Nucleotide-binding leucine-rich repeat (NLR) proteins have a pivotal role in plant immunity by recognizing pathogen effectors^1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity^3,4. Unlike most NLRs, plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation⁵. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers, and higher-order oligomers at elevated concentrations. Cryo-electron microscopy (cryo-EM) reveals an inactive conformation of SlNRC2 within these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or inter-dimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana (N.) benthamiana. The cryo-EM structures unexpectedly reveal inositol hexakisphosphate (IP₆) or pentakisphosphate (IP₅) bound to the inner surface of SlNRC2’s C-terminal LRR domain as confirmed by mass spectrometry. Mutations at the IP-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Together, our study unveils a novel negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.

核苷酸结合富含亮氨酸重复序列 (NLR) 蛋白通过识别病原体效应子，在植物免疫中发挥着关键作用^1,2 。维持平衡的免疫反应至关重要，因为过度的 NLR 表达可能会导致意外的自身免疫^3,4 。与大多数 NLR 不同，细胞死亡所需的植物 NLR 2 (NRC2) 属于一个小 NLR 群体，其特征是组成型高表达而无自我激活⁵ 。 NRC2 自动抑制和激活的机制尚不清楚。在这里，我们展示了番茄NRC2 ( Sl NRC2) 在升高的浓度下形成二聚体和四聚体以及更高阶的寡聚体。冷冻电子显微镜（cryo-EM）揭示了这些寡聚物中Sl NRC2的非活性构象。二聚和寡聚不仅稳定非活性状态，而且隔离S1 NRC2以防止组装成活性形式。二聚体或二聚体间界面的突变增强了本塞姆氏烟草中病原体诱导的细胞死亡和免疫。冷冻电镜结构出乎意料地揭示了肌醇六磷酸 (IP ₆ ) 或五磷酸 (IP ₅ ) 与Sl NRC2 的 C 端 LRR 结构域的内表面结合，经质谱分析证实。 IP结合位点的突变会损害本塞姆氏烟草中Sl NRC2的磷酸肌醇结合和病原体诱导的Sl NRC2介导的细胞死亡。总之，我们的研究揭示了 NLR 激活的一种新的负调节机制，并表明肌醇磷酸盐是 NRC 的辅助因子。