Nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune receptors that activate innate immune responses upon sensing pathogen attack. However, the molecular mechanisms by which NLR proteins initiate downstream signal transduction pathways to counteract pathogen invasion remain poorly understood. In this study, we identified the wheat (Triticum aestivum) NLR protein Resistance Gene Analogs3 (TaRGA3), which was significantly up-regulated during Puccinia striiformis f. sp. tritici (Pst) infection. TaRGA3 and its coiled-coil (CC) domain, localized to the cytoplasm and nucleus, can induce cell death in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) and overexpression suggested that TaRGA3 contributed to wheat resistance to stripe rust by facilitating reactive oxygen species (ROS) accumulation. Yeast two-hybrid, luciferase complementation imaging, and co-immunoprecipitation assays revealed that TaRGA3 interacted with wheat protein Ascorbate Peroxidase 6 (TaAPX6). Further analysis showed that TaAPX6 specifically targeted the CC domain of TaRGA3. The TaRGA3-TaAPX6 interplay led to reduced enzyme activity of TaAPX6. Notably, TaAPX6 negatively regulated wheat resistance to Pst by removing excessive ROS accompanying Pst-induced hypersensitive responses. Our findings reveal that TaRGA3 responding to Pst infection confers enhanced wheat resistance to stripe rust, possibly by suppressing TaAPX6-modulated ROS scavenging, and demonstrate that TaRGA3 can be used to engineer stripe rust resistance in wheat.

中文翻译：

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小麦 CC-NBS-LRR 蛋白 TaRGA3 通过抑制抗坏血酸过氧化物酶 6 活性赋予对条锈病的抵抗力


核苷酸结合亮氨酸富集重复序列 （NLR） 蛋白是细胞内免疫受体，在感应到病原体攻击时激活先天免疫反应。然而，NLR 蛋白启动下游信号转导途径以对抗病原体入侵的分子机制仍然知之甚少。在这项研究中，我们鉴定了小麦 （Triticum aestivum） NLR 蛋白抗性基因类似物 3 （TaRGA3），该基因在 Puccinia striiformis f. sp. tritici （Pst） 感染期间显著上调。TaRGA3 及其位于细胞质和细胞核的卷曲螺旋 （CC） 结构域可诱导本氏烟草的细胞死亡。病毒诱导的基因沉默 （VIGS） 和过表达表明，TaRGA3 通过促进活性氧 （ROS） 积累促进小麦对条锈病的抗性。酵母双杂交、荧光素酶互补成像和免疫共沉淀测定显示，TaRGA3 与小麦蛋白抗坏血酸过氧化物酶 6 （TaAPX6） 相互作用。进一步分析表明，TaAPX6 特异性靶向 TaRGA3 的 CC 结构域。TaRGA3-TaAPX6 相互作用导致 TaAPX6 的酶活性降低。值得注意的是，TaAPX6 通过去除伴随 Pst 诱导的超敏反应的过量 ROS 来负向调节小麦对 Pst 的抗性。我们的研究结果表明，TaRGA3 响应 Pst 感染，可能通过抑制 TaAPX6 调制的 ROS 清除来增强小麦对条锈病的抗性，并证明 TaRGA3 可用于设计小麦的条锈病抗性。