Data_Sheet_1_Panax notoginseng WRKY Transcription Factor 9 Is a Positive Regulator in Responding to Root Rot Pathogen Fusarium solani.docx

Panax notoginseng (Burk) F.H. Chen is a rare and valuable Chinese herb, but root rot mainly caused by Fusarium solani severely affects the yield and quality of P. notoginseng herbal materials. In this study, we isolated 30 P. notoginseng WRKY transcription factors (TFs), which were divided into three groups (I, II, and III) on the basis of a phylogenetic analysis. The expression levels of 10 WRKY genes, including PnWRKY9, in P. notoginseng roots increased in response to a methyl jasmonate (MeJA) treatment and the following F. solani infection. Additionally, PnWRKY9 was functionally characterized. The PnWRKY9 protein was localized to the nucleus. The overexpression of PnWRKY9 in tobacco (Nicotiana tabacum) considerably increased the resistance to F. solani, whereas an RNAi-mediated decrease in the PnWRKY9 expression level in P. notoginseng leaves increased the susceptibility to F. solani. The RNA sequencing and hormone content analyses of PnWRKY9-overexpression tobacco revealed that PnWRKY9 and the jasmonic acid (JA) signaling pathway synergistically enhance disease resistance. The PnWRKY9 recombinant protein was observed to bind specifically to the W-box sequence in the promoter of a JA-responsive and F. solani resistance-related defensin gene (PnDEFL1). A yeast one-hybrid assay indicated that PnWRKY9 can activate the transcription of PnDEFL1. Furthermore, a co-expression assay in tobacco using β-glucuronidase (GUS) as a reporter further verified that PnWRKY9 positively regulates PnDEFL1 expression. Overall, in this study, we identified P. notoginseng WRKY TFs and demonstrated that PnWRKY9 positively affects plant defenses against the root rot pathogen. The data presented herein provide researchers with fundamental information regarding the regulatory mechanism mediating the coordinated activities of WRKY TFs and the JA signaling pathway in P. notoginseng responses to the root rot pathogen.

三七（Panax notoginseng (Burk) F.H. Chen）是一种珍稀名贵的中药材，但由茄病镰刀菌（Fusarium solani）为主要致病菌引发的根腐病，严重制约了三七药材的产量与品质。本研究共分离得到30个三七WRKY转录因子（WRKY transcription factors, TFs），通过系统发育分析将其划分为I、II、III共3个组群。对三七根部的10个WRKY基因（包含PnWRKY9）的表达水平检测结果显示，这些基因在经甲基茉莉酸（methyl jasmonate, MeJA）处理以及后续茄病镰刀菌侵染后，表达量均显著上调。

此外，本研究对PnWRKY9开展了功能鉴定：PnWRKY9蛋白定位于细胞核；在烟草（Nicotiana tabacum）中过表达PnWRKY9可显著提升其对茄病镰刀菌的抗性，而在三七叶片中通过RNA干扰（RNA interference, RNAi）降低PnWRKY9的表达水平，则会增强植株对该病原菌的感病性。

对过表达PnWRKY9的烟草进行转录组测序及激素含量分析后发现，PnWRKY9与茉莉酸（jasmonic acid, JA）信号通路协同增强植物的抗病性。实验证实，PnWRKY9重组蛋白可特异性结合茉莉酸响应且与茄病镰刀菌抗性相关的防御素基因PnDEFL1启动子区域的W-box顺式作用元件。酵母单杂交实验表明，PnWRKY9能够激活PnDEFL1的转录；进一步以β-葡萄糖苷酸酶（β-glucuronidase, GUS）为报告基因，在烟草中开展的共表达实验进一步验证了PnWRKY9可正向调控PnDEFL1的表达。

综上，本研究成功鉴定了三七WRKY转录因子家族，并证实PnWRKY9可正向调控植株抵御根腐病病原菌的防卫反应。本研究所得数据可为解析三七WRKY转录因子与茉莉酸信号通路协同调控植株响应根腐病病原菌的分子机制提供基础理论依据。