Key message

Here, we reported that a pathogen- and herbivore-induced Kunitz trypsin inhibitor gene, NaKTI2, is required for herbivore resistance, and transcriptionally regulated mainly by NaWRKY3 and NaWRKY6 but not Jasmonate signaling.

Abstract

Plant protease inhibitor (PI) occurs widely in plant species, and is considered as an important part of plant defense arsenal against herbivores. Transcriptome analysis of Nicotiana attenuata leaves revealed that a Kunitz trypsin inhibitor gene, NaKTI2, was highly elicited after inoculation of Alternaria alternata (tobacco pathotype). However, the roles of NaKTI2 in pathogen- and herbivore resistance and its regulation were unclear. NaKTI2 had typical domains of Kunitz trypsin inhibitors and exhibited a high level of trypsin protease inhibitor activities when transiently over-expressed. The transcripts of NaKTI2 could be induced by A. alternata and Spodoptera litura oral secretions (OS). Silencing NaKTI2 via virus-induced gene silencing technique has no influence on lesion diameters developed on N. attenuata leaves after A. alternata inoculation, but S. litura larvae gained more mass and had higher survivorship on NaKTI2-silenced plants. Meanwhile, the expression of NaPI, a PI gene essential for herbivore resistance previously identified in N. attenuata, was not affected in NaKTI2-silenced plants. Unlike NaPI, which was predominantly regulated by jasmonate (JA) signaling, OS-elicited NaKTI2 transcripts were only slightly reduced in JA-deficient plants, but were dramatically decreased in NaWRKY3- and NaWRKY6- silenced plants, respectively. Further electromobility shift assays indicated that NaWRKY3 and NaWRKY6 could directly bind to the promoter regions of NaKTI2 in vitro. Taken together, our results demonstrate that in addition to NaPI, NaKTI2, a pathogen- and herbivore-induced Kunitz trypsin inhibitor gene, is also required for herbivore resistance, and mainly regulated by NaWRKY3 and NaWRKY6.

中文翻译：

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NaKTI2，一种由 NaWRKY3 和 NaWRKY6 转录调控的 Kunitz 胰蛋白酶抑制剂，是烟草中草食动物抗性所必需的

关键信息

在这里，我们报道了一种病原体和食草动物诱导的 Kunitz 胰蛋白酶抑制剂基因 NaKTI2，它是食草动物抗性所必需的，并且主要受 NaWRKY3 和 NaWRKY6 的转录调节，而不是 Jasmonate 信号传导。

抽象的

植物蛋白酶抑制剂（PI）广泛存在于植物物种中，被认为是植物防御草食动物的重要组成部分。Nicotiana attenuata叶子的转录组分析显示，在接种交链格孢（烟草致病型）后，高度引发了 Kunitz 胰蛋白酶抑制剂基因NaKTI2 。然而，NaKTI2在病原体和食草动物抗性中的作用及其调节尚不清楚。NaKTI2 具有 Kunitz 胰蛋白酶抑制剂的典型结构域，并且在瞬时过表达时表现出高水平的胰蛋白酶抑制剂活性。NaKTI2的转录本可以由A. alternata和Spodoptera litura诱导口腔分泌物（OS）。通过病毒诱导的基因沉默技术沉默NaKTI2对A. alternata接种后N. attenuata叶片上的病变直径没有影响，但S. litura幼虫在NaKTI2沉默的植物上获得了更多的质量和更高的存活率。同时，先前在N. attenuata中鉴定的草食动物抗性所必需的 PI 基因NaPI的表达在NaKTI2沉默的植物中不受影响。与主要受茉莉酸 (JA) 信号调节的NaPI不同，OS 引发的NaKTI2JA 缺陷植物中的转录本仅略有减少，但在NaWRKY3 - 和NaWRKY6 - 沉默的植物中分别显着减少。进一步的电迁移试验表明，NaWRKY3 和NaWRKY6在体外可以直接与 NaKTI2 的启动子区域结合。总之，我们的研究结果表明，除了NaPI 外，NaKTI2是一种病原体和食草动物诱导的 Kunitz 胰蛋白酶抑制剂基因，也是食草动物抗性所必需的，主要受 NaWRKY3 和 NaWRKY6 调节。