RNA interference (RNAi) is an across-kingdom gene regulatory and defense mechanism. However, little is known about how organisms sense initial cues to mobilize RNAi. Here, we show that wounding to Nicotiana benthamiana cells during virus intrusion activates RNAi-related gene expression through calcium signaling. A rapid wound-induced elevation in calcium fluxes triggers calmodulin-dependent activation of calmodulin-binding transcription activator-3 (CAMTA3), which activates RNA-dependent RNA polymerase-6 and Bifunctional nuclease-2 (BN2) transcription. BN2 stabilizes mRNAs encoding key components of RNAi machinery, notably AGONAUTE1/2 and DICER-LIKE1, by degrading their cognate microRNAs. Consequently, multiple RNAi genes are primed for combating virus invasion. Calmodulin-, CAMTA3-, or BN2-knockdown/knockout plants show increased susceptibility to geminivirus, cucumovirus, and potyvirus. Notably, Geminivirus V2 protein can disrupt the calmodulin-CAMTA3 interaction to counteract RNAi defense. These findings link Ca²⁺ signaling to RNAi and reveal versatility of host antiviral defense and viral counter-defense.

RNA干扰（RNAi）是一种跨界基因调控和防御机制。然而，人们对生物体如何感知初始线索来动员 RNAi 知之甚少。在这里，我们发现病毒入侵期间对本塞姆氏烟草细胞的伤害会通过钙信号传导激活 RNAi 相关基因的表达。伤口引起的钙通量快速升高会触发钙调蛋白依赖性的钙调蛋白结合转录激活因子 3 (CAMTA3) 激活，从而激活RNA 依赖性 RNA 聚合酶 6和双功能核酸酶 2 ( BN2 ) 转录。 BN2 通过降解同源 microRNA 来稳定编码 RNAi 机制关键组件的 mRNA，尤其是AGONAUTE1 / 2和DICER-LIKE1 。因此，多种 RNAi 基因已准备好对抗病毒入侵。钙调蛋白-、 CAMTA3-或BN2-敲低/敲除植物表现出对双生病毒、黄瓜病毒和马铃薯Y病毒的敏感性增加。值得注意的是，Geminivirus V2 蛋白可以破坏钙调蛋白-CAMTA3 相互作用，从而抵消 RNAi 防御。这些发现将 Ca ²⁺信号传导与 RNAi 联系起来，并揭示了宿主抗病毒防御和病毒反防御的多功能性。