Key Laboratory of Applied Genetics of Universities in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Agricultural College, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Int J Mol Sci. 2022 Feb 28;23(5):2656. doi: 10.3390/ijms23052656.
CabZIP63 and CaWRKY40 were previously found to be shared in the pepper defense response to high temperature stress (HTS) and to inoculation (RSI), forming a transcriptional cascade. However, how they activate the two distinct defense responses is not fully understood. Herein, using a revised genetic approach, we functionally characterized CabZIP23 in the CabZIP63-CaWRKY40 cascade and its context specific pepper immunity activation against RSI by interaction with CabZIP63. CabZIP23 was originally found by immunoprecipitation-mass spectrometry to be an interacting protein of CabZIP63-GFP; it was upregulated by RSI and acted positively in pepper immunity against RSI by virus induced gene silencing in pepper plants, and transient overexpression in plants. By chromatin immunoprecipitation (ChIP)-qPCR and electrophoresis mobility shift assay (EMSA), CabZIP23 was found to be directly regulated by CaWRKY40, and CabZIP63 was directly regulated by CabZIP23, forming a positive feedback loop. CabZIP23-CabZIP63 interaction was confirmed by co-immunoprecipitation (CoIP) and bimolecular fluorescent complimentary (BiFC) assays, which promoted CabZIP63 binding immunity related target genes, including CaPR1, CaNPR1 and CaWRKY40, thereby enhancing pepper immunity against RSI, but not affecting the expression of thermotolerance related CaHSP24. All these data appear to show that CabZIP23 integrates in the CabZIP63-CaWRKY40 cascade and the context specifically turns it on mounting pepper immunity against RSI.
先前发现 CabZIP63 和 CaWRKY40 共同参与了辣椒对高温胁迫（HTS）和接种（RSI）的防御反应，形成了一个转录级联反应。然而，它们如何激活这两种不同的防御反应还不完全清楚。在此，我们使用改良的遗传方法，对 CabZIP63-CaWRKY40 级联反应中的 CabZIP23 进行了功能表征，并通过与 CabZIP63 的相互作用，研究了其在辣椒对 RSI 的特定免疫激活中的作用。CabZIP23 最初是通过免疫沉淀-质谱法发现的 CabZIP63-GFP 的互作蛋白；它受 RSI 上调，并通过病毒诱导的基因沉默在辣椒植株中对 RSI 的辣椒免疫发挥正向作用，在 植物中瞬时过表达也能发挥正向作用。通过染色质免疫沉淀（ChIP）-qPCR 和电泳迁移率变动分析（EMSA），发现 CabZIP23 被 CaWRKY40 直接调控，CabZIP63 被 CabZIP23 直接调控，形成一个正反馈回路。CabZIP23-CabZIP63 相互作用通过共免疫沉淀（CoIP）和双分子荧光互补（BiFC）实验得到了验证，该相互作用促进了 CabZIP63 结合免疫相关靶基因，包括 CaPR1、CaNPR1 和 CaWRKY40，从而增强了辣椒对 RSI 的免疫反应，但不影响热胁迫相关 CaHSP24 的表达。所有这些数据似乎表明，CabZIP23 整合到了 CabZIP63-CaWRKY40 级联反应中，并在特定的情况下开启了针对 RSI 的辣椒免疫。
