A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana

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.新近鉴定的钙调素类蛋白（calmodulin-like protein）是一种内源RNA沉默抑制因子，能够抑制正义RNA诱导的转录后基因沉默（sense-RNA induced post-transcriptional gene silencing, S-PTGS）并促进病毒侵染，但其介导S-PTGS抑制的具体分子机制仍不明确。本研究揭示，本氏烟草（Nicotiana benthamiana）来源的钙调素类蛋白（NbCaM）可与基因沉默抑制因子3（Suppressor of Gene Silencing 3, NbSGS3）发生相互作用。缺失分析结果表明，NbSGS3与NbCaM相互作用所必需的结构域，同时也是NbSGS3实现亚细胞定位以及NbCaM发挥沉默抑制活性所必需的结构元件。过表达NbCaM可通过降解细胞质中的NbSGS3蛋白、降低其积累量，进而减少NbSGS3相关颗粒的数量。该NbCaM介导的NbSGS3降解过程对自噬抑制剂3-甲基腺嘌呤（3-methyladenine）和E64d敏感，而当磷脂酰肌醇3-激酶（PI3K）复合物的关键自噬基因被敲低后，该降解过程会受到显著抑制。同时，沉默PI3K复合物内的关键自噬基因可抑制双生病毒（geminivirus）的侵染。综上，本研究结果表明，NbCaM可通过自噬通路降解NbSGS3蛋白，进而作为RNA沉默抑制因子发挥功能。