本氏烟中与番茄斑萎病毒N蛋白互作的寄主因子的筛选

苗淑月; 高晓晓; 郑立敏; 陈建斌; 赵星月; 程菊娥; 陈莎; 章松柏; 刘勇

doi:10.19303/j.issn.1008-0384.2021.02.013

本氏烟中与番茄斑萎病毒N蛋白互作的寄主因子的筛选

doi: 10.19303/j.issn.1008-0384.2021.02.013

苗淑月^{1, 2,},
高晓晓¹,
郑立敏²,
陈建斌²,
赵星月³,
程菊娥²,
陈莎⁴,
章松柏^1, ,,
刘勇^{1, 2, ,}

1.
长江大学农学院，湖北荆州　434025
2.
湖南省农业科学院植物保护研究所，湖南长沙　410125
3.
西南林业大学，云南昆明　650032
4.
湖南工业大学生命科学与化学学院，湖南株洲　412007

基金项目: 国家自然科学基金（31871935）；国家重点研发计划政府间/港澳台重点专项项目（2018YFE0112600）；湖南省农业科技创新资金项目（2020CX38）；湖南省自然科学基金（2019JJ50143）；湖南省教育厅科学研究项目（18C0491、20C0588）；长沙市杰出创新青年培养计划

详细信息

作者简介:
苗淑月（1995−），女，硕士研究生，研究方向：植物病毒与分子生物学（E-mail：1724711842@qq.com）

通讯作者:
章松柏（1978-），男，博士，副教授，研究方向：分子病毒学（E-mail：yangtze2008@126.com）

刘勇（1966-），男，博士，研究员，研究方向：主要从事蔬菜病害致害机制及绿色综合防控技术研究（E-mail：liuyong@hunaas.cn）

中图分类号: S 436.412.1+1
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出版历程
- 收稿日期: 2020-10-30
- 修回日期: 2020-12-02
- 网络出版日期: 2021-02-08
- 刊出日期: 2021-02-28

Host Factors in Tobacco Interacting with N Protein of Tomato Spotted Wilt Virus

MIAO Shuyue^{1, 2
,},
GAO Xiaoxiao¹,
ZHENG Limin²,
CHEN Jianbin²,
ZHAO Xingyue³,
CHENG Jue²,
CHEN Sha⁴,
ZHANG Songbai^{1
, ,},
LIU Yong^{1, 2
, ,}

1.
Agricultural School, Yangtze University, Jingzhou, Hubei 　434025, China
2.
Institute of Plant Protection, Hunan Academy of Agricultural Science, Changsha, Hunan 　410125, China
3.
Southwest Forestry University, Kunming, Yunnan 　650032, China
4.
School of Life Sciences and Chemistry, Hunan University of Technology, Zhuzhou, Hunan 　412007, China

摘要

摘要: 目的番茄斑萎病毒（Tomato spotted wilt virus, TSWV）是植物多分体负义链RNA病毒的代表性成员。TSWV的N蛋白在病毒感染的寄主植物内表达量高，可能调控病毒对寄主的感病，探究N蛋白通过影响哪些寄主蛋白的表达来完成病毒的侵染，以期为深入解析寄主蛋白调控病毒的分子机制提供理论基础，为后续有效的防控TSWV提供新的思路。方法以TSWV N蛋白作为诱饵蛋白，采用酵母双杂交（Yeast two-hybrid, Y2H）的方法筛选本氏烟内与TSWV N相互作用的寄主蛋白。结果共筛选获得15种与TSWV N相互作用的寄主蛋白。结论这些介体因子主要参与色素的生物合成、类囊体膜的组装、植物防御反应和核糖体生物发生的过程，调节脂质代谢和细胞功能，可能是翻译调控的辅助蛋白，在植物发育和对非生物胁迫的反应中发挥着重要作用。
- 番茄斑萎病毒（TSWV） /
- N蛋白 /
- 酵母双杂交 /
- 寄主蛋白
Abstract: Objective Host factors in Nicotiana benthamiana that interact with the N protein of tomato spotted wilt virus (TSWV), a representative member of plant negative stranded RNA viruses, were identified in preparation for further analysis on the regulation mechanism and in search for effective prevention and control of TSWV-induced disease on plants. Method Using the yeast two-hybrid (Y2H) method, proteins in N. benthamiana that interacted with the bait N protein of TSWV were screened. Result Fifteen host proteins were identified. Conclusion These identified proteins are known to be associated with pigment biosynthesis, thylakoid membrane assembly, plant defense response, ribosome biogenesis, lipid metabolism, and cellular functions in plants. Being upregulated in N. benthamiana infected by TSWV, these proteins might also act as auxiliary proteins in translating the regulation playing an important role in the development and response to abiotic stress of the plant.
- Tomato spotted wilt virus (TSWV) /
- N protein /
- yeast two-hybrid /
- host protein

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图 1 N蛋白在酵母细胞中的自激活验证

注：a. 诱饵载体pGBKT7-N和空载体pGBKT7、阳性对照、阴性对照在SD/-Trp/-Leu营养缺陷型培养基上的生长情况。b. 诱饵载体pGBKT7-N和空载体pGBKT7、阳性对照、阴性对照在SD/-Trp/-Leu/-His/-Ade营养缺陷型培养基上的生长情况。c. 诱饵载体pGBKT7-N和空载体pGBKT7、阳性对照、阴性对照SD/-Trp/-Leu/-His/-Ade/X-ɑ-gal营养缺陷型培养基上的生长情况。A为pGBKT7-53和pGADT7-T；B为pGBKT7-N和pGADT7；C为pGBKT7-Lam和pGADT7-T。

Figure 1. Verification of self-activation of N protein in yeast cells

Note: a: Growth of bait vector pGBKT7-N, empty vector pGBKT7, and positive and negative controls on SD/-Trp/-Leu nutrient-deficient medium. b: Growth of bait vector pGBKT7-N, empty vector pGBKT7, and positive and negative controls on SD/-Trp/-Leu/-His/-Ade nutrient-deficient medium. c: Growth of bait vector pGBKT7-N, empty vector pGBKT7, and positive and negative controls on SD/-Trp/-Leu/-His/-Ade/X-α-gal nutrient-deficient medium. A: pGBKT7-53 and pGADT7-T. B: pGBKT7-N and pGADT7. C: pGBKT7 Lam and pGADT7-T.

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图 2 Western blot检测N蛋白在酵母内的表达情况

注：M为17-180 kDa蛋白分子标记。

Figure 2. Expression of N protein in yeast as detected by Western blot

Note: M: 17-180kDa protein molecular marker.

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图 3 酵母结合体

Figure 3. Yeast complexes

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图 4 酵母菌在四缺培养基上的生长情况

注：a为酵母菌在SD/-Trp/-Leu/-His/-Ade营养缺陷型培养基上的生长情况；b为酵母菌在SD/-Trp/-Leu/-His/-Ade/X-α-Gal营养缺陷型培养基上的生长情况；c为酵母菌在SD/-Trp/-Leu/-His/-Ade/X-α-Gal/AbA营养缺陷型培养基上的生长情况。

Figure 4. Growth of yeast on 4 nutrient-deficient culture media

Note: a: Growth of yeast on SD/-Trp/-Leu/-His/-Ade nutrient-deficient medium. b: Growth of yeast on SD/-Trp/- Leu/-His/-Ade/X-α-gal nutrient-deficient medium. C: Growth of yeast on SD/-Trp/-Leu/-His/-Ade/X-α-gal/AbA nutrient-deficient medium.

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图 5 阳性克隆的PCR检测

注：M为DNA分子标记2K plus Ⅱ；1-14为酵母阳性克隆。

Figure 5. PCR detection of positive clones

Note: M: DNA molecular marker, 2K plus Ⅱ. 1-14: Positive clones of yeast.

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表 1 与TSWV N蛋白互作的15个候选蛋白

Table 1. Fifteen candidate proteins that interacted with N protein of TSWV

蛋白种类 Protein types	蛋白大小/aa Length of protein	GeneBank登录号 GeneBank No.
叶绿素a-b结合蛋白16 Chlorophyll a-b binding protein 16	266	XP_016448384.1
叶绿素a-b结合蛋白Chlorophyll a-b binding protein	270	XP_016501042.1
叶绿素a-b结合蛋白50 Chlorophyll a-b binding protein 50,	267	XP_016445667.1
叶绿素a-b结合蛋白CP 26 Chlorophyll a-b binding protein CP 26	285	NP_001312267.1
二磷酸核酮糖羧化加氧酶小链Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase small chain Rubisco	123	1RLC_S
叶绿素a-b结合蛋白21 Chlorophyll a-b binding protein 21	265	XP_016487828.1
30S核糖体蛋白S6 30S ribosomal protein S6 alpha	199	XP_016444523.1
光系统I反应中心亚基II Photosystem I reaction center subunit II	214	XP_016455922.1
酰基辅酶A结合蛋白Acyl-coa-binding protein	89	XP_016464154.1
脂质转移蛋白Lipid transfer protein	120	AAA21437.1
非特异性脂转移蛋白2 Non-specific lipid-transfer protein 2-like	121	XP_016490998.1
多蛋白桥接因子1b Multiprotein-bridging factor 1b-like	140	XP_016470347.1
衰减核糖体生物发生蛋白BRX1同源物Ribosome biogenesis protein BRX1 homolog	315	XP_016510339.1
细胞分裂周期蛋白48同源物Cell division cycle protein 48 homolog	811	XP_009782020.1
硫胺噻唑合酶Thiamine thiazole synthase	359	XP_016477305.1

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参考文献(33)

[1]	DAVISON A, SIDDELL S, MUSHEGIAN A, et al. Virus Taxonomy[S]. International Committee on Taxonomy of Viruse, 2019.
[2]	PARRELLA G, GOGNALONS P, GEBRE-SELASSIE K, et al. An update of the host range of tomato spotted wilt virus [J]. Journal of Plant Pathology, 2003, 85(4): 227−264.
[3]	SCHOLTHOF K B G, ADKINS S, CZOSNEK H, et al. Top 10 plant viruses in molecular plant pathology [J]. Molecular Plant Pathology, 2011, 12(9): 938−954. doi: 10.1111/j.1364-3703.2011.00752.x
[4]	PRINS M, GOLDBACH R. The emerging problem of Tospovirus infection and nonconventional methods of control [J]. Trends in Microbiology, 1998, 6(1): 31−35. doi: 10.1016/S0966-842X(97)01173-6
[5]	CULBREATH A K, CSINOS A S, BERTRAND P F, et al. Tomato spotted wilt virus epidemic in fluecured tobacco in Georgia [J]. Plant Disease, 1991, 75: 483−485. doi: 10.1094/PD-75-0483
[6]	HU Z Z, FENG Z K, ZHANG Z J, et al. Complete genome sequence of a tomato spotted wilt virus isolate from China and comparison to other TSWV isolates of different geographic origin [J]. Archives of Virology, 2011, 156(10): 1905−1908. doi: 10.1007/s00705-011-1078-9
[7]	LIAN S, LEE J S, CHO W K, et al. Phylogenetic and recombination analysis of tomato spotted wilt virus [J]. PLoS One, 2013, 8(5): e63380. doi: 10.1371/journal.pone.0063380
[8]	SIVPRASAD B J, GUBBA A. Isolation and molecular characterization of Tomato spotted wilt virus (TSWV) isolates occurring in South Africa [J]. African Journal of Agricultural Research, 2008(3): 428−434.
[9]	NAGATA T, INOUE-NAGATA A K, PRINS M, et al. Impeded Thrips Transmission of Defective Tomato spotted wilt virus Isolates [J]. Phytopathology, 2000, 90(5): 454−459. doi: 10.1094/PHYTO.2000.90.5.454
[10]	TURINA M, KORMELINK R, RESENDE R O. Resistance to tospoviruses in vegetable crops: Epidemiological and molecular aspects [J]. Annual Review of Phytopathology, 2016, 54: 347−371. doi: 10.1146/annurev-phyto-080615-095843
[11]	WHITFIELD A E, KUMAR N K K, ROTENBERG D, et al. A soluble form of the Tomato spotted wilt virus (TSWV) glycoprotein G(N) (G(N)-S) inhibits transmission of TSWV by Frankliniella occidentalis [J]. Phytopathology, 2008, 98(1): 45−50. doi: 10.1094/PHYTO-98-1-0045
[12]	GUO Y, LIU B C, DING Z Z, et al. Distinct mechanism for the formation of the ribonucleoprotein complex of tomato spotted wilt virus [J]. Journal of Virology, 2017, 91(23): e00892. doi: 10.1128/jvi.00892-17
[13]	RICHMOND K E, CHENAULT K, SHERWOOD J L, et al. Characterization of the nucleic acid binding properties of tomato spotted wilt virus nucleocapsid protein [J]. Virology, 1998, 248(1): 6−11. doi: 10.1006/viro.1998.9223
[14]	SOELLICK T R, UHRIG J F, BUCHER G L, et al. The movement protein NSm of tomato spotted wilt Tospovirus (TSWV): RNA binding, interaction with the TSWV N protein, and identification of interacting plant proteins [J]. PNAS, 2000, 97(5): 2373−2378. doi: 10.1073/pnas.030548397
[15]	RIBEIRO D, BORST J W, GOLDBACH R, et al. Tomato spotted wilt virus nucleocapsid protein interacts with both viral glycoproteins Gn and Gc in planta [J]. Virology, 2009, 383(1): 121−130. doi: 10.1016/j.virol.2008.09.028
[16]	KORMELINK R, STORMS M, VAN LENT J, et al. Expression and subcellular location of the NSM protein of tomato spotted wilt virus (TSWV), a putative viral movement protein [J]. Virology, 1994, 200(1): 56−65. doi: 10.1006/viro.1994.1162
[17]	FENG Z K, CHEN X J, BAO Y Q, et al. Nucleocapsid of Tomato spotted wilt Tospovirus forms mobile particles that traffic on an actin/endoplasmic Reticulum network driven by myosin XI-K [J]. The New Phytologist, 2013, 200(4): 1212−1224. doi: 10.1111/nph.12447
[18]	MARIS P C, JOOSTEN N N, GOLDBACH R W, et al. Restricted Spread of Tomato spotted wilt virus in Thrips-Resistant Pepper [J]. Phytopathology, 2003, 93(10): 1223−1227. doi: 10.1094/PHYTO.2003.93.10.1223
[19]	JAN F J, FAGOAGA C, PANG S Z, et al. A minimum length of N gene sequence in transgenic plants is required for RNA-mediated Tospovirus resistance [J]. The Journal of General Virology, 2000, 81(1): 235−242.
[20]	DE BUCK E, LEBEAU I, VAN MELLAERT L, et al. The use of the cMyc epitope tag can be problematic for protein detection in Legionella pneumophila [J]. Journal of Microbiological Methods, 2004, 59(1): 131−134. doi: 10.1016/j.mimet.2004.05.010
[21]	GREEN B R, PICHERSKY E, KLOPPSTECH K. Chlorophyll a/b-binding proteins: An extended family [J]. Trends in Biochemical Sciences, 1991, 16(5): 181−186.
[22]	CROCE R, CANINO G, ROS F, et al. Chromophore organization in the higher-plant photosystem II antenna protein CP26 [J]. Biochemistry, 2002, 41(23): 7334−7343. doi: 10.1021/bi0257437
[23]	SCHWARTE S, TIEDEMANN R. A gene duplication/loss event in the ribulose-1,5-bisphosphate-carboxylase/oxygenase (rubisco) small subunit gene family among accessions of Arabidopsis thaliana [J]. Molecular Biology and Evolution, 2011, 28(6): 1861−1876. doi: 10.1093/molbev/msr008
[24]	欧志远. 叶绿素含量与植物抗病性的关系 [J]. 安徽农学通报, 2007, 13(6):134−135. doi: 10.3969/j.issn.1007-7731.2007.06.075 OU Z Y. Relationship of chlorophyll contents and plant disease-resistance [J]. Auhui Agricultural Science Bulletin, 2007, 13(6): 134−135.(in Chinese) doi: 10.3969/j.issn.1007-7731.2007.06.075
[25]	TILLER N, WEINGARTNER M, THIELE W, et al. The plastid-specific ribosomal proteins of Arabidopsis thaliana can be divided into non-essential proteins and genuine ribosomal proteins [J]. The Plant Journal, 2012, 69(2): 302−316. doi: 10.1111/j.1365-313X.2011.04791.x
[26]	BOGENGRUBER E, BRIZA P, DOPPLER E, et al. Functional analysis in yeast of the Brix protein superfamily involved in the biogenesis of ribosomes [J]. FEMS Yeast Research, 2003, 3(1): 35−43. doi: 10.1016/S1567-1356(02)00193-9
[27]	COHEN Y, STEPPUHN J, HERRMANN R G, et al. Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing [J]. The EMBO Journal, 1992, 11(1): 79−85. doi: 10.1002/j.1460-2075.1992.tb05030.x
[28]	LUNG S C, CHYE M L. The binding versatility of plant acyl-CoA-binding proteins and their significance in lipid metabolism [J]. Biochimica et Biophysica Acta, 2016, 1861(9 Pt B): 1409−1421.
[29]	SAFI H, SAIBI W, ALAOUI M M, et al. A wheat lipid transfer protein (TdLTP4) promotes tolerance to abiotic and biotic stress in Arabidopsis thaliana [J]. Plant Physiology and Biochemistry, 2015, 89: 64−75. doi: 10.1016/j.plaphy.2015.02.008
[30]	ZHAO J, WANG S S, QIN J J, et al. The lipid transfer protein OsLTPL159 is involved in cold tolerance at the early seedling stage in rice [J]. Plant Biotechnology Journal, 2020, 18(3): 756−769. doi: 10.1111/pbi.13243
[31]	ZHANG L L, WANG Y X, ZHANG Q K, et al. Overexpression of HbMBF1a, encoding multiprotein bridging factor 1 from the halophyte Hordeum brevisubulatum, confers salinity tolerance and ABA insensitivity to transgenic Arabidopsis thaliana [J]. Plant Molecular Biology, 2020, 102(1/2): 1−17.
[32]	LIANG K, PAREDES R, CARMODY R, et al. Human TRIB₂ oscillates during the cell cycle and promotes ubiquitination and degradation of CDC25C [J]. International Journal of Molecular Sciences, 2016, 17(9): 1378. doi: 10.3390/ijms17091378
[33]	FENG X X, YANG S X, TANG K Q, et al. GmPGL1, a thiamine thiazole synthase, is required for the biosynthesis of thiamine in soybean [J]. Frontiers in Plant Science, 2019, 10: 1546. doi: 10.3389/fpls.2019.01546