本氏烟半胱氨酸蛋白酶基因家族特征及其在TMV侵染中的功能

doi:10.3864/j.issn.0578-1752.2022.21.008

摘要/Abstract

摘要：

【目的】明确抗病毒化合物氯吲哚酰肼（chloroinconazide，CHI）诱导的本氏烟（Nicotiana benthamiana）半胱氨酸蛋白酶（cysteine proteinase，CP）在烟草花叶病毒（tobacco mosaic virus，TMV）侵染过程中的作用及其基因家族特征，为理解茄科作物抗病毒分子机制及化学调控提供理论依据。【方法】利用全基因组策略，从茄科作物基因组数据库Sol Genomics Network中检索获得本氏烟NbCP的全家族基因序列，利用生物信息学分析NbCP基因家族的进化关系、Motif基序及启动子顺式作用元件。根据生物信息分析结果，利用实时荧光定量PCR技术分析TMV侵染后NbCP基因的表达，以此筛选出潜在抗病蛋白。使用烟草脆裂病毒（tobacco rattle virus，TRV）介导的基因沉默技术和马铃薯X病毒（potato virus X，PVX）介导的基因过表达技术验证该关键蛋白对TMV-GFP侵染的影响，结合实时荧光定量PCR手段探索该关键蛋白的抗病毒机制。【结果】NbCP家族共有24个成员，根据其染色体定位命名为NbCP1—NbCP24。进化关系分析表明NbCP分成5个亚家族，其中Group V含有7个NbCP，而Group I仅含2个NbCP;Motif分析表明不同分支NbCP具有相似的motif分布;启动子顺式作用元件分析表明NbCP家族基因受光调控，并且多个NbCP家族基因启动子含有茉莉酸甲酯、水杨酸甲酯、脱落酸、赤霉素和生长素等激素响应元件。结合生物信息学分析，从上述各个亚家族中分别筛选1个关键潜在抗病相关NbCP基因（NbCP8、NbCP12、NbCP13、NbCP18、NbCP22），并利用实时荧光定量PCR技术分析上述基因在TMV侵染第5天的表达，发现NbCP8表达差异性最大，上升了2.6倍，NbCP8在叶中的表达量最高，其次是茎和根，在花中的表达量最低。TRV介导的NbCP8沉默能显著增加TMV-GFP的侵染，而PVX介导的NbCP8过表达能显著抑制TMV-GFP侵染，表明NbCP8作为植物正调控因子抑制病毒侵染。沉默NbCP8显著抑制了水杨酸信号途径相关基因PR1以及茉莉酸信号途径相关基因MYC2的表达，但对NPR1、COI1以及脱落酸信号途径相关基因ABA1和NCED1的表达无显著影响;而过表达NbCP8则显著提高了PR1以及ABA1的表达，但对NPR1、MYC2、COI1和NCED1的表达无显著影响。【结论】本氏烟NbCP参与了植物胁迫防御，其中NbCP8在抗TMV防御中起显著作用，其分子机制是通过诱导水杨酸信号途径参与抗病防御，研究结果可为茄科作物抗病毒的分子机制提供证据。

关键词: 本氏烟, 全基因组, 半胱氨酸蛋白酶, NbCP8, 烟草花叶病毒

Abstract:

【Objective】The objective of this study is to identify the effects of Nicotiana benthamiana cysteine proteinase (CP) induced by the antiviral compound chloroinconazide (CHI) on the infection of tobacco mosaic virus (TMV) and its gene family characteristics, and to provide a theoretical basis for understanding the antiviral molecular mechanism and chemical regulation of Solanaceae crops.【Method】Genome-wide analysis of NbCP genes was identified from Sol Genomics Network and the evolutionary relationships, motifs and promoter cis-acting elements of NbCP gene family were analyzed by bioinformatics. According to the results of bioinformatics analysis, the expression of NbCP genes after TMV infection was analyzed by real-time fluorescence quantitative PCR (qRT-PCR), so as to screen out potential disease-resistant protein. The effect of this key protein on TMV-GFP infection was verified by gene silencing mediated by tobacco rattle virus (TRV) and gene overexpression mediated by potato virus X (PVX). qRT-PCR and biochemical methods were combined to determine the antiviral mechanism of this key protein.【Result】There were 24 NbCP genes in N. benthamiana, which were named NbCP1-NbCP24 according to their chromosome location. The phylogenetic analysis showed that the NbCPs were divided into five subfamilies, of which Group V contained seven NbCPs, while Group I contained only two NbCPs. Motif analyses showed that NbCPs of different branches had similar motif distribution. Promoter cis-acting elements analysis showed that NbCP family genes were all regulated by light, and several NbCP family gene promoters contained hormone response elements such as methyl jasmonate (MeJA), methyl salicylate (MeSA), abscisic acid (ABA), gibberellin (GA) and auxin (IAA). Combined with bioinformatics analysis, five key potential disease-resistant related NbCP genes (NbCP8, NbCP12, NbCP13, NbCP18, and NbCP22) were screened from the above subfamilies, and the expression of these NbCP genes under TMV infection at the 5th day was analyzed by qRT-PCR, and it was found that the expression difference of NbCP8 was the greatest, which increased 2.6 times. The expression of NbCP8 was highest in leaves, followed by stems and roots, and lowest in flowers. TRV-mediated NbCP8 silencing significantly increased TMV-GFP infection, while PVX-mediated NbCP8 overexpression significantly inhibited TMV-GFP infection, suggesting that NbCP8 acts as a positive regulatory factor in plant to inhibit virus infection. Furthermore, silencing NbCP8 significantly inhibited the expression of salicylic acid (SA) signaling pathway related gene PR1 and jasmonic acid (JA) signaling pathway related gene MYC2, while the expressions of NPR1, COI1, and ABA related genes ABA1, NCED1 were not significantly affected. The overexpression of NbCP8 enhanced the expression of PR1 and ABA1, but had no significant effect on expression of NPR1, MYC2, COI1 and NCED1. 【Conclusion】N. benthamiana NbCPs are involved in plant stress defense, while NbCP8 plays a key role in TMV defense, which can induce SA signaling pathway to participate in the defense. The research results will provide evidence for the molecular mechanism of antiviral in Solanaceae crops.

Key words: Nicotiana benthamiana, genome-wide, cysteine proteinase (CP), NbCP8, tobacco mosaic virus (TMV)

裴悦宏,李凤巍,刘维娜,温玉霞,朱鑫,田绍锐,樊光进,马小舟,孙现超. 本氏烟半胱氨酸蛋白酶基因家族特征及其在TMV侵染中的功能[J]. 中国农业科学, 2022, 55(21): 4196-4210.

PEI YueHong,LI FengWei,LIU WeiNa,WEN YuXia,ZHU Xin,TIAN ShaoRui,FAN GuangJin,MA XiaoZhou,SUN XianChao. Characteristics of Cysteine Proteinase Gene Family in Nicotiana benthamiana and Its Function During TMV Infection[J]. Scientia Agricultura Sinica, 2022, 55(21): 4196-4210.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.21.008

https://www.chinaagrisci.com/CN/Y2022/V55/I21/4196

图/表 10

表1

表2

NbCP基因家族鉴定与特征分析"

名称 Name	基因定位 Gene locus ID	蛋白质编码区长度 CDS (bp)	分子量 MW (Da)	等电点 pI	分子式 Formula
NbCP1	Niben101Scf00712g02010.1	1068	39921.29	5.74	C₁₇₉₀H₂₇₅₆N₄₆₈O₅₃₈S₁₅
NbCP2	Niben101Scf00973g01002.1	1113	40808.02	5.95	C₁₈₀₁H₂₇₈₆N₄₉₈O₅₅₁S₁₈
NbCP3	Niben101Scf01369g00024.1	1068	39907.19	5.64	C₁₇₈₂H₂₇₄₆N₄₇₀O₅₄₀S₁₆
NbCP4	Niben101Scf01631g00002.1	999	36623.07	5.29	C₁₆₁₄H₂₄₈₆N₄₂₈O₅₁₂S₁₇
NbCP5	Niben101Scf02159g01012.1	1086	40558.46	5.78	C₁₈₀₀H₂₇₅₅N₄₈₉O₅₅₄S₁₄
NbCP6	Niben101Scf02336g00010.1	1311	48758.34	5.71	C₂₁₇₅H₃₃₄₉N₅₇₃O₆₅₆S₂₃
NbCP7	Niben101Scf02336g00012.1	1482	55407.91	5.82	C₂₄₆₀H₃₇₇₁N₆₆₁O₇₃₆S₃₂
NbCP8	Niben101Scf02763g05012.1	1059	38333.52	7.00	C₁₇₁₂H₂₆₅₈N₄₆₄O₅₁₁S₁₃
NbCP9	Niben101Scf02832g01014.1	1074	38845.02	5.09	C₁₇₀₅H₂₆₇₉N₄₇₃O₅₂₇S₁₉
NbCP10	Niben101Scf02853g06021.1	1086	40508.59	6.20	C₁₇₉₈H₂₇₆₅N₄₉₁O₅₄₈S₁₅
NbCP11	Niben101Scf02976g00007.1	1086	39876.46	6.00	C₁₇₈₁H₂₇₃₃N₄₈₁O₅₂₀S₂₁
NbCP12	Niben101Scf03867g02046.1	1092	40222.77	5.67	C₁₈₀₁H₂₇₃₉N₄₇₉O₅₂₆S₂₂
NbCP13	Niben101Scf04209g00004.1	1023	37952.64	6.67	C₁₆₇₄H₂₅₆₆N₄₆₀O₅₁₅S₁₈
NbCP14	Niben101Scf04653g01012.1	1059	39469.28	6.23	C₁₇₄₇H₂₆₉₁N₄₈₃O₅₃₇S₁₃
NbCP15	Niben101Scf04934g02006.1	1035	38571.66	8.89	C₁₇₁₁H₂₆₂₈N₄₆₈O₅₁₃S₁₉
NbCP16	Niben101Scf05035g01005.1	2052	74678.53	5.18	C₃₂₆₁H₄₉₇₃N₈₉₁O₁₀₃₂S₄₇
NbCP17	Niben101Scf05047g04012.1	987	36843.67	5.72	C₁₆₄₉H₂₅₂₂N₄₃₄O₄₉₆S₁₅
NbCP18	Niben101Scf05135g02002.1	1125	42280.54	6.73	C₁₈₆₁H₂₈₈₃N₅₂₃O₅₇₂S₁₇
NbCP19	Niben101Scf05469g01014.1	336	11947.10	4.37	C₅₀₅H₇₇₈N₁₄₀O₁₇₈S₉
NbCP20	Niben101Scf05469g01024.1	1623	59602.30	4.45	C₂₆₃₈H₃₉₆₄N₆₈₂O₈₄₀S₂₉
NbCP21	Niben101Scf08921g02020.1	1584	59286.05	8.87	C₂₆₃₂H₄₀₆₉N₇₂₉O₇₈₉S₂₃
NbCP22	Niben101Scf10490g00007.1	990	36122.34	4.81	C₁₅₉₇H₂₄₄₆N₄₂₀O₅₀₉S₁₄
NbCP23	Niben101Scf11030g00003.1	720	26115.98	7.95	C₁₁₄₆H₁₇₈₆N₃₁₀O₃₄₄S₂₂
NbCP24	Niben101Scf15022g00015.1	1113	40804.10	6.01	C₁₇₉₇H₂₇₉₀N₅₀₂O₅₄₈S₁₉

表2

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参考文献 53

[1]	SCHOLTHOF K B, ADKINS S, CZOSNEK H, PALUKAITIS P, JACQUOT E, HOHN T, HOHN B, SAUNDERS K, CANDRESSE T, AHLQUIST P, HEMENWAY C, FOSTER G. Top 10 plant viruses in molecular plant pathology. Molecular Plant Pathology, 2011, 12(9): 938-954.
[2]	CREAGER A N, SCHOLTHOF K B, CITOVSKY V, SCHOLTHOF H B. Tobacco mosaic virus: Pioneering research for a century. The Plant Cell, 1999, 11(3): 301-308.
[3]	SHINDO T, MISAS-VILLAMIL J C, HÖRGER A C, SONG J, VAN DER HOORN R A L. A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14. PLoS ONE, 2012, 7(1): e29317.
[4]	BAR-ZIV A, LEVY Y, HAK H, METT A, BELAUSOV E, CITOVSKY V, GAFNI Y. The tomato yellow leaf curl virus (TYLCV) V2 protein interacts with the host papain-like cysteine protease CYP1. Plant Signaling & Behavior, 2012, 7(8): 983-989.
[5]	MISAS-VILLAMIL J C, VAN DER HOORN R A L, DOEHLEMANN G. Papain-like cysteine proteases as hubs in plant immunity. New Phytologist, 2016, 212(4): 902-907.
[6]	BAR-ZIV A, LEVY Y, CITOVSKY V, GAFNI Y. The tomato yellow leaf curl virus (TYLCV) V2 protein inhibits enzymatic activity of the host papain-like cysteine protease CYP1. Biochemical and Biophysical Research Communications, 2015, 460(3): 525-529.
[7]	BERNOUX M, TIMMERS T, JAUNEAU A, BRIÈRE C, DE WIT P, MARCO Y, DESLANDES L. RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector. The Plant Cell, 2008, 20(8): 2252-2264.
[8]	MUELLER A N, ZIEMANN S, TREITSCHKE S, ASSMANN D, DOEHLEMANN G. Compatibility in the Ustilago maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2. PLoS Pathogens, 2013, 9(2): e1003177.
[9]	VAN DER LINDE K, MUELLER A N, HEMETSBERGER C, KASHANI F, VAN DER HOORN R A L, DOEHLEMANN G. The maize cystatin CC9 interacts with apoplastic cysteine proteases. Plant Signaling & Behavior, 2012, 7(11): 1397-1401.
[10]	VAN DER LINDE K, HEMETSBERGER C, KASTNER C, KASCHANI F, VAN DER HOORN R A L, KUMLEHN J, DOEHLEMANN G. A maize cystatin suppresses host immunity by inhibiting apoplastic cysteine proteases. The Plant Cell, 2012, 24(3): 1285-1300.
[11]	LV X, XIANG S, WANG X, WU L, LIU C, YUAN M, GONG W, WIN H, HAO C, XUE Y, MA L, CHENG D, SUN X. Synthetic chloroinconazide compound exhibits highly efficient antiviral activity against tobacco mosaic virus. Pest Management Science, 2020, 76(11): 3636-3648.
[12]	LV X, YUAN M, PEI Y, LIU C, WANG X, WU L, CHENG D, MA X, SUN X. The enhancement of antiviral activity of chloroinconazide by aglinate-based nanogel and its plant growth promotion effect. Journal of Agricultural and Food Chemistry, 2021, 69(17): 4992-5002.
[13]	LIU Y, SCHIFF M, MARATHE R, DINESH-KUMAR S P. Tobacco Rar1, EDS1and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. The Plant Journal, 2002, 30(4): 415-429.
[14]	FERNANDEZ-POZO N, MENDA N, EDWARDS J D, SAHA S, TECLE I Y, STRICKLER S R, BOMBARELY A, FISHER-YORK T, PUJAR A, FOERSTER H, YAN A, MUELLER L A. The Sol Genomics Network (SGN)—From genotype to phenotype to breeding. Nucleic Acids Research, 2015, 43(Database issue): D1036-D1041.
[15]	LIU C, PENG H, LI X, LIU C, LV X, WEI X, ZOU A, ZHANG J, FAN G, MA G, MA L, SUN X. Genome-wide analysis of NDR1/ HIN1-like genes in pepper (Capsicum annuum L.) and functional characterization of CaNHL4 under biotic and abiotic stresses. Horticulture Research, 2020, 7: 93.
[16]	LETUNIC I, KHEDKAR S, BORK P. SMART: Recent updates, new developments and status in 2020. Nucleic Acids Research, 2021, 49(D1): D458-D460.
[17]	OLONEN A, KALKKINEN N, PAULIN L. A new type of cysteine proteinase inhibitor—The salarin gene from Atlantic salmon (Salmo salar L.) and Arctic charr (Salvelinus alpinus). Biochimie, 2003, 85(7): 677-681.
[18]	YAMAMOTO Y, KURATA M, WATABE S, MURAKAMI R, TAKAHASHI S Y. Novel cysteine proteinase inhibitors homologous to the proregions of cysteine proteinases. Current Protein & Peptide Science, 2002, 3(2): 231-238.
[19]	PENG H, PU Y, YANG X, WU G, QING L, MA L, SUN X. Overexpression of a pathogenesis-related gene NbHIN1 confers resistance to tobacco mosaic virus in Nicotiana benthamiana by potentially activating the jasmonic acid signaling pathway. Plant Science, 2019, 283: 147-156.
[20]	TAMURA K, STECHER G, KUMAR S. MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 2021, 38(7): 3022-3027.
[21]	LIU C, PU Y, PENG H, LV X, TIAN S, WEI X, ZHANG J, ZOU A, FAN G, SUN X. Transcriptome sequencing reveals that photoinduced gene IP-L affects the expression of PsbO to response to virus infection in Nicotiana benthamiana. Physiological and Molecular Plant Pathology, 2021, 114: 101613.
[22]	LESCOT M, DÉHAIS P, THIJS G, MARCHAL K, MOREAU Y, VAN DE PEER Y, ROUZÉ P, ROMBAUTS S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Research, 2002, 30(1): 325-327.
[23]	CHEN C, CHEN H, ZHANG Y, THOMAS H R, FRANK M H, HE Y, XIA R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Molecular Plant, 2020, 13(8): 1194-1202.
[24]	BAILEY T L, BODEN M, BUSKE F A, FRITH M, GRANT C E, CLEMENTI L, REN J, LI W W, NOBLE W S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Research, 2009, 37(Web Server issue): W202-W208.
[25]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method. Method, 2001, 25(4): 402-408.
[26]	LIU Y, NAKAYAMA N, SCHIFF M, LITT A, IRISH V F, DINESH- KUMAR S P. Virus induced gene silencing of a DEFICIENS ortholog in Nicotiana benthamiana. Plant Molecular Biology, 2004, 54(5): 701-711.
[27]	FERNANDEZ-POZO N, ROSLI H G, MARTIN G B, MUELLER L A. The SGN VIGS tool: User-friendly software to design virus-induced gene silencing (VIGS) constructs for functional genomics. Molecular Plant, 2015, 8(3): 486-488.
[28]	LIU C, TIAN S, LV X, PU Y, PENG H, FAN G, MA X, MA L, SUN X. Nicotiana benthamiana asparagine synthetase associates with IP-L and confers resistance against tobacco mosaic virus via the asparagine-induced salicylic acid signalling pathway. Molecular Plant Pathology, 2022, 23(1): 60-77.
[29]	彭浩然, 潘琪, 魏周玲, 蒲运丹, 张永至, 吴根土, 青玲, 孙现超. 番茄抗性相关基因SlHin1的克隆、表达与抗病毒功能. 中国农业科学, 2017, 50(7): 1242-1251.
	PENG H R, PAN Q, WEI Z L, PU Y D, ZHANG Y Z, WU G T, QING L, SUN X C. Cloning,expression and anti-virus function analysis of tomato resistance-related gene SlHin1. Scientia Agricultura Sinica, 2017, 50(7): 1242-1251. (in Chinese)
[30]	ZHANG J, ZOU A, WEN Y, WEI X, LIU C, LV X, MA X, FAN G, SUN X. SlCML55, a novel Solanum lycopersicum calmodulin-like gene, negatively regulates plant immunity to Phytophthora pathogens. Scientia Horticulturae, 2022, 299: 111049.
[31]	HUANG C. From player to pawn: Viral avirulence factors involved in plant immunity. Viruses, 2021, 13(4): 688.
[32]	刘昌云, 李欣羽, 田绍锐, 王靖, 裴悦宏, 马小舟, 樊光进, 汪代斌, 孙现超. 番茄SlN-like的克隆、表达与抗病毒功能. 中国农业科学, 2021, 54(20): 4348-4357.
	LIU C Y, LI X Y, TIAN S R, WANG J, PEI Y H, MA X Z, FAN G J, WANG D B, SUN X C. Cloning,expression and anti-virus function analysis of Solanum lycopersicum SlN-like. Scientia Agricultura Sinica, 2021, 54(20): 4348-4357. (in Chinese)
[33]	WHITHAM S, DINESH-KUMAR S P, CHOI D, HEHL R, CORR C, BAKER B. The product of the tobacco mosaic virus resistance gene N: Similarity to toll and the interleukin-1 receptor. Cell, 1994, 78(6): 1101-1115.
[34]	MARATHE R, ANANDALAKSHMI R, LIU Y, DINESH-KUMAR S P. The tobacco mosaic virus resistance gene, N. Molecular Plant Pathology, 2002, 3(3): 167-172.
[35]	张洁琳, 付畅. 半胱氨酸蛋白酶及半胱氨酸蛋白酶抑制剂对逆境胁迫的应答. 分子植物育种, 2018, 16(13): 4453-4459.
	ZHANG J L, FU C. Response of cysteine protease and cysteine protease inhibitor to adverse stress. Molecular Plant Breeding, 2018, 16(13): 4453-4459. (in Chinese)
[36]	UEDA T, SEO S, OHASHI Y, HASHIMOTO J. Circadian and senescence-enhanced expression of a tobacco cysteine protease gene. Plant Molecular Biology, 2000, 44(5): 649-657.
[37]	张炳慧, 高翔, 杨明明, 王宪国, 何庆梦, 姚晓露, 李晓燕, 赵万春, 董剑. 小麦TaCP3基因的克隆及其参与非生物胁迫的表达分析. 麦类作物学报, 2019, 39(5): 513-519.
	ZHANG B H, GAO X, YANG M M, WANG X G, HE Q M, YAO X L, LI X Y, ZHAO W C, DONG J. Cloning of TaCP3gene and its expression in abiotic stress. Journal of Triticeae Crops, 2019, 39(5): 513-519. (in Chinese)
[38]	闫龙凤, 杨青川, 韩建国, 刘志鹏. 植物半胱氨酸蛋白酶研究进展. 草业学报, 2005, 14(5): 11-19.
	YAN L F, YANG Q C, HAN J G, LIU Z P. Summary of cysteine protease in plants. Acta Prataculturae Sinica, 2005, 14(5): 11-19. (in Chinese)
[39]	NARUSAKA Y, NAKASHIMA K, SHINWARI Z K, SAKUMA Y, FURIHATA T, ABE H, NARUSAKA M, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses. The Plant Journal, 2003, 34(2): 137-148.
[40]	YAMAGUCHI-SHINOZAKI K, SHINOZAKI K. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. The Plant Cell, 1994, 6(2): 251-264.
[41]	UNO Y, FURIHATA T, ABE H, YOSHIDA R, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(21): 11632-11637.
[42]	BAKER S S, WILHELM K S, THOMASHOW M F. The 5′-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Molecular Biology, 1994, 24(5): 701-713.
[43]	ZANG Q W, WANG C X, LI X Y, GUO Z A, JING R L, ZHAO J, CHANG X P. Isolation and characterization of a gene encoding a polyethylene glycol-induced cysteine protease in common wheat. Journal of Biosciences, 2010, 35(3): 379-388.
[44]	马岩岩, 张军, 陈娇, 张凌云, 朱世平, 闫树堂, 钟广炎. 柑橘半胱氨酸蛋白酶基因CsCysP的分离、亚细胞定位及表达分析. 园艺学报, 2014, 41(4): 621-630.
	MA Y Y, ZHANG J, CHEN J, ZHANG L Y, ZHU S P, YAN S T, ZHONG G Y. Isolation, subcellular localization and expression analysis of a citrus cysteine protease gene, CsCysP. Acta Horticulturae Sinica, 2014, 41(4): 621-630. (in Chinese)
[45]	HAO L, HSIANG T, GOODWIN P H. Role of two cysteine proteinases in the susceptible response of Nicotiana benthamiana to Colletotrichum destructivum and the hypersensitive response to Pseudomonas syringae pv. tomato. Plant Science, 2006, 170(5): 1001-1009.
[46]	LI M, LI C, JIANG K, LI K, ZHANG J, SUN M, WU G, QING L. Characterization of pathogenicity-associated V2 protein of tobacco curly shoot virus. International Journal of Molecular Sciences, 2021, 22(2): 923.
[47]	温玉霞, 张坚, 王琴, 王靖, 裴悦宏, 田绍锐, 樊光进, 马小舟, 孙现超. 本氏烟NbMBF1c的克隆、表达及在TMV侵染过程中的功能. 中国农业科学, 2022, 55(18): 3543-3555.
	WEN Y X, ZHANG J, WANG Q, WANG J, PEI Y H, TIAN S R, FAN G J, MA X Z, SUN X C. Cloning,expression and anti-TMV function analysis of Nicotiana benthamiana NbMBF1c. Scientia Agricultura Sinica, 2022, 55(18): 3543-3555. (in Chinese)
[48]	PIETERSE C M J, LEON-REYES A, VAN DER ENT S, VAN WEES S C M. Networking by small-molecule hormones in plant immunity. Nature Chemical Biology, 2009, 5(5): 308-316.
[49]	DING P, DING Y. Stories of salicylic acid: A plant defense hormone. Trends in Plant Science, 2020, 25(6): 549-565.
[50]	BOL J F, LINTHORST H J M, CORNELISSEN B J C. Plant pathogenesis-related proteins induced by virus infection. Annual Review of Phytopathology, 1990, 28: 113-138.
[51]	CUTT J R, HARPSTER M H, DIXON D C, CARR J P, DUNSMUIR P, KLESSIG D F. Disease response to tobacco mosaic virus in transgenic tobacco plants that constitutively express the pathogenesis- related PR1b gene. Virology, 1989, 173(1): 89-97.
[52]	魏周玲, 彭浩然, 潘琪, 张永至, 蒲运丹, 吴根土, 青玲, 孙现超. 核糖体失活蛋白(α-MC)亚细胞定位及对TMV的抑制作用. 中国农业科学, 2017, 50(5): 840-848.
	WEI Z L, PENG H R, PAN Q, ZHANG Y Z, PU Y D, WU G T, QING L, SUN X C. Subcellular localization of the ribosome-inactivating protein α-MC and its antiviral effect on TMV. Scientia Agricultura Sinica, 2017, 50(5): 840-848. (in Chinese)
[53]	RAUSCHER M, ÁDÁM A L, WIRTZ S, GUGGENHEIM R, MENDGEN K, DEISING H B. PR-1 protein inhibits the differentiation of rust infection hyphae in leaves of acquired resistant broad bean. The Plant Journal, 1999, 19(6): 625-633.

引物名称 Primers name		引物序列F Primer sequence F (5′-3′)	引物序列R Primer sequence R (5′-3′)
PCR	NbCP8	ATGTCTCGTTTCTCACTTCTATTGGC	TCCTACCCTGTCGTTGCCTGA
qRT-PCR	CP8	TCGCTTTGCTCACAGGTATG	TGGTGGCTGAACAGTTTTGA
	CP12	GAAAGCTGCACTGAACCCTC	GTGCTACAGTTAGGCCAAGC
	CP22	CGGCAGTGAGAGAGCATTTC	CCACCCTTCCTCATAAGCCA
	CP18	CCCTTATCGCGGTGTTGAAG	GATGCTTCAATGGCTACGCA
	CP13	TAAGAACCAAGGGCAATGCG	GAGACCTCCTTGACAACCCA
	MYC2	CGGTTCTTCTTCCGTCTTCTT	ACGCTGTTGAAGGGTTTCT
	TMV MP	TTAGGTTCCCTGACGGTGAC	ACCGTTGCGTCGTCTACTCT
	ABA1	GCCGTTGTTTGTTCATCTC	ACTGTATCACCTTGCCTCC
	NCED1	ACCCGACTCGATTTTCAATG	AACTTTTGGCCATGGTTCTG
	COI1	CAGCAGCCCATTGTTTCTTAC	TACTGGCCAAGTACTTCCAATC
	PR1	CGTGAAGATGTGGGTCAATG	CCATACGGACGTTGTCCTCT
	NPR1	TGTTGCTGCCATGAGAAAAG	TAACCGATCCTTTGGAGCAG
	Actin	CGGTGCCCACTTTCCGATCT	TCCTCACCGTCAGCCATTTT
TRV	TRV:NbCP8	CGCGGATCCGGCTGCAATGGTGGGCT	CCGCTCGAGTACATCCATGGGAGTGTTGC
PVX	PVX:NbCP8	ACGCGTCGACATGTCTCGTTTCTCACTTCTATTGGC	CCATCGATCATGTCTCGTTTCTCACTTCTATTGGC