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

【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 stressrelated physiological parameters (including O2 - 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 the transcriptional activity of VvNPR1, as the fluorescence signal in the tobacco leaves co-injected with 35S::VvWRKY5 and ProVvNPR1-LUC (35S::VvWRKY5 + ProVvNPR1-LUC) was significantly stronger than the control groups (35S + LUC, 35S::VvWRKY5 + LUC, 35S + ProVvNPR1-LUC).【Conclusion】In conclusion, VvWRKY5 is a typical WRKY transcription factor associated with resistance to white rot in grape, which is significantly upregulated under C. diplodiella induction, and its overexpression positively regulates the resistance of grape to C. diplodiella. Moreover, its nuclear localization and tissue- specific expression (highest in leaves/fruits) align with its role in defending against foliar and fruit pathogens. Additionally, this study reveals a novel mechanism by which VvWRKY5 enhances grape resistance to white rot through activation of VvNPR1 transcription. This expands our understanding of WRKY-mediated immunity in grape and provides a candidate gene for breeding disease-resistant cultivars.