• CaWRKY40 in pepper (Capsicum annuum) promotes immune responses to Ralstonia solanacearum infection (RSI) and to high-temperature, high-humidity (HTHH) stress, but how it interacts with upstream signalling components remains poorly understood.
• Here, using approaches of reverse genetics, biochemical and molecular biology we functionally characterised the relationships among the WRKYGMK-containing WRKY protein CaWRKY27b, the calcium-dependent protein kinase CaCDPK29, and CaWRKY40 during pepper response to RSI or HTHH.
• Our data indicate that CaWRKY27b is upregulated and translocated from the cytoplasm to the nucleus upon phosphorylation of Ser137 in the nuclear localisation signal by CaCDPK29. Using electrophoretic mobility shift assays and microscale thermophoresis, we observed that, due to the replacement of Q by M in the conserved WRKYGQK, CaWRKY27b in the nucleus failed to bind to W-boxes in the promoters of immunity- and thermotolerance-related marker genes. Instead, CaWRKY27b interacted with CaWRKY40 and promoted its binding and positive regulation of the tested marker genes including CaNPR1, CaDEF1 and CaHSP24. Notably, mutation of the WRKYGMK motif in CaWRKY27b to WRKYGQK restored the W-box binding ability.
• Our data therefore suggest that CaWRKY27b is phosphorylated by CaCDPK29 and acts as a transcriptional activator of CaWRKY40 during the pepper response to RSI and HTHH.

中文翻译：

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CaCDPK29–CaWRKY27b 模块促进 CaWRKY40 介导的辣椒对青枯菌的耐热性和免疫力

• 辣椒 ( Capsicum annuum ) 中的 CaWRKY40 促进对青枯菌感染 (RSI) 和高温高湿 (HTHH) 应激的免疫反应，但它如何与上游信号成分相互作用仍知之甚少。
• 在这里，使用反向遗传学、生化和分子生物学的方法，我们在功能上描述了在辣椒对 RSI 或 HTHH 的反应过程中，含有 WRKYGMK 的 WRKY 蛋白 CaWRKY27b、钙依赖性蛋白激酶 CaCDPK29 和 CaWRKY40 之间的关系。
• 我们的数据表明，CaCDPK29 在核定位信号中的 Ser137 磷酸化后，CaWRKY27b 被上调并从细胞质转移到细胞核。使用电泳迁移率变动分析和微尺度热泳，我们观察到，由于保守的 WRKYGQK 中的 Q 被 M 取代，细胞核中的 CaWRKY27b 未能与免疫和耐热相关标记基因的启动子中的 W-box 结合。相反，CaWRKY27b 与 CaWRKY40 相互作用并促进其对包括CaNPR1、CaDEF1和CaHSP24在内的测试标记基因的结合和正调控。值得注意的是，CaWRKY27b 中的 WRKYGMK 基序突变为 WRKYGQK 恢复了 W-box 结合能力。
• 因此，我们的数据表明 CaWRKY27b 被 CaCDPK29 磷酸化，并在辣椒对 RSI 和 HTHH 的反应过程中充当 CaWRKY40 的转录激活剂。