Disease resistance of SlJAZ7 and its interaction with SlTGA7 in tomato
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摘要:目的
细菌性斑点病是导致番茄Solanum lycopersicum减产的主要因素之一，丁香假单胞菌(Pseudomonas syringae pv. tomato DC3000，Pst DC3000)是细菌性斑点病的致病因子之一。瞬时沉默番茄JAZ7基因导致其对Pst DC3000的敏感性增加，然而，验证JAZ7基因抗细菌性斑点病的直接证据及其作用机理的报道较少。本研究从番茄叶片中克隆得到SlJAZ7基因，创制稳定遗传的过表达SlJAZ7的转基因番茄，分析其抗病功能和机理，研究其在转录水平和蛋白水平上与抗病性密切相关的SlTGA7的关系，为有效防治细菌性斑点病提供理论基础。
方法通过野生型和转基因番茄接种Pst DC3000的表型差异分析，鉴定SlJAZ7基因的抗病功能。使用RT-qPCR分析SlJAZ7和SlTGA7基因在Pst DC3000、茉莉酸甲酯(Methyl jasmonate，MeJA)和水杨酸(Salicylic acid，SA)处理下的表达模式和组织特异性。利用烟草瞬时表达的方法研究SIJAZ7和SlTGA7蛋白的亚细胞定位。利用酵母双杂交(Y2H)试验、双分子荧光互补(BiFC)试验和蛋白下拉(pull down)试验研究SlJAZ7蛋白与SlTGA7蛋白的互作关系，验证SlJAZ7的抗病功能和可能的抗病机理。
结果过表达SlJAZ7基因的转基因番茄叶片在Pst DC3000处理时受到的过氧化损伤较野生型更少，转基因株系中SlTGA7表达量升高，SlJAZ7基因在营养器官中高表达，且受到Pst DC3000诱导，响应MeJA、SA处理，SlTGA7基因在同样的处理下呈相反的变化趋势。SlJAZ7和SlTGA7蛋白均定位于细胞核。Y2H、BiFC和pull down试验同时证明SlJAZ7蛋白和SlTGA7蛋白存在互作关系。
结论过表达SlJAZ7基因有利于减少活性氧积累，提高番茄抗病性，同时SlJAZ7在转录水平正调控SlTGA7基因表达。SlJAZ7与SlTGA7存在互作，推测SIJAZ7基因可能通过提高Pst DC3000病原菌侵染时SITGA7基因的表达量，启动SITGA7下游抗病基因的表达来提高转基因番茄的抗病性，也可能通过与SITGA7蛋白结合，影响其调控MYC等转录因子的活性。这为进一步研究SlJAZ7的作用机制奠定了基础。
Abstract:ObjectiveBacterial spot disease is one of the main factors leading to tomato (Solanum lycopersicum) yield reduction, and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is one of the pathogenic factors of bacterial spot disease. Transient silencing of JAZ7 gene in tomato results in its increased susceptibility to Pst DC3000. At present, there are a few reports to verify the resistance function and mechanism of JAZ7 gene in tomato. In this study, we cloned SlJAZ7 gene in tomato leaves, and a stable genetic transgenic tomato with overexpression of SlJAZ7 was created to analyze its resistance function and mechanism, and the relationship between SlJAZ7 and SlTGA7, which is closely related to disease resistance, was studied at the transcriptional and protein levels, providing a theoretical basis for effective prevention and control of bacterial spot disease.
MethodThe resistance function of SlJAZ7 gene was determined by the phenotypic difference between wild type and transgenic tomato inoculated with Pst DC3000. RT-qPCR was used to analyze the tissue-specific expression of SlJAZ7 gene and SlTGA7 gene under Pst DC3000, methyl jasmonate (MeJA) and salicylic acid (SA) treatments. The subcellular localization of SIJAZ7 and SlTGA7 proteins was studied by transient expression of tobacco. The interaction between SlJAZ7 and SlTGA7 was verified by Y2H, BiFC and pull down experiments, to verify the disease resistance function and possible mechanism of SlJAZ7.
ResultTransgenic tomato leaves overexpressing SlJAZ7 gene had lower peroxidation damage than wild type when treated with pathogens, the expression level of SlTGA7 in transgenic lines increased. SlJAZ7 gene was highly expressed in nutrient tissues. SlJAZ7 gene was induced by Pst DC3000 and in response to MeJA and SA treatments. SlTGA7 gene showed opposite change trend under the same treatment. Both SlJAZ7 and SlTGA7 proteins were located in the nucleus. All Y2H, BiFC and pull down experiments proved the interaction between SlJAZ7 and SlTGA7.
ConclusionOverexpression of SlJAZ7 gene can reduce the accumulation of reactive oxygen species and improve tomato disease resistance. SlJAZ7 positively regulates SlTGA7 gene expression at the transcriptional level. SlJAZ7 interacts with SlTGA7. It is speculated that SIJAZ7 gene may improve the disease resistance of transgenic tomato by increasing the expression level of SITGA7 gene during pathogen infection, initiating the expression of downstream resistance genes of SITGA7, or by binding with SITGA7 protein, affecting its regulation of MYC and other transcription factors. The result lays a foundation for further research on the mechanism of SlJAZ7.
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Keywords:
- Tomato /
- SlJAZ7 /
- Protein interaction /
- Disease resistance /
- Methyl jasmonate /
- Salicylic acid
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图 1 转基因阳性植株SlJAZ7相对表达量
WT：野生型；OE1、5、6、7、12、14：过表达株系；“*”“**”分别表示过表达株系与野生型在P < 0.05和P < 0.01水平差异显著(t检验)
Figure 1. Relative expression level of SlJAZ7 in transgenic positive plants
WT: Wild type; OE1, 5, 6, 7, 12, 14: Overexpressed lines; “*” and “**” indicate signidicant differences between overexpressed lines and wild type at P < 0.05 and P < 0.01 levels (t test)
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图 2 Pst DC3000病原菌侵染野生型(WT)和过表达株系(OE)后的表型(A)及DAB染色结果(B)
Figure 2. Phenotype (A) and DAB staining results (B) after Pst DC3000 pathogens infection on wild type (WT) and overexpressed line (OE)
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图 3 Pst DC3000、MeJA和SA处理下SlTGA7 (A、B、C)和SlJAZ7 (D、E、F)的表达模式
“*”“**”分别表示其他时间点与0 h在P < 0.05和P < 0.01水平差异显著(t检验)
Figure 3. Expression patterns of SlTGA7 (A, B, C) and SlJAZ7 (D, E, F) under Pst DC3000, MeJA and SA treatments
“*” and “**” indicate significant differences between other time points and 0 h at P < 0.05 and P < 0.01 respectively (t test)
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图 4 SlTGA7和SlJAZ7在不同器官中的表达模式
1：青熟期果实，2：绿熟期果实，3：破色期果实，4：成熟红果，5：根，6：茎，7：幼叶，8：成熟叶，9：老叶，10：未完全展开的花；“*”“**”分别表示其他器官与青熟期果实在P < 0.05、P < 0.01水平差异显著(t检验)
Figure 4. Tissue specific expression profile of SlTGA7 and SlJAZ7
1: Fruit at cyan ripening stage, 2: Fruit at green ripening stage, 3: Fruit at green colour breaking stage, 4: Mature red fruit, 5: Root, 6: Stem, 7: Young leaf, 8: Mature leaf, 9: Old leaf, 10: Not fully unfolded flower; “*” and “**” indicate significant differences between other organs and fruits at cyan ripening stage at P < 0.05 and P < 0.01 levels respectively (t test)
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图 5 SlJAZ7过表达株系叶片中SlTGA7的表达模式
WT：野生型，OE1、5、6、7、12、14：过表达株系；“*”“**”分别表示过表达株系与野生型在P < 0.05和P < 0.01水平差异显著(t检验)
Figure 5. Expression pattern of SlTGA7 in the leaves of SlJAZ7 overexpressed strains
WT: Wild type, OE1, 5, 6, 7, 12, 14: Overexpressed lines; “*” and “**” indicate signidicant differences between overexpressed lines and wild type at P < 0.05 and P < 0.01 levels (t test)
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图 6 番茄SlJAZ7和SlTGA7的亚细胞定位
Figure 6. Subcellular localization of SlJAZ7 and SlTGA7 in tomato
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图 7 Y2H试验验证番茄SlJAZ7和SlTGA7蛋白的互作关系
pGADT7-T/pGBKT7-Lam为阴性对照；pGADT7-T/pGBKT7-53为阳性对照；0、10−1、10−2、10−3表示菌液的稀释梯度
Figure 7. Interaction between SlJAZ7 and SlTGA7 proteins verified by Y2H
pGADT7-T/pGBKT7-Lam is negative control; pGADT7-T/pGBKT7-53 is positive control; 0, 10−1, 10−2, 10−3 represent the dilution gradients of bacterial solution
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图 8 BiFC验证SlJAZ7和SlTGA7蛋白的互作关系(标尺：20 μm)
Figure 8. Interaction between SlJAZ7 and SlTGA7 verified by BiFC (scale: 20 μm)
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图 9 考马斯亮蓝染色检测SlJAZ7与SlTGA7蛋白的分子量
Figure 9. Relative molecular masses of SlJAZ7 and SlTGA7 determined by Kaumas bright blue staining
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图 10 Pull down试验验证SlJAZ7与SlTGA7的互作关系
Figure 10. Interaction between SlJAZ7与SlTGA7 verified by pull down experiment
表 1 本研究所用引物
Table 1 Primers used in this study
引物名称
Primer name引物序列1) (5′→3′)
Primer sequence目的
PurposeSlJAZ7-NC-F agtggtctctgtccagtcctATGGATTCAAGAATGGAGATAGATT 基因克隆 SlJAZ7-NC-R ggtctcagcagaccacaagtGTTTTCCCAATGAACGCTTGAC 基因克隆 SlJAZ7-EcoRI-F ccggaattcATGGATTCAAGAATGGAGATAGATT 互作关系验证 SlJAZ7-BamHI-R cgggatccGTTTTCCCAATGAACGCTTGAC 互作关系验证 SlTGA7-EcoRI-F ccggaattcATGAACATGAGTTCTACATCAACTC 互作关系验证 SlTGA7-BamHI-R cgggatccGGTAGGTTCACGAGGACGAG 互作关系验证 SlJAZ7-SalI-F acgcgtcgacATGGATTCAAGAATGGAGATAGATT BiFC试验 SlTGA7-SalI-F acgcgtcgacATGAACATGAGTTCTACATCAACTC BiFC试验 SlJAZ7-Q-F ATTATGCTAAAGGAGCACTTGCTAT RT-qPCR SlJAZ7-Q-R CGCTTGACGACGCCG RT-qPCR Sltublin-Q-F TTGGTTTTGCACCACTGACTTC RT-qPCR Sltublin-Q-R AAGCTCTGGCACTCTCAAAGC RT-qPCR 1)带下划线的碱基代表酶切位点序列
1) The underlined bases represent the sequences of enzyme cleavage site
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[1] LIN T, ZHU G T, ZHANG J H, et al. Genomic analyses provide insights into the history of tomato breeding[J]. Nature Genetics, 2014, 46(11): 1220-1226. doi: 10.1038/ng.3117
[2] FRIEDMAN M. Tomato glycoalkaloids: Role in the plant and in the diet[J]. Journal of Agricultural and Food Chemistry, 2002, 50(21): 5751-5780. doi: 10.1021/jf020560c
[3] WAI K P P, SIDDIQUE M I, MO H S, et al. Pathotypes of bacterial spot pathogen infecting capsicum peppers in Korea[J]. Plant Pathology Journal, 2015, 31(4): 428-432. doi: 10.5423/PPJ.NT.05.2015.0074
[4] XIN X F, HE S Y. Pseudomonas syringae pv. tomato DC3000: A model pathogen for probing disease susceptibility and hormone signaling in plants[J]. Annual Review of Phytopathology, 2013, 51: 473-498. doi: 10.1146/annurev-phyto-082712-102321
[5] BEALE M H, WARD J L. Jasmonates: Key players in the plant defence[J]. Natural Product Reports, 1998, 15(6): 533-548. doi: 10.1039/a815533y
[6] CREELMAN R A, MULLET J E. Oligosaccharins, brassinolides, and jasmonates: Nontraditional regulators of plant growth, development, and gene expression[J]. Plant Cell, 1997, 9(7): 1211-1223. doi: 10.1105/tpc.9.7.1211
[7] BROWSE J. Jasmonate passes muster: A receptor and targets for the defense hormone[J]. Annual Review of Plant Biology, 2009, 60: 183-205. doi: 10.1146/annurev.arplant.043008.092007
[8] ZHANG F, YAO J, KE J Y, et al. Structural basis of JAZ repression of MYC transcription factors in jasmonate signalling[J]. Nature, 2015, 525(7568): 269-273. doi: 10.1038/nature14661
[9] SUN Y G, LIU C X, LIU Z B, et al. Characterization and expression analysis of the JAZ gene family in resistance to gray leaf spots in tomato[J]. International Journal of Molecular Sciences, 2021, 22(18): 9974. doi: 10.3390/ijms22189974
[10] 裴童. JAZ3在番茄抗叶霉病免疫应答过程中的功能及调控机制研究[D]. 哈尔滨: 东北农业大学, 2021. [11] THATCHER L F, CEVIK V, GRANT M, et al. Characterization of a JAZ7 activation-tagged Arabidopsis mutant with increased susceptibility to the fungal pathogen Fusarium oxysporum[J]. Journal of Experimental Botany, 2016, 67(8): 2367-2386. doi: 10.1093/jxb/erw040
[12] ISHIGA Y, ISHIGA T, UPPALAPATI S R, et al. Jasmonate ZIM-domain (JAZ) protein regulates host and nonhost pathogen-induced cell death in tomato and Nicotiana benthamiana[J]. PLoS One, 2013, 8(9): e75728. doi: 10.1371/journal.pone.0075728
[13] ALLU A D, BROTMAN Y, XUE G P, et al. Transcription factor ANAC032 modulates JA/SA signalling in response to Pseudomonas syringae infection[J]. EMBO Reports, 2016, 17(11): 1578-1589. doi: 10.15252/embr.201642197
[14] ZHANG N L, ZHOU S, YANG D Y, et al. Revealing shared and distinct genes responding to JA and SA signaling in Arabidopsis by Meta-Analysis[J]. Frontiers in Plant Science, 2011, 11: 908.
[15] 田义, 张彩霞, 康国栋, 等. 植物TGA转录因子研究进展[J]. 中国农业科学, 2016, 49(4): 632-642. [16] KATAGIRI F, LAM E, CHUA N H. Two tobacco DNA-binding proteins with homology to the nuclear factor CREB[J]. Nature, 1989, 340(6236): 727-730. doi: 10.1038/340727a0
[17] SHEARER H L, WANG L P, DELONG C, et al. NPR1 enhances the DNA binding activity of the Arabidopsis bZIP transcription factor TGA7[J]. Botany, 2009, 87(6): 561-570. doi: 10.1139/B08-143
[18] ZHOU J M, TRIFA Y, SILVA H, et al. NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid[J]. Molecular Plant-Microbe Interactions, 2000, 13(2): 191-202. doi: 10.1094/MPMI.2000.13.2.191
[19] JOHNSON C, BODEN E, ARIAS J. Salicylic acid and NPR1 induce the recruitment of trans-activating TGA factors to a defense gene promoter in Arabidopsis[J]. Plant Cell, 2003, 15(8): 1846-1858. doi: 10.1105/tpc.012211
[20] ZANDER M, LA CAMERA S, LAMOTTE O, et al. Arabidopsis thaliana class-II TGA transcription factors are essential activators of jasmonic acid/ethylene-induced defense responses[J]. Plant Journal, 2009, 61(2): 200-210. doi: 10.1111/j.1365-313X.2009.04044.x
[21] JIANG H, GU S Y, LI K, et al. Two TGA transcription factor members from hyper-susceptible soybean exhibiting significant basal resistance to soybean mosaic virus[J]. International Journal of Molecular Sciences, 2021, 22(21): 11329. doi: 10.3390/ijms222111329
[22] LIU W, ZHAO C, LIU L, et al. Genome-wide identification of the TGA gene family in kiwifruit (Actinidia chinensis spp. ) and revealing its roles in response to Pseudomonas syringae pv. actinidiae (Psa) infection[J]. International Journal of Biological Macromolecules, 2022, 222: 101-113. doi: 10.1016/j.ijbiomac.2022.09.154
[23] OLSON B J S C. Assays for determination of protein concentration[J]. Current Protocols in Pharmacology, 2016, 73. doi: 10.1002/cpph.3.
[24] ZHANG Y, FAN W, KINKEMA M, et al. Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene[J]. Proceedings of the National Academy of Sciences of the United States of America, 1996, 96(11): 6523-6528.
[25] SUN T J, BUSTA L, ZHANG Q, et al. TGACG-BINDING FACTOR 1 (TGA1) and TGA4 regulate salicylic acid and pipecolic acid biosynthesis by modulating the expression of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g)[J]. New Phytologist, 2017, 217(1): 344-354.
[26] 段军叶. 转录因子TGA7参与拟南芥响应干旱胁迫的分子机制研究[D]. 北京: 中国农业大学, 2016. [27] 吴娟娟. 拟南芥转录因子TGA7参与植物响应干旱胁迫的机制研究[D]. 北京: 中国农业大学, 2014. [28] 侯佳音. 番茄TGA2转录因子在农药百菌清代谢降解中的功能及调控机制[D]. 杭州: 浙江大学, 2019. [29] SHEARER H L, CHENG Y T, WANG L P, et al. Arabidopsis clade I TGA transcription factors regulate plant defenses in an NPR1-independent fashion[J]. Molecular Plant-Microbe Interactions, 2012, 25(11): 1459-1468. doi: 10.1094/MPMI-09-11-0256
[30] 高苗苗, 郭鑫, 朱寿松, 等. 番茄SlJAZ11的表达分析及互作蛋白的筛选与验证[J]. 核农学报, 2023, 37(3): 483-494. [31] KOORNNEEF A, LEON-REYES A, RITSEMA T, et al. Pieterse, kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation[J]. Plant Physiology, 2008, 147(3): 1358-1368. doi: 10.1104/pp.108.121392
[32] NIKI T, MITSUHARA I, SEO S, et al. Antagonistic effect of salicylic acid and jasmonic acid on the expression of pathogenesis-related (PR) protein genes in wounded mature tobacco leaves[J]. Plant Cell Physiology, 1998, 39(5): 500-507. doi: 10.1093/oxfordjournals.pcp.a029397
[33] VAN WEES S C, DE SWART E A, VAN PELT J A, et al. Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana[J]. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(15): 8711-8716.
[34] HU Y, LIU Y, TAO J J, et al. GmJAZ3 interacts with GmRR18a and GmMYC2a to regulate seed traits in soybean[J]. Journal of Integrative Plant Biology, 2023, 65(8): 1983-2000. doi: 10.1111/jipb.13494
[35] CHINI A, GIMENEZ-IBANEZ S, GOOSSENS A, et al. Redundancy and specificity in jasmonate signalling[J], Current Opinion in Plant Biology, 2016, 33: 147-156.
[36] YANG J, DUAN G H, LI C Q, et al. The crosstalks between jasmonic acid and other plant hormone signaling highlight the involvement of jasmonic acid as a core component in plant response to biotic and abiotic stresses[J]. Frontiers in Plant Science, 2019, 10: 1349. doi: 10.3389/fpls.2019.01349
