A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana.
A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana.
复制标题
本塞姆氏烟草中钙调蛋白相关蛋白通过自噬途径降解 SGS3 来抑制 RNA 沉默并促进双生病毒感染
DOI：
10.1371/journal.ppat.1006213
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发表时间：
2017-02
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;Zhao N;Li Z;Xu X;Wang Y;Yang X;Liu SS;Wang A;Zhou X
一种最近被鉴定出的类钙调蛋白是一种内源性RNA沉默抑制因子，它能抑制正义RNA诱导的转录后基因沉默（S - PTGS）并增强病毒感染，但类钙调蛋白介导的S - PTGS抑制机制尚不清楚。在此，我们发现本氏烟草中的一种类钙调蛋白（NbCaM）与基因沉默抑制因子3（NbSGS3）相互作用。缺失分析表明，NbSGS3与NbCaM相互作用所必需的结构域，对于NbSGS3和NbCaM的亚细胞定位以及NbCaM的抑制活性也是必需的。NbCaM的过表达通过降低细胞质中NbSGS3蛋白的积累，减少了与NbSGS3相关的颗粒数量。这种由NbCaM介导的NbSGS3降解对自噬抑制剂3 - 甲基腺嘌呤和E64d敏感，并且当磷脂酰肌醇3 - 激酶（PI3K）复合体的关键自噬基因被敲低时，该降解过程会受到影响。同时，PI3K复合体内关键自噬基因的沉默抑制了双生病毒的感染。综合这些数据表明，NbCaM通过自噬途径降解NbSGS3，从而发挥RNA沉默抑制因子的作用。
转录后基因沉默（PTGS）是植物抵御病毒感染的一个精细调控过程。为了实现有效感染，与双生病毒相关的β卫星分子诱导产生高水平的内源性RNA沉默抑制因子——类钙调蛋白（CaM），以对抗宿主防御。然而，尽管CaM是最早被鉴定出的细胞RNA沉默抑制因子之一，但其对PTGS的抑制机制仍知之甚少。本研究表明，CaM与本氏烟草中的基因沉默抑制因子3（SGS3）相互作用并使其降解。我们发现，NbSGS3与NbCaM相互作用所必需的结构域，对于NbSGS3的亚细胞定位以及NbCaM的抑制活性也是必需的。此外，使用自噬抑制剂3 - 甲基腺嘌呤和E64d，以及敲低磷脂酰肌醇3 - 激酶（PI3K）复合体内关键的自噬相关基因，均可阻断NbCaM介导的NbSGS3蛋白降解。PI3K复合体的沉默也抑制了双生病毒的感染，这与自噬在RNA沉默抑制途径和双生病毒感染中发挥重要作用是一致的。
A recently characterized calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that a calmodulin-like protein from Nicotiana benthamiana (NbCaM) interacts with Suppressor of Gene Silencing 3 (NbSGS3). Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3-associated granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitors 3-methyladenine and E64d, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of key autophagy genes within the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway. Post-transcriptional gene silencing (PTGS) is an elaborately regulated process for defense against virus infection in plants. To achieve effective infection, a betasatellite molecule associated with geminivirus induced high levels of an endogenous RNA silencing suppressor, calmodulin-like protein (CaM), to counter host defenses. However, although CaM is one of the first identified cellular suppressors of RNA silencing, the mechanism of PTGS suppression is still poorly understood. This study demonstrates that CaM interacts with and degrades Suppressor of Gene Silencing 3 (SGS3) in Nicotiana benthamiana. We found that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and for NbCaM suppressor activity. Moreover, NbCaM mediated NbSGS3 protein degradation can be blocked using the autophagy inhibitors 3-methyladenine and E64d, and by knock-down of key autophagy-related genes within the phosphatidylinositol 3-kinase (PI3K) complex. Silencing of the PI3K complex also inhibited geminivirus infection, which is consistent with autophagy playing an important role in RNA silencing suppression pathway and geminivirus infection.