Suppression of RNA silencing by a plant DNA virus satellite requires a host calmodulin-like protein to repress RDR6 expression.
Suppression of RNA silencing by a plant DNA virus satellite requires a host calmodulin-like protein to repress RDR6 expression.
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DOI：
10.1371/journal.ppat.1003921
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发表时间：
2014-02
期刊：
影响因子：
6.7
通讯作者：
Zhou X
中科院分区：
文献类型：
--
作者：
Li F;Huang C;Li Z;Zhou X
在植物中，RNA沉默在抗病毒防御中起着关键作用。为了对抗宿主防御，植物病毒编码RNA沉默的病毒抑制因子（VSRs），这些抑制因子针对RNA沉默途径中的不同效应分子。有证据表明，植物也编码内源性RNA沉默抑制因子（ESRs），这些抑制因子在RNA沉默的适当调节中起作用。这些细胞蛋白可以被病毒破坏以阻止宿主防御的可能性很有趣，但尚未得到充分探索。本研究发现，与双病毒中国番茄黄卷叶病毒（TYLCCNV）相关的DNA卫星编码的唯一蛋白VSR βC1的功能需要烟叶钙调素样蛋白nbrg - cam。βC1上调Nbrgs-CaM的表达。过表达Nbrgs-CaM的转基因植物表现出与β c1相关的形态学改变相似的发育异常。在农杆菌浸润试验中，Nbrgs-CaM抑制RNA沉默，当过表达时，阻断tylccnv诱导的基因沉默。遗传证据表明，Nbrgs-CaM介导βC1的沉默抑制和症状调节功能，是有效感染病毒所必需的。此外，Nbrgs-CaM的烟草和番茄同源物也具有ESR活性，并通过betasatail诱导促进了这些茄科寄主的病毒感染。我们进一步证明，β c1诱导的Nbrgs-CaM可能通过抑制RNA依赖性RNA聚合酶6 （RDR6）的表达来抑制次级sirna的产生。缺乏rdr6的benthamiana植物抗病毒反应缺陷，对TYLCCNV感染过敏。更重要的是，当拟南芥携带RDR6突变时，TYLCCNV可以克服宿主范围限制感染拟南芥。这些发现证明了VSR通过篡夺宿主ESR来抑制PTGS的独特机制，并强调了RDR6在RNA沉默防御双病毒感染反应中的重要作用。在植物中，RNA沉默在发育调控和抗病毒防御中起着关键作用。为了成功感染寄主，植物病毒会编码沉默抑制子（VSRs）作为对抗防御措施。这些VSRs通过各种尚不清楚的机制禁用宿主抗病毒RNA沉默防御。在这里，我们报道了宿主钙调素样蛋白Nbrgs-CaM，它似乎是RNA沉默的内源性抑制因子，在抑制RNA沉默和VSR βC1诱导症状中发挥重要作用，VSR βC1是由双子星病毒相关DNA卫星编码的唯一蛋白。Nbrgs-CaM被中国番茄黄卷叶双病毒（TYLCCNV）编码的VSR βC1感染后表达上调或通过转基因稳定表达。进一步分析表明，βC1上调Nbrgs-CaM可能通过抑制RNA依赖性RNA聚合酶6 （RDR6）的表达来抑制RNA沉默。我们已经证明，rdr6介导的RNA沉默在拟南芥（Nicotiana benthamiana）的抗病毒防御中发挥重要作用，并限制拟南芥（Arabidopsis thaliana）对TYLCCNV感染的宿主范围。我们的研究表明，利用细胞抑制因子可能是病毒对抗宿主RNA沉默防御反应的有效机制。
In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection. In plants, RNA silencing plays a key role in developmental regulation and antiviral defense. To successfully infect their hosts, plant viruses encode silencing suppressors (VSRs) as counter-defense measures. These VSRs function to disable host antiviral RNA silencing defenses through various mechanisms that are not well understood. Here we report that a host calmodulin-like protein called Nbrgs-CaM, which appears to be an endogenous suppressor of RNA silencing, plays essential roles in suppression of RNA silencing and induction of symptoms by the VSR βC1, the sole protein encoded by a geminivirus-associated DNA satellite. The Nbrgs-CaM was up-regulated by Tomato yellow leaf curl China geminivirus (TYLCCNV)-encoded VSR βC1 upon virus infection or stable expression via a transgene. Further analyses revealed that up-regulation of Nbrgs-CaM by βC1 suppressed RNA silencing likely through repressing the expression of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6). We have demonstrated that RDR6-mediated RNA silencing plays an important role in antiviral defense in Nicotiana benthamiana and confers host range restriction against TYLCCNV infection on Arabidopsis thaliana. Our study suggests that exploiting a cellular suppressor can be an efficient mechanism for viruses to counteract host RNA silencing defense response.
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