Howard Hughes Medical Institute, Duke University, Durham, NC 27708, USA; University Program in Genetics and Genomics, Duke University, Durham, NC 27708, USA.
Howard Hughes Medical Institute, Duke University, Durham, NC 27708, USA.
Mol Plant. 2024 Oct 7;17(10):1558-1572. doi: 10.1016/j.molp.2024.08.008. Epub 2024 Aug 22.
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.
六十多年来，水杨酸（SA）一直被认为是植物免疫信号，是基础和系统获得性抗性所必需的。SA 通过 NPR1 重新编程约 20%的转录组来激活这些免疫反应。然而，由于研究这种转录共因子的困难，NPR1 信号枢纽中的成分（表现为核凝聚物）和 NPR1 信号级联仍然难以捉摸，其染色质关联是间接的，而且可能是短暂的。为了克服这一挑战，我们应用 TurboID 来揭示 NPR1 近蛋白组，该方法检测到几乎所有已知的 NPR1 相互作用物以及转录相关复合物的新成分。对新成分的测试表明，染色质重塑和组蛋白去甲基化有助于 SA 诱导的抗性。总体而言，NPR1 近蛋白组与参与 SA 合成的 GBPL3 的近蛋白组非常相似，除了相关的转录因子（TFs）外，这表明共同的调节模块被招募到通过结合独特的 TFs 来重新编程特定的转录组，就像 NPR1 一样，通过结合独特的 TFs 来重新编程特定的转录组。使用核酸酶进行逐步绿色荧光蛋白标记因子切割下的靶向和释放（greenCUT&RUN）分析表明，在 SA 诱导后，NPR1 主要通过与 TGACG 结合 TF 结合来启动转录级联，以诱导次要 TF 的表达，主要是 WRKYs。此外，鉴定出 WRKY54 和 WRKY70 在不与染色质上的 NPR1 相互作用的情况下在诱导免疫输出基因中发挥主要作用。此外，NPR1 凝聚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 信号枢纽和启动转录级联中的重要性。总的来说，这项研究展示了如何组合应用 TurboID 和逐步 greenCUT&RUN 超越传统的遗传方法，来全面绘制信号枢纽和转录级联，以进行深入探索。
