State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, People's Republic of China.
Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, People's Republic of China.
J Virol. 2018 Aug 29;92(18). doi: 10.1128/JVI.00036-18. Print 2018 Sep 15.
Cytosine DNA methylation is a conserved epigenetic silencing mechanism that defends against biotic stresses such as geminivirus infection. As a countermeasure, geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Previous studies showed that V2 protein of (TYLCV) functions as a TGS suppressor. However, how V2 mediates TGS suppression remains unknown. Here we show that V2 interacts directly with a histone deacetylase 6 (NbHDA6), a homolog of HDA6 (AtHDA6), known to be involved in gene silencing in cooperation with methyltransferase 1 (MET1). NbHDA6 genetically complemented a late-flowering phenotype and restored histone deacetylation of an AtHDA6 mutant. Furthermore, our investigation showed that NbHDA6 displayed histone deacetylase enzymatic activity, which was not inhibited by V2. Genetic analysis revealed that silencing of expression resulted in enhanced susceptibility to TYLCV infection. In addition, methylation-sensitive PCR and bisulfite sequencing analysis showed that silencing of expression caused reduced DNA methylation of the viral genome in infected plants. HDA6 was previously shown to recruit and physically interact with MET1 to function in gene silencing. Using competitive pulldown and coimmunoprecipitation assays, we demonstrated that V2 did not interact but competed with NbMET1 for direct binding to NbHDA6. These findings suggest that V2 interacts with host HDA6 and interferes with the recruitment of MET1 by HDA6, resulting in decreased methylation of the viral DNA genome by TGS with a concomitant increase in host susceptibility to TYLCV infection. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses. In turn, geminiviruses encode proteins that inhibit methylation by TGS. Previous studies showed that TYLCV V2 can efficiently suppress TGS, but the mechanism remains unknown. We showed that V2 interacted with NbHDA6 but did not inhibit its enzymatic activity. As HDA6 is known to be involved in gene silencing in cooperation with MET1, we explored the relationship between V2, NbMET1, and NbHDA6. Our investigation showed that V2 did not interact but competed with NbMET1 for direct binding to NbHDA6. To our knowledge, this is the first report that viral proteins inhibit TGS by interacting with histone deacetylase but not by blocking the methyl cycle. This work provides an additional mechanism for TGS suppression by geminiviruses.
胞嘧啶 DNA 甲基化是一种保守的表观遗传沉默机制，可抵御生物胁迫，如双生病毒感染。作为一种对策，双生病毒编码的蛋白质可以抑制甲基化和转录基因沉默（TGS）。先前的研究表明，（TYLCV）的 V2 蛋白具有 TGS 抑制作用。然而，V2 如何介导 TGS 抑制尚不清楚。在这里，我们显示 V2 与一个 组蛋白去乙酰化酶 6（NbHDA6）直接相互作用，NbHDA6 是 组蛋白去乙酰化酶 6（AtHDA6）的同源物，已知与甲基转移酶 1（MET1）一起参与基因沉默。NbHDA6 在遗传上互补了晚开花的表型，并恢复了 AtHDA6 突变体的组蛋白去乙酰化。此外，我们的研究表明，NbHDA6 显示出组蛋白去乙酰化酶的酶活性，而该酶活性不受 V2 的抑制。遗传分析表明，沉默 表达导致对 TYLCV 感染的敏感性增加。此外，甲基化敏感 PCR 和亚硫酸氢盐测序分析表明，沉默 表达导致感染植物中病毒基因组的 DNA 甲基化减少。HDA6 先前被证明可以募集并与 MET1 物理相互作用，以在基因沉默中发挥作用。使用竞争性下拉和共免疫沉淀测定，我们证明 V2 不相互作用，但与 NbMET1 竞争直接结合 NbHDA6。这些发现表明，V2 与宿主 HDA6 相互作用并干扰 HDA6 募集 MET1，导致 TGS 中病毒 DNA 基因组的甲基化减少，同时宿主对 TYLCV 感染的敏感性增加。植物利用抑制性病毒基因组甲基化作为抵御双生病毒的表观遗传防御。反过来，双生病毒编码的蛋白质可以通过 TGS 抑制甲基化。先前的研究表明，TYLCV V2 可以有效地抑制 TGS，但机制尚不清楚。我们表明 V2 与 NbHDA6 相互作用，但不抑制其酶活性。由于 HDA6 已知与 MET1 一起参与基因沉默，我们探讨了 V2、NbMET1 和 NbHDA6 之间的关系。我们的研究表明，V2 不相互作用，但与 NbMET1 竞争直接结合 NbHDA6。据我们所知，这是第一个报道病毒蛋白通过与组蛋白去乙酰化酶相互作用而不是通过阻断甲基循环来抑制 TGS 的报告。这项工作为双生病毒抑制 TGS 提供了另一种机制。
