Synthesis of Full-Length cDNA Infectious Clones ofSoybean Mosaic Virusand Functional Identification of a Key Amino Acid in the Silencing Suppressor Hc-Pro
Synthesis of Full-Length cDNA Infectious Clones ofSoybean Mosaic Virusand Functional Identification of a Key Amino Acid in the Silencing Suppressor Hc-Pro
复制标题
大豆花叶病毒全长cDNA感染性克隆的合成及沉默抑制子Hc-Pro关键氨基酸的功能鉴定
DOI：
10.3390/v12080886
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发表时间：
2020-08-01
期刊：
影响因子：
4.7
通讯作者：
Wuriyanghan, Hada
中科院分区：
文献类型：
--
作者：
Bao, Wenhua;Yan, Ting;Wuriyanghan, Hada
大豆花叶病毒（SMV）是马铃薯Y病毒科的一种重要病毒，可引起大豆产量和种子品质的显著下降。本研究利用吉布森组装法和酵母同源重组系统分别构建了SMV N1株的无标签和含报告基因绿色荧光蛋白（GFP）的感染性克隆。这两个侵染性无性系均适合于在模式寄主N.本塞姆氏菌对天然寄主大豆和其它几种豆科植物表现出较强的感染性。这两个侵染性无性系都是种子传播的，并在种子和后代植株上引起典型的病毒症状。我们使用SMV-GFP感染性克隆进一步研究沉默抑制子辅助组分-蛋白酶（Hc-Pro）中的关键氨基酸的作用。在12个氨基酸替换突变体中，FRNK（Phe-Arg-Asn-Lys）基序182位天冬酰胺→亮氨酸替换突变体2-的共表达减轻了SMV引起的病毒症状和宿主生长迟缓。此外，Hc-Prom 2突变体显示出比野生型Hc-Pro更强的寡聚化。两者合计，SMV感染性克隆将是有用的主机SMV的相互作用和功能基因在大豆和相关豆科植物物种，特别是在种子传播特性的研究。此外，SMV-GFP感染性克隆也将促进N. benthamiana瞬时表达系统。
Soybean mosaic virus (SMV), which belongs to the Potyviridae, causes significant reductions in soybean yield and seed quality. In this study, both tag-free and reporter gene green fluorescent protein (GFP)-containing infectious clones for the SMV N1 strain were constructed by Gibson assembly and with the yeast homologous recombination system, respectively. Both infectious clones are suitable for agroinfiltration on the model host N. benthamiana and show strong infectivity for the natural host soybean and several other legume species. Both infectious clones were seed transmitted and caused typical virus symptoms on seeds and progeny plants. We used the SMV-GFP infectious clone to further investigate the role of key amino acids in the silencing suppressor helper component-proteinase (Hc-Pro). Among twelve amino acid substitution mutants, the co-expression of mutant 2—with an Asparagine→Leucine substitution at position 182 of the FRNK (Phe-Arg-Asn-Lys) motif—attenuated viral symptoms and alleviated the host growth retardation caused by SMV. Moreover, the Hc-Prom2 mutant showed stronger oligomerization than wild-type Hc-Pro. Taken together, the SMV infectious clones will be useful for studies of host–SMV interactions and functional gene characterization in soybeans and related legume species, especially in terms of seed transmission properties. Furthermore, the SMV-GFP infectious clone will also facilitate functional studies of both virus and host genes in an N. benthamiana transient expression system.