Powers Jordan, Zhang Xing, Reyes Andres V, Zavaliev Raul, Xu Shou-Ling, Dong Xinnian
Howard Hughes Medical Institute, Duke University, Durham, NC 27708, USA.
University Program in Genetics and Genomics, Duke University, Durham, NC 27708, USA.
bioRxiv. 2024 Jan 4:2024.01.03.574047. doi: 10.1101/2024.01.03.574047.
For over 60 years, salicylic acid (SA) has been known as a plant immune signal required for both basal and systemic acquired resistance (SAR). SA activates these immune responses by reprogramming up to 20% of the transcriptome through the function of NPR1. However, components in the NPR1-signaling hub, which appears as nuclear condensates, and the NPR1- signaling cascade remained elusive due to difficulties in studying transcriptional cofactors whose chromatin associations are often indirect and 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, NPR1-proxiome shares a striking similarity to GBPL3-proxiome involved in SA synthesis, except 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 greenCUT&RUN analyses showed that, upon SA-induction, NPR1 initiates the transcriptional cascade primarily through association with TGA TFs to induce expression of secondary TFs, predominantly WRKYs. WRKY54 and WRKY70 then play a major role in inducing immune-output genes without interacting with NPR1 at the chromatin. Moreover, a loss of NPR1 condensate formation decreases its chromatin-association and transcriptional activity, indicating the importance of condensates in organizing the NPR1- signaling hub and initiating the transcriptional cascade. This study demonstrates how combinatorial applications of TurboID and stepwise greenCUT&RUN transcend traditional genetic methods to globally map signaling hubs and transcriptional cascades.
60多年来，水杨酸（SA）一直被认为是基础抗性和系统获得性抗性（SAR）所需的植物免疫信号。SA通过NPR1的功能对高达20%的转录组进行重新编程，从而激活这些免疫反应。然而，由于研究染色质关联通常间接且短暂的转录辅因子存在困难，NPR1信号枢纽中的成分（表现为核凝聚物）以及NPR1信号级联仍不清楚。为了克服这一挑战，我们应用TurboID来揭示NPR1近端蛋白质组，它检测到了几乎所有已知的NPR1相互作用蛋白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近端蛋白质组与参与SA合成的GBPL3近端蛋白质组有显著相似性，除了相关转录因子（TFs），这表明通过与独特的TFs结合，像NPR1这样的转录辅因子会招募共同的调控模块来重新编程特定的转录组。逐步的greenCUT&RUN分析表明，在SA诱导后，NPR1主要通过与TGA TFs结合来启动转录级联反应，以诱导次级TFs（主要是WRKYs）的表达。然后，WRKY54和WRKY70在诱导免疫输出基因方面发挥主要作用，而不在染色质上与NPR1相互作用。此外，NPR1凝聚物形成的丧失会降低其与染色质的结合及转录活性，表明凝聚物在组织NPR1信号枢纽和启动转录级联反应中的重要性。这项研究展示了TurboID和逐步的greenCUT&RUN的组合应用如何超越传统遗传方法，以全局方式绘制信号枢纽和转录级联反应。
