Soybean mosaic virus (SMV) causes severe yield losses and seed quality reduction in soybean (Glycine max) production worldwide. Rsc4 from cultivar Dabaima is a dominant genetic locus for SMV resistance, and its mapping interval contains three nucleotide-binding domain leucine-rich repeat-containing (NLR) candidates (Rsc4-1, Rsc4-2, and Rsc4-3). The NLR-type resistant proteins were considered as important intracellular pathogen sensors in the previous studies. In this study, based on transient expression assay in Nicotiana benthamiana leaves, we found that the longest transcript of Rsc4-3 is sufficient to confer resistance to SMV, and CRISPR/Cas9-mediated editing of Rsc4-3 in resistant cultivar Dabaima compromised the resistance. Interestingly, Rsc4-3 encodes a cell-wall-localized NLR-type resistant protein. We found that the internal polypeptide region responsible for apoplastic targeting of Rsc4-3 and the putative palmitoylation sites on the N terminus are essential for the resistance. Furthermore, we showed that viral-encoded cylindrical inclusion (CI) protein partially localizes to the cell wall and can interact with Rsc4-3. Virus-driven or transient expression of CI protein of avirulent SMV strains is enough to induce resistance response in the presence of Rsc4-3, suggesting that CI is the avirulent gene for Rsc4-3-mediated resistance. Taken together, our work identified a unique NLR that recognizes plant virus in the apoplast, and provided a simple and effective method for identifying resistant genes against SMV infection.

大豆花叶病毒(SMV) 导致全球大豆 ( Glycine max ) 生产严重减产和种子质量下降。来自栽培品种大白马的Rsc4是 SMV 抗性的显性基因位点，其定位区间包含三个核苷酸结合域富含亮氨酸重复 (NLR) 候选物（Rsc4-1、Rsc4-2和Rsc4-3）。在先前的研究中，NLR 型抗性蛋白被认为是重要的细胞内病原体传感器。在本研究中，基于本氏烟草叶片中的瞬时表达测定，我们发现Rsc4-3的最长转录本足以赋予对 SMV 的抗性，并且 CRISPR/Cas9 介导的抗性品种大白马中Rsc4-3的编辑损害了抗性。有趣的是，Rsc4-3编码一种定位于细胞壁的 NLR 型抗性蛋白。我们发现负责 Rsc4-3 的质外体靶向的内部多肽区域和 N 末端推定的棕榈酰化位点对于抗性至关重要。此外，我们发现病毒编码的圆柱形包涵体 (CI) 蛋白部分定位于细胞壁，并且可以与 Rsc4-3 相互作用。无毒 SMV 毒株 CI 蛋白的病毒驱动或瞬时表达足以在 Rsc4-3 存在下诱导抗性反应，表明 CI 是Rsc4-3的无毒基因介导的抗性。总之，我们的工作确定了一种独特的 NLR，它可以识别质外体中的植物病毒，并为识别 SMV 感染的抗性基因提供了一种简单有效的方法。