因柑橘抗溃疡病的机制解析[J]. 中国农业科学, 2021, 54(18): 3871-3880.

ZHANG JingYun,LIU YuNuo,WANG ZhaoHao,PENG AiHong,CHEN ShanChun,HE YongRui. Analysis of Resistance Mechanism of CiNPR4 Transgenic Plants to Citrus Canker[J]. Scientia Agricultura Sinica, 2021, 54(18): 3871-3880.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.18.007

https://www.chinaagrisci.com/CN/Y2021/V54/I18/3871

图/表 4

表1

图1

图2

图3

参考文献 32

[1]	PITINO M, ARMSTRONG C M, DUAN Y. Rapid screening for citrus canker resistance employing pathogen-associated molecular pattern- triggered immunity responses. Horticulture Research, 2015, 2:15042. doi: 10.1038/hortres.2015.42
[2]	杨枫, 陈传武, 范七君, 石春梅, 谢宗周, 郭大勇, 刘继红. 温度和多胺对柑橘溃疡病发生的影响及作用机制. 中国农业科学, 2018, 51(10): 1899-1907.
	YANG F, CHEN C W, FAN Q J, SHI C M, XIE Z Z, GUO D Y, LIU J H. Influence of temperature and polyamines on occurrence of citrus canker disease and underlying mechanisms. Scientia Agricultura Sinica, 2018, 51(10): 1899-1907. (in Chinese)
[3]	姚廷山, 胡军华, 唐科志, 冉春, 李中安, 周常勇. 利用rep-PCR技术研究我国9省柑橘溃疡病菌遗传多样性. 果树学报, 2010, 27(5): 819-822.
	YAO T S, HU J H, TANG K Z, RAN C, LI Z A, ZHOU C Y. Primary analysis on genomic diversities of Xanthomonas axnopodis pv. citri in nine provinces of China. Journal of Fruit Science, 2010, 27(5): 819-822. (in Chinese)
[4]	向旭. 柑桔抗病分子育种研究进展. 分子植物育种, 2006, 4(2): 262-268.
	XIANG X. Progresses on molecular breeding for citrus disease resistance. Molecular Plant Breeding, 2006, 4(2): 262-268. (in Chinese)
[5]	彭爱红. 根癌农杆菌介导甲型肝炎病毒(HAV)衣壳蛋白融合基因转化柑桔的研究[D]. 重庆: 西南大学, 2006.
	PENG A H. Agrobacterium-mediated transformation of citrus with hepatitis A virus capsid protein fusion gene[D]. Chongqing: Southwest University, 2006. (in Chinese)
[6]	李鼎立. 柑橘遗传转化受体系统优化与抗溃疡病转基因植株培育[D]. 武汉: 华中农业大学, 2008.
	LI D L. Optimization of transformation system and production of transgenic plants with Xa21 gene in citrus[D]. Wuhan: Huazhong Agricultural University, 2008. (in Chinese)
[7]	STICHER L, MAUCH-MANI B, METRAUX J P. Systemic acquired resistance. Annual Review of Phytopathology, 1997, 35:235-270. doi: 10.1146/annurev.phyto.35.1.235
[8]	AN C, MOU Z. Salicylic acid and its function in plant immunity. Journal of Integrative Plant Biology, 2011, 53(6): 412-428. doi: 10.1111/jipb.2011.53.issue-6
[9]	FU Z Q, YAN S, SALEH A, WANG W, RUBLE J, OKA N, MOHAN R, SPOEL S H, TADA Y, ZHENG N, DONG X. NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature, 2012, 486(7402): 228-232. doi: 10.1038/nature11162
[10]	WU Y, ZHANG D, CHU J Y, BOYLE P, WANG Y, BRINDLE I D, DE LUCA V, DESPRÉS C. The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid. Cell Reports, 2012, 1(6): 639-647. doi: 10.1016/j.celrep.2012.05.008
[11]	DING Y, SUN T, AO K, PENG Y, ZHANG Y, LI X, ZHANG Y. Opposite roles of salicylic acid receptors NPR1 and NPR3/NPR4 in transcriptional regulation of plant immunity. Cell, 2018, 173(6): 1454-1467. doi: 10.1016/j.cell.2018.03.044
[12]	MOU Z, FAN W, DONG X. Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell, 2003, 113(7): 935-944. doi: 10.1016/S0092-8674(03)00429-X
[13]	DESPRES C, DELONG C, GLAZE S, LIU E, FOBERT P R. The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. The Plant Cell, 2000, 12(2): 279-290. doi: 10.1105/tpc.12.2.279
[14]	ZHANG X, FRANCIS M I, DAWSON W O, GRAHAM J H, ORBOVIĆ V, TRIPLETT E W, MOU Z. Over-expression of the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker. European Journal of Plant Pathology, 2010, 128:91-100. doi: 10.1007/s10658-010-9633-x
[15]	DUTT M, BARTHE G, IREY M, GROSSER J. Transgenic citrus expressing an Arabidopsis NPR1 gene exhibit enhanced resistance against Huanglongbing (HLB; citrus greening). PLoS ONE, 2015, 10(9): e0137134. doi: 10.1371/journal.pone.0137134
[16]	CHEN X, BARNABY J Y, SREEDHARAN A, HUANG X, ORBOVIĆ V, GROSSER J W, WANG N, DONG X, SONG W Y. Over-expression of the citrus gene CtNH1 confers resistance to bacterial canker disease. Physiological and Molecular Plant Pathology, 2013, 84:115-122. doi: 10.1016/j.pmpp.2013.07.002
[17]	LIU G, HOLUB E B, ALONSO J M, ECKER J R, FOBERT P R. An Arabidopsis NPR1-like gene, NPR4, is required for disease resistance. The Plant Journal, 2005, 41(2): 304-318. doi: 10.1111/tpj.2005.41.issue-2
[18]	ZHANG Y, CHENG Y T, QU N, ZHAO Q, BI D, LI X. Negative regulation of defense responses in Arabidopsis by two NPR1 paralogs. The Plant Journal, 2006, 48(5): 647-656. doi: 10.1111/tpj.2006.48.issue-5
[19]	WANG Y, ZHOU L, YU X, STOVER E, LUO F, DUAN Y. Transcriptome profiling of Huanglongbing (HLB) tolerant and susceptible citrus plants reveals the role of basal resistance in HLB tolerance. Frontiers in Plant Science, 2016, 7:933.
[20]	PENG A, ZOU X, HE Y, CHEN S, LIU X, ZHANG J, ZHANG Q, XIE Z, LONG J, ZHAO X. Overexpressing a NPR1-like gene from Citrus paradisi enhanced Huanglongbing resistance in C. sinensis. Plant Cell Reports, 2021, 40(3): 529-541. doi: 10.1007/s00299-020-02648-3
[21]	DAS A K. Citrus canker-A review. Journal of Applied Horticulture, 2003, 5(1): 52-60. doi: 10.37855/jah.2003.v05i01.15
[22]	PENG A, XU L, HE Y, LEI T, YAO L, CHEN S, ZOU X. Efficient production of marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system. Plant Cell, Tissue and Organ Culture, 2015, 123:1-13. doi: 10.1007/s11240-015-0799-y
[23]	DUAN S, JIA H, PANG Z, TEPER D, WHITE F, JONES J, ZHOU C, WANG N. Functional characterization of the citrus canker susceptibility gene CsLOB1. Molecular Plant Pathology, 2018, 19(8): 1908-1916. doi: 10.1111/mpp.2018.19.issue-8
[24]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-∆∆Ct method. Methods, 2001, 25(4): 402-408. doi: 10.1006/meth.2001.1262
[25]	XU Q, CHEN L L, RUAN X A, CHEN D J, ZHU A D, CHEN C L, BERTRAND D, JIAO W B, HAO B H, LYON M P, et al. The draft genome of sweet orange (Citrus sinensis). Nature Genetics, 2013, 45(1): 59-66. doi: 10.1038/ng.2472
[26]	GÓMEZ-MUÑOZ N, VELÁZQUEZ K, VIVES M C, RUIZ-RUIZ S, PINA J A, FLORES R, MORENO P, GUERRI J. The resistance of sour orange to citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways. Molecular Plant Pathology, 2017, 18(9): 1253-1266. doi: 10.1111/mpp.2017.18.issue-9
[27]	SHU L J, LIAO J Y, LIN N C, CHUNG C L. Identification of a strawberry NPR-like gene involved in negative regulation of the salicylic acid-mediated defense pathway. PLoS ONE, 2018, 13(10): e0205790. doi: 10.1371/journal.pone.0205790
[28]	LIU X, LIU Z, NIU X, XU Q, YANG L. Genome-wide identification and analysis of the NPR1-like gene family in bread wheat and its relatives. International Journal of Molecular Science, 2019, 20(23): 5974. doi: 10.3390/ijms20235974
[29]	YAMADA S, KANO A, TAMAOKI D, MIYAMOTO A, SHISHIDO H, MIYOSHI S, TANIGUCHI S, AKIMITSU K, GOMI K. Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice. Plant and Cell Physiology, 2012, 53(12): 2060-2072. doi: 10.1093/pcp/pcs145
[30]	YUAN Y, ZHONG S, LI Q, ZHU Z, LOU Y, WANG L, WANG J, WANG M, LI Q, YANG D, HE Z. Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility. Plant Biotechnology Journal, 2007, 5(2): 313-324. doi: 10.1111/pbi.2007.5.issue-2
[31]	SPOEL S H, KOORNNEEF A, CLAESSENS S M C, KORZELIUS J P, VAN PELT J A, MUELLER M J, BUCHALA A J, MÉTRAUX J P, BROWN R, KAZAN K, VAN LOON L C, DONG X, PIETERSE C M J. NPR1 modulates cross-talk between salicylate- and jasmonate- dependent defense pathways through a novel function in the cytosol. The Plant Cell, 2003, 15(3): 760-770. doi: 10.1105/tpc.009159
[32]	ZHANG Y, FAN W, KINKEMA M, LI X, DONG X. Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR1 gene. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(11): 6523-6528.

引物名称 Primer name	引物序列 Primer sequence (5′→3′)	用途 Usage
CsPR1-f	AAATGTGGGTGAATGAGAAAGC	CsPR1的表达分析 Expression analysis of CsPR1
CsPR1-r	ATTATTGTTGCACGTCACCTTG	CsPR1的表达分析 Expression analysis of CsPR1
CsPDF1.2-f	CAGTGGCAGAAGCAAAACAA	CsPDF1.2的表达分析 Expression analysis of CsPDF1.2
CsPDF1.2-r	CCGGGGAAGTCGTAGTGGC	CsPDF1.2的表达分析 Expression analysis of CsPDF1.2
Actin-f	CATCCCTCAGCACCTTCC	柑橘actin的表达分析 Expression analysis of citrus actin
Actin-r	CCAACCTTAGCACTTCTCC	柑橘actin的表达分析 Expression analysis of citrus actin
CiNPR4-f	CATGGAGGCCGAATTATGGTTGAGAAGGCTCTCGTGG	CiNPR4的cDNA扩增 Amplification of cDNA from CiNPR4
CiNPR4-r	GGATCCCCGGGAATTTTAATTGCTCGCTGCCGAGG	CiNPR4的cDNA扩增 Amplification of cDNA from CiNPR4
CsTGA2-f	GCCATGGAGGCCAGTGAATTCATGGAGAATGCTGTTGACCT	CsTGA2的cDNA扩增 Amplification of cDNA from CsTGA2
CsTGA2-r	GCAGCTCGAGCTCGATGGATCCTCACTCCCGTGGCCGGGCCA	CsTGA2的cDNA扩增 Amplification of cDNA from CsTGA2
CsTGA6-f	GCCATGGAGGCCAGTGAATTCATGCCGAGCTTTGATTCTC	CsTGA6的cDNA扩增 Amplification of cDNA from CsTGA6
CsTGA6-r	GCAGCTCGAGCTCGATGGATCCTCACTCTCTTGGGCGGGC	CsTGA6的cDNA扩增 Amplification of cDNA from CsTGA6