For over 60 years, salicylic acid (SA) has been known as a plant immune signal required for basal and systemic acquired resistance. SA activates these immune responses by reprogramming ∼20% of the transcriptome through NPR1. However, components in the NPR1 signaling hub, which appears as nuclear condensates, and the NPR1 signaling cascade have remained elusive due to difficulties in studying this transcriptional cofactor, whose chromatin association is indirect and likely transient. To overcome this challenge, we applied TurboID to divulge the NPR1 proxiome, which detected almost all known NPR1 interactors as well as new components of transcription-related complexes. Testing of new components showed that chromatin remodeling and histone demethylation contribute to SA-induced resistance. Globally, the NPR1 proxiome has a striking similarity to the proxiome of GBPL3 that is involved in SA synthesis, except for associated transcription factors (TFs), suggesting that common regulatory modules are recruited to reprogram specific transcriptomes by transcriptional cofactors, like NPR1, through binding to unique TFs. Stepwise green fluorescent protein-tagged factor cleavage under target and release using nuclease (greenCUT&RUN) analyses showed that, upon SA induction, NPR1 initiates the transcriptional cascade primarily through association with TGACG-binding TFs to induce expression of secondary TFs, predominantly WRKYs. Further, WRKY54 and WRKY70 were identified to play a major role in inducing immune-output genes without interacting with NPR1 at the chromatin. Moreover, loss of condensate formation function of NPR1 decreases its chromatin association and transcriptional activity, indicating the importance of condensates in organizing the NPR1 signaling hub and initiating the transcriptional cascade. Collectively, this study demonstrates how combinatorial applications of TurboID and stepwise greenCUT&RUN transcend traditional genetic methods to globally map signaling hubs and transcriptional cascades for in-depth explorations.

中文翻译：

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水杨酸信号转导枢纽和转录级联的下一代定位


60 多年来，水杨酸 （SA） 一直被认为是基础和全身获得性耐药所需的植物免疫信号。SA 通过 NPR1 重编程 ∼20% 的转录组来激活这些免疫反应。然而，由于难以研究这种转录辅因子，NPR1 信号传导中枢（表现为核凝聚物）和 NPR1 信号级联的成分仍然难以捉摸，其染色质结合是间接的，并且可能是短暂的。为了克服这一挑战，我们应用 TurboID 来揭示 NPR1 近邻组，它检测到几乎所有已知的 NPR1 相互作用子以及转录相关复合物的新组分。新成分的测试表明，染色质重塑和组蛋白去甲基化有助于 SA 诱导的耐药性。在全球范围内，NPR1 近源组与参与 SA 合成的 GBPL3 近源组具有惊人的相似性，除了相关的转录因子 （TFs），这表明通过与独特的 TFs 结合，转录辅因子（如 NPR1）募集了共同的调节模块来重编程特定转录组。使用核酸酶 （greenCUT&RUN） 分析在靶点下逐步切割和释放绿色荧光蛋白标记的因子表明， SA 诱导后，NPR1 主要通过与 TGACG 结合 TF 结合来启动转录级联反应，以诱导次级 TF 的表达，主要是 WRKYs。此外，WRKY54 和 WRKY70 被确定在诱导免疫输出基因中起主要作用，而不与染色质上的 NPR1 相互作用。 此外，NPR1 的凝聚物形成功能的丧失降低了其染色质结合和转录活性，表明凝聚物在组织 NPR1 信号转导枢纽和启动转录级联反应中的重要性。总的来说，本研究展示了 TurboID 和逐步 greenCUT&RUN 的组合应用如何超越传统的遗传方法，以全局映射信号传导中心和转录级联进行深入探索。