Effect of NbMVKa Interacting with AV2 Protein of Tomato leaf curl Hsinchu virus on Virus Pathogenicity
LI Ding-Shan1, HAN Xing1,2, LIN Wen-Zhong1, ZHA Qing-Chen1, ZHANG Wen-Wen1, DU Zhen-Guo1, ZHANG Jie1,*, WU Zu-Jian1
1 Institute of Plant Virology/State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2 Jiangxi Agricultural Technology Promotion Center, Nanchang 330046, China
Abstract：Monopartite geminiviral V2 protein plays an important role in the process of virus infection, while the research on the function of bipartite geminiviral AV2 protein is relatively few. To reveal the function of the AV2 protein of bipartite geminiviral Tomato leaf curl Hsinchu virus (ToLCHsV) by using the technique of proximity-dependent biotin identification (BioID)-mass spectrometry, several host proteins proximal to AV2 were preliminarily screened in Nicotiana benthamiana. The interaction between mevalonate kinase a (NbMVKa) and ToLCHsV-AV2 was verified by yeast two hybrid (YTH) and bimolecular fluorescence complementation (BiFC). Using the Tobacco rattle virus (TRV)-induced gene silencing technique, ToLCHsV infectious clone was inoculated in N. benthamiana plants. The results showed that 10 d post inoculation (dpi), NbMVKa silenced N. benthamiana plants showed no obvious symptoms, while the non-silenced plants showed leaf curl. Meanwhile, using the Southern blot analysis, the accumulation of viral DNA in silenced plants was relatively decreased. After 20 dpi, symptoms of NbMVKa silenced N. benthamiana plants were basically the same as those of the non-silenced plants. The results revealed that NbMVKa might play a positive role in regulating the pathogenesis of the virus by interacting with ToLCHsV-AV2. These results provide theoretical basis for further elucidating the function of bipartite geminiviral AV2 protein.
[1] 冯细华, 郭安平, 郭运玲, 等. 2008. 利用PCR方法从苎麻中检测出粉虱传双生病毒[J]. 热带作物学报, 29(4): 518-521. (Feng X H, Guo A P, Guo Y L, et al.2008. PCR detection of whitefly-transmitted geminiviruses from ramie[J]. Chinese Journal of Tropical Crops, 29(4): 518-521.) [2] 芮鹏环. 2021. 寄主因子FvZFP与SVBV P6互作影响森林草莓抗病性的分子机制[D]. 硕士学位论文, 安徽农业大学, 导师: 江彤, pp. 27-29. (Rui P.2021. Molecular mechanism of interaction between host factor FvZFP and SVBV P6 on disease resistance of Fragaria vesca [D]. Thesis for M.S., Anhui Agricultural University, Supervisor: Jiang T, pp. 27-29.) [3] 杨彩霞. 2009. 福建省六种双生病毒的分子鉴定及RaMoV NSP互作蛋白的筛选[D]. 博士学位论文, 福建农林大学, 导师: 谢联辉. pp. 34-36. (Yang C X.2009. Molecular identification of six begomoviruses in Fujian and selection the interactive proteins of RaMoV NSP[D]. Thesis for Ph.D., Fujian Agriculture and Forestry University, Supervisor: Xie L H, pp. 34-36.) [4] 张雨欣, 丁明, 柳军. 2022. 基于生物素连接酶的邻近标记技术在蛋白质组学中的研究进展[J]. 中国药科大学学报, 53(1): 18-24. (Zhang Y X, Ding M, Liu J.2022. Research progress of proximity labeling technology based on biotin ligase in proteomics[J]. Journal of China Pharmaceutical University, 53(1): 18-24.) [5] 庄军. 2015. BBTV抗菌蛋白与血蓝蛋白抗菌肽的抑菌活性研究[D]. 博士学位论文, 福建农林大学, 导师: 谢联辉, pp. 18. (Zhuang J.2015. The antagonistic activity studies on BBTV antifungal peotein and hemocyanin-derived antimicrobial peptide[D]. Thesis for Ph.D., Fujian Agriculture Forestry University, Supervisor: Xie L, pp. 18.) [6] Aguilar E, Gomez B G, Lozano-duran R.2020. Recent advances on the plant manipulation by geminiviruses[J]. Current Opinion in Plant Biology, 56: 56-64. [7] Amin I, Hussain K, Akbergenov R, et al.2011. Suppressors of RNA silencing encoded by the components of the cotton leaf curl begomovirus-betasatellite complex[J]. Molecular Plant-microbe Interactions, 24(8): 973-983. [8] Atsumi G, Kagaya U, Tabayashi N, et al.2018. Analysis of the mechanisms regulating the expression of isoprenoid biosynthesis genes in hydroponically-grown Nicotiana benthamiana plants using virus-induced gene silencing[J]. Scientific Reports, 8(1):14804. [9] Champenoy S, Tourte M.1998. Expression of the yeast mevalonate kinase gene in trangenic tobacco[J]. Molecular Breeding, 4: 29-300. [10] Chang L Y, Chen Y J, Fan C Y, et al.2017. Identification of siglec ligands using a proximity labeling method[J]. Journal of Proteome Research, 16(10): 3929-3941. [11] Cho K F, Branon T C, Rajeev S, et al.2020. Split-TurboID enables contact-dependent proximity labeling in cells[J]. Proceedings of The National Academy of Sciences of the USA, 117(22): 12143-12154. [12] Chu X, Li D.2003. Cloning expression and purification of Histagged ratmevabnate kinase[J]. Protein Expression and Purification, 27(1): 165-170. [13] Cota-Sánchez J H, Remarchuk K, Ubayasena K.2006. Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue[J]. Plant Molecular Biology Reporter, 24(2): 161-167. [14] Conlan B, Stoll T, Gorman J J, et al.2018. Development of a rapid in planta BioID system as a probe for plasma membrane-associated immunity proteins[J]. Frontiers in Plant Science, 9: 1182. [15] Das P P, Macharia M W, Lin Q S, et al.2019. In planta proximity-dependent biotin identification (BioID) identifies a TMV replication co-chaperone NbSGT1 in the vicinity of 126 kDa replicase[J]. Journal of Proteomics, 204: 103402. [16] Fiallo-Olive E, Lett J M, Martin D P, et al.2021. ICTV Virus taxonomy profile: Geminiviridae 2021[J]. Journal of General Virology, 102(12): 001696. [17] Fields S, Song O.1989. A novel genetic system to detect protein-protein interactions[J]. Nature, 340(6230): 245-246. [18] Fondong V N.2013. Geminivirus protein structure and function[J]. Molecular Plant Pathology, 14(6): 635-649. [19] Fukunaga R, Doudna J A.2009. dsRNA with 5' overhangs contributes to endogenous and antiviral RNA silencing pathways in plants[J]. The EMBO Journal, 28(5): 545-555. [20] Glick E, Zrachya A, Levy Y, et al.2008. Interaction with host SGS3 is required for suppression of RNA silencing by Tomato yellow leaf curl virus V2 protein[J]. Proceedings of The National Academy of Sciences of the USA, 105(1): 157-161. [21] Go C D, Knight J D R, Rajasekharan A, et al.2021. A proximity-dependent biotinylation map of a human cell[J]. Nature, 595(7865): 120-124. [22] Hanssen I M, Lapidot M, Thomma B P.2010. Emerging viral diseases of tomato crops[J]. Molecular Plant-microbe Interactions, 23(5): 539-548. [23] Huang A B, Tang Y, Shi X T, et al.2020. Proximity labeling proteomics reveals critical regulators for inner nuclear membrane protein degradation in plants[J]. Nature Communications, 11(1): 3284. [24] Hyodo K, Hashimoto K, Kuchitsu K, et al.2017. Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus[J]. Proceedings of the National Academy of Sciences of the USA, 114(7): E1282-E1290. [25] Kaido M, Inoue Y, Takeda Y, et al.2007. Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana[J]. Molecular Plant-microbe Interactions, 20(6): 671-681. [26] Khan M, Youn J Y, Gingras A C, et al.2018. In planta proximity dependent biotin identification (BioID)[J]. Scientific Reports, 8(1): 9212. [27] Kido K, Yamanaka S, Nakano S, et al.2020. AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions[J]. Elife, 9: e54983. [28] Kim D I, Birendra K C, Zhu W H, et al.2014. Probing nuclear pore complex architecture with proximity-dependent biotinylation[J]. Proceedings of the National Academy of Sciences of the USA, 111(24): e2453-2461. [29] Li J, Zhang X Y, Qian Y J.2009. Molecular characterization of Ramie mosaic virus isolates detected in Jiangsu and Zhejiang provinces, China[J]. Acta Virologica, 54(3): 225-228. [30] Lin Q P, Zhou Z J, Luo W B, et al.2017. Screening of proximal and interacting proteins in rice protoplasts by proximity-dependent biotinylation[J]. Frontiers in Plant Science, 8: 749. [31] Liu D, Shi L, Han C, et al.2012. Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR[J]. PLOS ONE, 7(9): e46451. [32] Macharia M W, Tan W Y Z, Das P P, et al.2019. Proximity-dependent biotinylation screening identifies NbHYPK as a novel interacting partner of ATG8 in plants[J]. BMC Plant Biology, 19(1): 326. [33] Mubin M, Briddon R W, Mansoor S.2019. The V2 protein encoded by a monopartite Begomovirus is a suppressor of both post-transcriptional and transcriptional gene silencing activity[J]. Gene, 686: 43-48. [34] Padidam M, Beachy R N, Fauquet C M.1996. The role of AV2 ("precoat") and coat protein in viral replication and movement in Tomato leaf curl geminivirus[J]. Virology, 224(2): 390-404. [35] Roshan P, Kulshreshtha A, Kumar S, et al.2018. AV2 protein of Tomato leaf curl Palampur virus promotes systemic necrosis in Nicotiana benthamiana and interacts with host Catalase2[J]. Scientific Reports, 8(1): 1273. [36] Rouhibakhsh A, Haq Q, Malathi V G.2011. Mutagenesis in ORF AV2 affects viral replication in Mungbean yellow mosaic India virus[J]. Journal of Biosciences, 36(2): 329-340. [37] Roux K J, Kim D I, Raida M, et al.2012. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in manmalian cells[J]. Journal of Cell Biology, 196(6): 801-810. [38] Sharma P, Ikegami M, Kon T.2010. Identification of the virulence factors and suppressors of posttranscriptional gene silencing encoded by Ageratum yellow vein virus, a monopartite begomovirus[J]. Virus Research, 149(1): 19-27. [39] Sharma P, Ikegami M.2010. Tomato leaf curl Java virus V2 protein is a determinant of virulence, hypersensitive response and suppression of posttranscriptional gene silencing[J]. Virology, 396(1): 85-93. [40] Wang B, Li F F, Huang C J, et al.2013. V2 of Tomato yellow leaf curl virus can suppress methylation-mediated transcriptional gene silencing in plants[J]. Journal of General Virology, 95(1): 225-230. [41] Wang B, Yang X L, Wang Y Q, et al.2018. Tomato yellow leaf curl virus V2 interacts with host histone deacetylase 6 to suppress methylation-mediated transcriptional gene silencing in plants[J]. Journal of Virology, 92(18): e00036-18. [42] Wang Y J, Wu Y Y, Gong Q, et al.2019. Geminiviral V2 protein suppresses transcriptional gene silencing through interaction with AGO4[J]. Journal of Virology, 93(6): e01675-18. [43] Wu X, Xiong E, Wang W, et al.2014. Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis[J]. Nature Protocols, 9(2): 362-374. [44] Yang X X, Wen Z Y, Zhang D L, et al.2021. Proximity labeling: An emerging tool for probing in plant a molecular interactions[J]. Plant Communications, 2(2): 100137.
