Arabidopsis Double-Stranded RNA Binding Protein DRB3 Participates in Methylation-Mediated Defense against Geminiviruses
Arabidopsis Double-Stranded RNA Binding Protein DRB3 Participates in Methylation-Mediated Defense against Geminiviruses
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DOI：
10.1128/jvi.02305-13
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发表时间：
2014-03-01
影响因子：
5.4
通讯作者：
Bisaro, David M.
中科院分区：
文献类型：
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作者：
Raja, Priya;Jackel, Jamie N.;Bisaro, David M.
拟南芥编码5个双链RNA结合蛋白。DRB 1和DRB 2参与microRNA（miRNA）的生物合成，而DRB 4在细胞质转录后小干扰RNA（siRNA）途径中起作用。DRB 3和DRB 5不参与双链RNA（dsRNA）加工，但有助于沉默DRB 2相关miRNA靶向的转录物。这项研究的目的是确定哪些DRB蛋白（如果有的话）也可能参与导致双生病毒基因组甲基化的核siRNA途径。在这里，我们证明了DRB 3与Dicer样3（DCL 3）和Argonaute 4（AGO 4）在甲基化介导的抗病毒防御中起作用。植物采用抑制性病毒基因组甲基化作为对双生病毒的表观遗传防御，使用RNA指导的DNA甲基化（RdDM）途径，其类似于用于抑制内源性侵入DNA如转座子的途径。染色质甲基化抑制病毒的复制和转录，甲基化缺陷的宿主植物对双生病毒的感染非常敏感。使用一组drb突变体，我们发现drb3植物独特地表现出类似的超敏反应，并且与野生型植物相比，drb3中的病毒基因组甲基化显著降低。此外，像dcl3和ago4突变体一样，drb3植物不能从感染中恢复，也不能完成病毒基因组的超甲基化，而这在无症状的恢复组织中总是可以观察到。小RNA分析，双分子荧光互补和免疫共沉淀实验表明，DRB 3的siRNA生物合成的下游行为，并建议它与DCL 3和AGO 4在不同的亚核区室。这些研究表明，除了其先前建立的作用在miRNA途径，DRB 3也在抗病毒RdDM的功能。重要的是，植物使用RNA指导的DNA甲基化（RdDM）作为对双生病毒的表观遗传防御。拟南芥中的RNA沉默途径包括与果蝇R2D2和哺乳动物TRBP和PACT相关的五种双链RNA结合蛋白（DRB）。虽然DRB蛋白在miRNA和细胞质siRNA途径中具有明确的作用，但在核RdDM中的作用是难以捉摸的。在这里，我们使用双生病毒系统来证明DRB 3参与甲基化介导的抗病毒防御。从一组拟南芥DRB突变体开始，我们证明了DRB 3植物独特地表现出对双生病毒的易感性增强。此外，像dcl3和ago4突变体一样，drb3植物不能使病毒基因组高度甲基化，这是宿主恢复的一个必要条件。我们还表明，DRB 3物理相互作用的RdDM途径组件DCL 3和AGO 4在细胞核中。这项工作突出了双生病毒作为从头RdDM模型的实用性，并将DRB 3蛋白置于这一基本的表观遗传途径中。
Arabidopsis encodes five double-stranded RNA binding (DRB) proteins. DRB1 and DRB2 are involved in microRNA (miRNA) biogenesis, while DRB4 functions in cytoplasmic posttranscriptional small interfering RNA (siRNA) pathways. DRB3 and DRB5 are not involved in double-stranded RNA (dsRNA) processing but assist in silencing transcripts targeted by DRB2-associated miRNAs. The goal of this study was to determine which, if any, of the DRB proteins might also participate in a nuclear siRNA pathway that leads to geminivirus genome methylation. Here, we demonstrate that DRB3 functions with Dicer-like 3 (DCL3) and Argonaute 4 (AGO4) in methylation-mediated antiviral defense. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses, using an RNA-directed DNA methylation (RdDM) pathway similar to that used to suppress endogenous invasive DNAs such as transposons. Chromatin methylation inhibits virus replication and transcription, and methylation-deficient host plants are hypersusceptible to geminivirus infection. Using a panel of drb mutants, we found that drb3 plants uniquely exhibit a similar hypersensitivity and that viral genome methylation is substantially reduced in drb3 compared to wild-type plants. In addition, like dcl3 and ago4 mutants, drb3 plants fail to recover from infection and cannot accomplish the viral genome hypermethylation that is invariably observed in asymptomatic, recovered tissues. Small RNA analysis, bimolecular fluorescence complementation, and coimmunoprecipitation experiments show that DRB3 acts downstream of siRNA biogenesis and suggest that it associates with DCL3 and AGO4 in distinct subnuclear compartments. These studies reveal that in addition to its previously established role in the miRNA pathway, DRB3 also functions in antiviral RdDM.IMPORTANCEPlants use RNA-directed DNA methylation (RdDM) as an epigenetic defense against geminiviruses. RNA silencing pathways in Arabidopsis include five double-stranded RNA binding proteins (DRBs) related to Drosophila R2D2 and mammalian TRBP and PACT. While DRB proteins have defined roles in miRNA and cytoplasmic siRNA pathways, a role in nuclear RdDM was elusive. Here, we used the geminivirus system to show that DRB3 is involved in methylation-mediated antiviral defense. Beginning with a panel of Arabidopsis drb mutants, we demonstrated that drb3 plants uniquely show enhanced susceptibility to geminiviruses. Further, like dcl3 and ago4 mutants, drb3 plants fail to hypermethylate the viral genome, a requirement for host recovery. We also show that DRB3 physically interacts with the RdDM pathway components DCL3 and AGO4 in the nucleus. This work highlights the utility of geminiviruses as models for de novo RdDM and places DRB3 protein in this fundamental epigenetic pathway.