葡萄VvWRKY5转录因子正调控 VvNPR1提高白腐病抗病性

VvWRKY5 transcription factor positively regulates VvNPR1 to enhance white rot resistance in grape

摘要

【目的】探究WRKY转录因子VvWRKY5在葡萄响应白腐病过程中的功能，并解析其调控抗病性的分子机制，为葡萄抗病分子育种提供理论依据。【方法】以红地球葡萄（ Vitis vinifera‘Red Globe’）为试材，克隆 VvWRKY5（GSVIVT01019419001）并构建系统进化树；以 VvWRKY5瞬时表达的葡萄叶片和稳定转化的葡萄愈伤组织为材料，通过白腐病菌接种试验，探究 VvWRKY5是否调控葡萄对白腐病的抗性；通过qRT-PCR分析其对关键抗病基因表达水平的调控；并利用酵母单杂交和萤光素酶报告试验研究VvWRKY5与 VvNPR1（nonexpressor of pathogenesis-related genes 1）启动子的结合及对其转录活性的影响。【结果】 VvWRKY5的开放阅读框为972 bp，编码323个氨基酸，预测的蛋白质分子质量是35.70 kD，等电点为5.85，含有1个WRKY保守结构域和C2H2锌指结构域。系统进化树分析表明，VvWRKY5与拟南芥AtWRKY22同源性最高。 VvWRKY5在葡萄各组织中均有表达，其中，叶片和果实是最主要的表达部位。亚细胞定位显示VvWRKY5蛋白定位于细胞核中。功能分析表明，过表达 VvWRKY5的葡萄叶片和愈伤组织的发病面积明显小于对照组，受伤害程度较轻，并能促进抗病关键基因 VvNPR1（水杨酸免疫信号转导途径核心基因）和 VvPR1（pathogenesis-related protein 1，病程防御相关蛋白1）的表达。相反，沉默 VvWRKY5的葡萄叶片的发病面积明显大于对照组，病害程度较重， VvNPR1和 VvPR1的表达量低于对照。酵母单杂交和萤光素酶报告试验发现，VvWRKY5能够与 VvNPR1启动子结合并激发其转录活性。【结论】VvWRKY5可能通过提高 VvNPR1的转录活性进而增强葡萄对白腐病的抗性。

Abstract

Abstract:【Objective】Grapevine ( Vitis vinifera L.) is one of the most economically important fruit crops globally, valued for its use in fresh consumption, winemaking, and processed products. However, its productivity and quality are severely threatened by grape white rot, a devastating fungal disease caused by Coniella diplodiella (Speg.) Sacc ( C. diplodiella). This pathogen primarily infects berries, leaves, and shoots, leading to fruit rot rates exceeding 50% in severe outbreaks and significant economic losses. Current management strategies rely heavily on chemical fungicides, which pose risks of environmental pollution, pathogen resistance, and food safety concerns. Thus, identifying host resistance genes and elucidating their molecular mechanisms is critical for developing sustainable disease management strategies through molecular breeding. Plant transcription factors (TFs) play pivotal roles in regulating immune responses by orchestrating the expression of defense-related genes. The WRKY family, characterized by the conserved WRKYGQ motif and zinc finger domains, is well-documented for its involvement in biotic stress responses, but their functions in grape white rot remain poorly understood. Here, we cloned and characterized VvWRKY5 (GSVIVT01019419001), a WRKY TF gene from the grape cultivar Red Globe, to investigate its role in white rot resistance and underlying regulatory mechanisms.【Methods】A phylogenetic tree was constructed using MEGA7.0 with the neighbor-joining method (1000 bootstrap replicates), including VvWRKY5 and WRKY proteins from Arabidopsis, Oryza sativa, and Malus domestica. For subcellular localization, the coding sequence (CDS) of VvWRKY5 was fused to GFP in the pRI101 vector and transiently expressed in onion epidermal cells via Agrobacterium tumefaciens-mediated transformation. Fluorescence was observed using a confocal laser scanning microscope. To identify the potential roles of VvWRKY5 in resistance to white rot, Agrobacterium tumefaciens carrying recombinant plasmids (VvWRKY5-pRI and VvWRKY5-TRV2) were infiltrated into Red Globe leaves and Red Gamay grape callus, respectively, and their resistance was subsequently evaluated following inoculating the C. diplodiella. Subsequently, we observed the phenotypes of VvWRKY5-transgenic and non-transgenic materials after inoculation with the white rot pathogen, measured stress-related physiological parameters (including O ₂ ^- and MDA contents, etc.), and analyzed the expression levels of the key stress response genes VvNPR1 (nonexpressor of pathogenesis-related genes 1, a core gene in the salicylic acid signal transduction pathway) and VvPR1 (encoding pathogenesis-related protein 1) using qRT-PCR technology. A yeast one-hybrid was performed to validate the interaction between VvWRKY5 and VvNPR1 promoter by observing the growth status of the yeast cells cotransformed with VvWRKY5-pGADT7 and ProVvNPR1-pHIS2 on the SD/-Trp/-His/-Leu defined medium supplemented with the 3-amino-1, 2, 4-triazole (3-AT). In addition, the effect of VvWRKY5 on VvNPR1 transcriptional activity was studied using the luciferase reporter assay. The coding region sequence of VvWRKY5 was inserted into the pRI101-AN vector and the promoter fragment of VvNPR1 was introduced into pGreenII 0800-LUC vector. One-month-old tobacco ( N. benthamiana) leaves were infected with Agrobacterium tumefaciens strain GV3101 carrying the recombinant plasmids. After the plants were kept in the dark for 48 h, luciferase activity was detected using the live imaging analysis system.【Results】Sequence analysis indicated that the open reading frame of VvWRKY5 was 972 bp, which encoded 323 amino acids. The predicted molecular weight of the protein was 35.70 kD, the theoretical pI was 5.85, and the protein contained a WRKY conserved domain and a C2H2 zinc finger domain. Phylogenetic analysis showed VvWRKY5 shared 78% amino acid identity with AtWRKY22 from Arabidopsis thaliana, indicating a close evolutionary relationship. qRT-PCR results showed that VvWRKY5 expression was highly induced after C. diplodiella inoculation and was ubiquitously expressed in all tissues, with the highest expression levels in leaves (3.0-fold higher than roots) and fruits (2.7-fold higher than roots). Subcellular localization assays confirmed that VvWRKY5-GFP fluorescence was exclusively localized to the nucleus, consistent with its role as a TF. Functional analysis indicated that the lesion area of grape leaves and callus overexpressing VvWRKY5 was significantly smaller than that of the control, the disease severity was reduced, and it could promote the expression of key disease resistance genes VvNPR1 and VvPR1. On the contrary, the lesion area of grape leaves with VvWRKY5 silenced was significantly larger than the control, the disease severity was significantly higher and the expression levels of VvNPR1 and VvPR1 were lower than the control. Yeast one-hybrid assay demonstrated that VvWRKY5 directly bound to the VvNPR1 promoter containing a W-box motif (-270 bp), as yeast cells co-transformed with bait ( ProVvNPR1-pHIS2) and prey (VvWRKY5-pGADT7) plasmids grew well on selective medium. Furthermore, the luciferase reporter assay revealed that VvWRKY5 could promote

关键词

葡萄 / 白腐病 / WRKY转录因子 / VvNPR1

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基金资助

宁夏自然科学基金项目(2025AAC050041)