甘蔗黄叶病毒P0蛋白分子特性及其抑制RNA沉默活性

doi:10.3864/j.issn.0578-1752.2014.23.008

摘要/Abstract

摘要： 【目的】甘蔗黄叶病（yellow leaf，YL）是一种主要的甘蔗病毒病害，在全球多数甘蔗种植国家或地区普遍发生，已对甘蔗生产造成严重威胁。其病原为甘蔗黄叶病毒（Sugarcane yellow leaf virus，SCYLV），属于黄症病毒科马铃薯卷叶病毒属成员。研究SCYLV编码的沉默抑制子P0蛋白的分子特征、保守结构域及其在自然寄主甘蔗上抑制RNA沉默的功能，为下一步从RNA沉默水平解析SCYLV致病分子机理奠定基础。【方法】利用RT-PCR克隆技术获得SCYLV中国分离物CHN-FJ4编码的RNA沉默抑制子基因P0及其两个缺失突变体P0^Δ2-15（N端缺失15个氨基酸）和P0^Δ155-256（C端缺失102个氨基酸），然后定向克隆到单子叶植物表达载体pUbi-nos（空载体）上，获得重组质粒。利用甘蔗嫩叶组织瞬时表达系统鉴定病毒RNA沉默抑制子技术，结合ImageJ软件定量分析P0及其两个缺失突变体抑制外源基因EYFP瞬时表达活性的变化。番茄丛矮病毒（Tomato bushy stunt virus，TBSV）编码的P19作为沉默抑制子阳性对照。【结果】P0核苷酸序列的遗传进化分析结果显示，所获得的SCYLV病毒分离物CHN-FJ4为BRA基因型，与其他BRA基因型分离物氨基酸序列一致性为96.1%—98.4%。利用MEME在线软件预测P0蛋白的保守结构域，结果表明P0蛋白含有3个显著的保守区。分别位于第1—60、76—125和161—210氨基酸残基。通过Datamonkey在线服务器（http://www.datamonkey.org）提供的5种运算方法分析P0蛋白氨基酸位点的选择压力，结果显示P0蛋白具有8个氨基酸正向选择位点。将含有P0及其缺失突变体的重组质粒连同含EYFP报告基因的pTEM12质粒采用基因枪轰击法分别导入甘蔗嫩叶组织细胞，轰击后48—120 h，P0和P19逐渐地提高EYFP荧光表达点数和荧光表达水平；在120 h，与pUbi-nos空载体（不含沉默抑制子）对照组相比，P0和P19均显著地提高甘蔗嫩叶组织EYFP荧光表达点数和荧光表达水平，分别达1.6倍和4.0倍以上。P0与P19抑制RNA沉默活性水平没有显著差异（P>0.05）。P0蛋白N端缺失15个氨基酸（P0^Δ2-15）和C端缺失102个氨基酸（P0^Δ155-256）均丧失了沉默抑制子的功能，荧光表达与不含沉默抑制子对照组相当。【结论】在甘蔗嫩叶组织EYFP瞬时表达体系上，P0能够显著地提高外源基因EYFP表达水平。P0蛋白N端15个氨基酸和C端102个氨基酸含有显著的保守区域，是P0发挥沉默抑制子功能所必需的。

关键词: 甘蔗黄叶病毒, P0蛋白, RNA沉默抑制子, 黄色荧光蛋白

Abstract: 【Objective】Yellow leaf (YL) disease of sugarcane is one of viral diseases in major sugarcane-producing regions in China and worldwide, causing a serious threat to sugarcane industry. Sugarcane yellow leaf virus (SCYLV), a member of the genus Polerovirus and family Luteoviridae, is the causal agent of YL. The aim of this study is to characterize the conserved regions of P0 protein encoded by SCYLV, and to test its functionality as a RNA silencing suppressor in natural host sugarcane (Saccharum spp. hybrids). The findings will contribute to further investigate the molecular mechanism of SCYLV pathogenesis at RNA silencing level. 【Method】P0 and its two deletion mutants, P0^?2-15(15-aa deletion in N terminal) and P0^?155-256(102-aa deletion in C terminal) from SCYLV CHN-FJ4 isolate were obtained by RT-PCR and cloned into an expression vector under control of the maize ubiquitin 1 promoter and Agrobacterium nopaline synthase terminator. Each of P0 or its deletion mutant constructs were co-bombarded with the reporter gene coding for enhance yellow fluorescent protein (EYFP) into sugarcane young leaf segments, and transient EYFP expression was quantified using ImageJ software. The Tomato bushy stunt virus-encoded P19 was included as a model suppressor.【Result】Phylogenetic analysis revealed that the CHN-FJ4 isolate clustered with isolates of the BRA genotype and shared 96.1%-98.4% aa identified with them. MEME software online revealed that the P0 proteins contained three significant conserved regions positioned at 1-60, 76-125, and 161-210 aa residues. Besides, eight positive sites were detected using five different approaches under the datamonkey web-server. P0, P0^?2-15 and P0^?155-256 were co-bombarded with EYFP into sugarcane young leaf segments, respectively. In young leaf segments co-expressing a suppressor P0 or P19, EYFP foci account and EYFP expression level were increased at 48-120 h post-DNA introduction compared with that in the absence of a suppressor. At 120 h post-bombardment, EYFP foci account and of EYFP expression levelincreased by more than 1.6-fold and 4.0-fold, respectively. No significant differences (P>0.05) in EYFP expression were found in presence of P0 or P19. However, EYFP expression was not increased by P0^?2-15 or P0^?155-256. The reason would be 15-aa deletion in N-terminal or 102-aa deletion in C-terminal of P0 protein impaired and even disabled the function of RNA silencing suppressor.【Conclusion】The P0 RNA silencing suppressor proved to be efficient in enhancing reporter gene expression in transient assay using sugarcane young leaf segments. The conserved regions of P0, 15-aa in N terminal and 102-aa in C terminal, are necessary for the RNA silencing suppressor activity of P0.

Key words: Sugarcane yellow leaf virus, P0 protein, RNA silencing suppressor, EYFP

林艺华，肖胜华，刘营航，陈建生，傅华英，陈如凯，高三基. 甘蔗黄叶病毒P0蛋白分子特性及其抑制RNA沉默活性[J]. 中国农业科学, 2014, 47(23): 4627-4636.

LIN Yi-hua, XIAO Sheng-hua, LIU Ying-hang, CHEN Jian-sheng, FU Hua-ying, CHEN Ru-kai, GAO San-ji. Molecular Characterization and Analysis of Suppressing RNA Silence of P0 Protein Encoded by Sugarcane yellow leaf virus[J]. Scientia Agricultura Sinica, 2014, 47(23): 4627-4636.

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.0578-1752.2014.23.008

https://www.chinaagrisci.com/CN/Y2014/V47/I23/4627

参考文献

[1] Schenck S. Yellow leaf syndrome-a new sugarcane disease. Hawaiian Sugar Planters Association: Annual Report, 1990: 38.

[2] Wang M Q, Xu D L, Li R, Zhou G H. Genotype identification and genetic diversity of Sugarcane yellow leaf virus in China. Plant Pathology, 2012, 61(5): 986-993.

[3] Mangwende T, Wang M L, Borth W, Hu J, Moore P H, Mirkov T E, Albert H H. The P0 gene of Sugarcane yellow leaf virus encodes an RNA silencing suppressor with unique activities. Virology, 2009, 384(45): 38-50.

[4] Scagliusi S M, Lockhart B E L. Transmission, characterization and serology of a luteovirus associated with yellow leaf syndrome of sugarcane. Phytopathology, 2000, 90(2): 120-124.

[5] Vega J, Scagluisi S M, Ulian E C. Sugarcane yellow leaf disease in Brazil: evidence of association with a Luteovirus. Plant Disease,1997, 81(1): 21-26.

[6] Pazhouhandeh M, Dieterle M, Marrocco K, Lechner E, Berry B, Brault V, Hemmer O, Kretsch T, Richards K E, Genschik P, Ziegler-Graff V. F-box-like domain in the polerovirus protein P0 is required for silencing suppressor function. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(6): 1994-1999.

[7] Bortolamiol D, Pazhouhandeh M, Marrocco K, Genschik P, Ziegler- Graff V. The polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing. Current Biology, 2007, 17(18): 1615-1621.

[8] 霍晓辉, 申永梅, 刘国富, 曹雪松. 马铃薯卷叶病毒P0抑制RNA沉默及其与AGO蛋白的相互作用. 生物技术通报, 2013(3): 70-76.

Huo X H, Shen Y M, Liu G F, Cao X S. P0 of Potato leafroll virus suppress RNA silencing and its interaction with AGO protein. Biotechnology Bulletin, 2013(3): 70-76. (in Chinese)

[9] ElSayed A I, Komor E. Investigation of ORF0 as a sensitive alternative diagnostic segment to detect Sugarcane yellow leaf virus. Journal of General Plant Pathology, 2012, 78(3): 207-216.

[10] Lin Y H, Gao S J, Damaj M B, Fu H Y, Chen R K, Mirkov T E. Genome characterization of Sugarcane yellow leaf virus from China reveals a novel recombinant genotype. Archives of Virology, 2014, 159(6): 1421-1429.

[11] Roth B M, Pruss G J, Vance V B. Plant viral suppressors of RNA silencing. Virus Research, 2004, 102(16): 97-108.

[12] Burgyán J. Role of silencing suppressor proteins. Methods in Molecular Biology, 2008, 451(46): 69-79.

[13] Alvarado V, Scholthof H B. Plant responses against invasive nucleic acids: RNA silencing and its suppression by plant viral pathogens. Seminars in Cell & Developmental Biology, 2009, 20(21): 1032-1040.

[14] Chiera J M, Lindbo J A, Finer J J. Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing. Transgenic Research, 2008, 17(21): 1143-1154.

[15] Dhillon T, Chiera J M, Lindbo J A, Finer J J. Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression. Plant Cell Reports, 2009, 28(38): 639-647.

[16] Gao S J, Damaj M B, Park J W, Beyene G, Buenrostro-Nava M T, Molina J, Wang X F, Ciomperlik J J, Manabayeva S A, Alvarado V Y, Rathore K S, Scholthof H B, Mirkov T E. Enhanced transgene expression in sugarcane by co-expression of virus-encoded RNA silencing suppressors. PloS ONE, 2013, 8(6): e66046.

[17] BaileyT L, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers//Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology. Menlo Park, California: AAAI Press, 1994: 28-36.

[18] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.

[19] Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 1987, 4(4): 406-425.

[20] Moonan F, Molina J, Mirkov T E. Sugarcane yellow leaf virus: an emerging virus that has evolved by recombination between luteoviral and poleroviral ancestors. Virology,2000, 269(1): 156-171.

[21] Smith G R, Borg Z, Lockhart B E L, Braithwaite K S, Gibbs M J. Sugarcane yellow leaf virus: a novel member of the Luteoviridae that probably arose by inter-species recombination. Journal of General Virology, 2000, 81(7): 1865-1869.

[22] Abu Ahmad Y, Rassaby L, Royer M, Borg Z, Braithwaite K S, Mirkov T E, Irey M S, Perrier X, Smith G R, Rott P. Yellow leaf of sugarcane is caused by at least three different genotypes of Sugarcane yellow leaf virus, one of which predominates on the Island of Reunion. Archives of Virology,2006, 151(7): 1355-1371.

[23] ElSayed A I, Weig A R, Komor E. Molecular characterization of Hawaiian Sugarcane yellow virus leaf genotypes and their phylogenetic relationship to strains from other sugarcane-growing countries. European Journal of Plant Pathology, 2011, 129(3): 399-412.

[24] Moonan F, Mirkov T E. Analyses of genotypic diversity among North, South, and Central American isolates of Sugarcane yellow leaf virus: evidence for Colombian origins and for intraspecific spatial phylogenetic variation. Journal of Virology, 2002, 76(3): 1339-1348.

[25] Chinnaraja C, Viswanathan R, Karuppaiah R, Bagyalakshmi K, Malathi P, Parameswari B. Complete genome characterization of Sugarcane yellow leaf virus from India: Evidence for RNA recombination. European Journal of Plant Pathology, 2013, 135(2): 335-349.

[26] Wang M Q, Zhou G H. A near-complete genome sequence of a distinct isolate of Sugarcane yellow leaf virus from China, representing a sixth new genotype. Virus Genes, 2010, 41(2): 268-272.

[27] Abu Ahmad Y, Royer M, Daugrois J H, Costet L, Lett J M, Victoria J I, Girard J C, Rott P. Geographical distribution of four Sugarcane yellow leaf virus genotypes. Plant Disease,2006, 90(9): 1156-1160.

[28] Abu Ahmad Y, Costet L, Daugrois J H, Nibouche S, Letourmy P, Girard J C, Rott P. Variation in infection capacity and in virulence exists between genotypes of Sugarcane yellow leaf virus. Plant Disease, 2007, 91(3): 253-259.

[29] Sérémé D, Lacombe S, Konaté M, Bangratz M, Pinel-Galzi A, Fargette D, Traoré A S, Konaté G, Brugidou C. Sites under positive selection modulate the RNA silencing suppressor activity of rice yellow mottle virus movement protein P1. Journal of General Virology, 2014, 95(Pt 1): 213-218.

[30] Kipreos E T, Pagano M. The F-box protein family. Genome Biology, 2000, 1(5): 3002.1-3002.7.

[31] Zhuo T, Li Y Y, Xiang H Y, Wu Z Y, Wang X B, Wang Y, Zhang Y L, Li D W, Yu J L, Han C G. Amino acid sequence motifs essential for P0-mediated suppression of RNA silencing in an isolate of Potato leaf roll virus from Inner Mongolia. Molecular Plant-Microbe Interactions, 2014, 27(6): 515-527.