Phytophthora blight of pepper, which is caused by the notorious oomycete pathogen Phytophthora capsici, is a serious disease in global pepper production regions. Our previous study had identified two WRKY transcription factors (TFs), CaWRKY01-10 and CaWRKY08-4, which are prominent modulators in the resistant pepper line CM334 against P. capsici infection. However, their functional mechanisms and underlying signaling networks remain unknown. Herein, we determined that CaWRKY01-10 and CaWRKY08-4 are localized in plant nuclei. Transient overexpression assays indicated that both CaWRKY01-10 and CaWRKY08-4 act as positive regulators in pepper resistance to P. capsici. Besides, the stable overexpression of CaWRKY01-10 and CaWRKY08-4 in transgenic Nicotiana benthamiana plants also significantly enhanced the resistance to P. capsici. Using comprehensive approaches including RNA-seq, CUT&RUN-qPCR, and dual-luciferase reporter assays, we revealed that overexpression of CaWRKY01-10 and CaWRKY08-4 can activate the expressions of the same four Capsicum annuum defense-related genes (one PR1, two PR4, and one pathogen-related gene) by directly binding to their promoters. However, we did not observe protein–protein interactions and transcriptional amplification/inhibition effects of their shared target genes when coexpressing these two WRKY TFs. In conclusion, these data suggest that both of the resistant line specific upregulated WRKY TFs (CaWRKY01-10 and CaWRKY08-4) can confer pepper’s resistance to P. capsici infection by directly activating a cluster of defense-related genes and are potentially useful for genetic improvement against Phytophthora blight of pepper and other crops.

中文翻译：

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CaWRKY01-10 和 CaWRKY08-4 通过直接激活防御相关基因簇赋予辣椒对辣椒疫霉感染的抵抗力


辣椒疫病是由臭名昭著的卵菌病原体辣椒疫霉引起的，是全球辣椒产区的严重病害。我们之前的研究已经鉴定出两个 WRKY 转录因子 (TF)：CaWRKY01-10 和 CaWRKY08-4，它们是抵抗辣椒疫病菌感染的抗性辣椒品系 CM334 中的重要调节剂。然而，它们的功能机制和底层信号网络仍然未知。在此，我们确定 CaWRKY01-10 和 CaWRKY08-4 位于植物细胞核中。瞬时过表达测定表明，CaWRKY01-10 和 CaWRKY08-4 在辣椒对辣椒疫病菌的抗性中起着正调节作用。此外，CaWRKY01-10和CaWRKY08-4在转基因本塞姆氏烟草植物中的稳定过表达也显着增强了对辣椒疫霉的抗性。使用包括 RNA-seq、CUT&RUN-qPCR 和双荧光素酶报告基因检测在内的综合方法，我们发现 CaWRKY01-10 和 CaWRKY08-4 的过表达可以激活相同的四个辣椒防御相关基因（一个 PR1、两个）的表达。 PR4 和一种病原体相关基因）通过直接结合到它们的启动子上。然而，当共表达这两个 WRKY TF 时，我们没有观察到蛋白质-蛋白质相互作用以及它们共享靶基因的转录放大/抑制效应。总之，这些数据表明，两种抗性品系特异性上调的 WRKY TF（CaWRKY01-10 和 CaWRKY08-4）都可以通过直接激活一组防御相关基因来赋予辣椒对辣椒疫病菌感染的抗性，并且可能对遗传研究有用。改善辣椒和其他作物的疫病。