Chongqing Key Laboratory of Plant Disease Biology, College of Plant Protection, Southwest University, Chongqing, 400716, China.
Virus Res. 2020 May;281:197939. doi: 10.1016/j.virusres.2020.197939. Epub 2020 Mar 18.
Geminiviruses are single-stranded DNA viruses that cause devastating diseases in many crops worldwide. The replication enhancer proteins (REn), encoded by the C3 (AC3, and AL3) ORFs of geminiviruses, have critical roles in viral DNA accumulation and symptom development in infected plants. In the current study, we have constructed an infectious clone of the Tobacco curly shoot virus (TbCSV) C3 mutant, TbCSV, that contains two start codon mutations that abrogated C3 ORF expression, but did not alter the amino acid sequence of the C2 ORF. As predicted, the absence of the C3 protein reduced TbCSV DNA accumulation, and over-expression of the C3 protein enhanced TbCSV DNA accumulation in infected leaves of Nicotiana benthamiana. The C3 mutation reduced the expression levels of both virion- and complementary-sense TbCSV genes whereas over-expression of the C3 protein increased TbCSV gene expression. Furthermore, the expression of the wild-type and site-directed mutants of C3 proteins using the potato virus X (PVX) system showed that Y93A mutation reduced the replication enhancement activity of the C3 protein in N. benthamiana. All the available evidence demonstrates that the C3 protein is tightly coupled with TbCSV DNA accumulation. However, the TbCSV mutant was nearly as infectious in N. benthamiana as TbCSV and only had slightly delayed and attenuated symptom expression. Our findings demonstrate that TbCSV C3 protein enhances viral replication and gene expression, but has only moderate effects on symptom development in N. benthamiana.
双生病毒是单链 DNA 病毒，会导致全球许多作物发生严重病害。双生病毒的复制增强蛋白（REn）由 C3（AC3 和 AL3）ORF 编码，在病毒 DNA 积累和感染植物症状发展中起关键作用。在本研究中，我们构建了烟草曲叶病毒（TbCSV）C3 突变体的传染性克隆 TbCSV，其包含两个起始密码子突变，使 C3 ORF 表达失活，但不改变 C2 ORF 的氨基酸序列。正如预测的那样，C3 蛋白的缺失降低了 TbCSV DNA 的积累，而 C3 蛋白的过表达增强了感染的 Nicotiana benthamiana 叶片中的 TbCSV DNA 积累。C3 突变降低了病毒和互补 sense TbCSV 基因的表达水平，而 C3 蛋白的过表达增加了 TbCSV 基因的表达。此外，使用马铃薯 X 病毒（PVX）系统表达 C3 蛋白的野生型和定点突变体表明，Y93A 突变降低了 C3 蛋白在 N. benthamiana 中的复制增强活性。所有现有证据表明，C3 蛋白与 TbCSV DNA 积累紧密相关。然而，与 TbCSV 相比，TbCSV 突变体在 N. benthamiana 中的传染性几乎相同，只是症状表达略有延迟和减弱。我们的研究结果表明，TbCSV C3 蛋白增强了病毒复制和基因表达，但对 N. benthamiana 中症状发展的影响仅为中等。
