VvWRKY5 enhances white rot resistance in grape by promoting the jasmonic acid pathway

Abstract Grape white rot is a disease caused by Coniella diplodiella (Speg.) Sacc. (Cd) can drastically reduce the production and quality of grape (Vitis vinifera). WRKY transcription factors play a vital role in the regulation of plant resistance to pathogens, but their functions in grape white rot need to be further explored. Here, we found that the expression of the WRKY IIe subfamily member VvWRKY5 was highly induced by Cd infection and jasmonic acid (JA) treatment. Transient injection and stable overexpression (in grape calli and Arabidopsis) demonstrated that VvWRKY5 positively regulated grape resistance to white rot. We also determined that VvWRKY5 regulated the JA response by directly binding to the promoters of VvJAZ2 (a JA signaling suppressor) and VvMYC2 (a JA signaling activator), thereby inhibiting and activating the transcription of VvJAZ2 and VvMYC2, respectively. Furthermore, the interaction between VvJAZ2 and VvWRKY5 enhanced the suppression and promotion of VvJAZ2 and VvMYC2 activities by VvWRKY5, respectively. When VvWRKY5 was overexpressed in grape, JA content was also increased. Overall, our results suggested that VvWRKY5 played a key role in regulating JA biosynthesis and signal transduction as well as enhancing white rot resistance in grape. Our results also provide theoretical guidance for the development of elite grape cultivars with enhanced pathogen resistance.


Introduction
Grapes (Vitis vinifera) are a popular fruit worldwide; however, they are extremely vulnerable to many diseases, which can result in significant crop losses in viticulture.Grape white rot caused by Coniella diplodiella (Speg.)Sacc.(Cd) is one of the most serious fungal diseases affecting grape [1].White rot can infect leaves, berries, and new shoots, among other tissues.The most common entry sites for white-rot pathogens are wounds caused by weather-related incidents, insects, or other fungal illnesses [1].In many areas aff licted by this disease, grape yields have decreased by at least 16.3% [2].Fungicides are widely used in viticulture; however, they can have negative effects on food safety, cause environmental pollution, and increase production costs [3].Therefore, characterizing the genes responsible for grape defense against white rot can offer useful information for breeding resistant varieties.
When plant cells have low concentrations of JA, a group of JAZ (JASMONATEZIM DOMAIN) proteins can combine with the corepressor TOPLESS to form inhibitory complexes that prevent MYC2, a basic helix-loop-helix (bHLH) protein, from triggering the expression of JA-responsive genes [15].Additionally, when plants are stimulated by stress, JA accumulates in plant cells, relieving JAZ protein inhibition of MYC2 and activating the expression of JA-responsive genes [16].
WRKY TFs are essential for plant resistance to various diseases.For instance, WRKY transcription factors regulate resistance to diseases, such as witches' broom disease in Chinese jujube, canker disease in citrus, Glomerella leaf spot in apple, downy mildew in grape, and Ralstonia solanacearum infection in tobacco [17][18][19][20][21].To date, 70 WRKY proteins have been identified in Arabidopsis [22], as well as more than 100 in rice [23].WRKY domains and zincfinger motifs are the two most noticeable structural features of WRKY proteins [24].Based on the number of WRKY domains and the pattern of the zinc-finger motif, WRKY proteins can be categorized into three primary groups (I-III).Furthermore, Group II is then classified into five subgroups (IIa-IIe) [25].In addition to the WRKY domains and zinc finger motifs shared by these TFs, some WRKY members have proline-rich regions, glutaminerich regions, leucine-zipper domains, kinase domains, or nuclear localization signals [26].This distinctive structural arrangement allows the WRKY TFs to perform various regulatory functions.
In Arabidopsis, the WRKY IIa member WRKY18 enhances developmentally regulated defense responses [27].Group III members WRKY53, WRKY38, and WRKY62 [28][29][30] have all been found to display more severe sensitivity to Pst (Pseudomonas syringae pv.tomato) DC3000; however, WRKY41 showed increased resistance [31].Overexpression of the group III member AtWRKY70 increases resistance to necrotrophic infections by inhibiting JA signaling and activating SA signaling [32,33].Functional studies have previously revealed that the WRKY III members OsWRKY13 [34], OsWRKY53 [35], OsWRKY89 [36], OsWRKY31 [37], and OsWRKY45 [38] play positive regulatory roles in rice blast resistance.Interestingly, overexpression of the group IIa member OsWRKY62 reduced resistance to Xanthomonas oryzae pv.oryzae [39].In grapevine, many WRKY TFs have been shown to be closely associated with plant disease resistance.For example, overexpression of VvWRKY1 (WRKY IIc) increases the resistance of transgenic lines to gray mold and downy mildew [40].VqWRKY31 (WRKY IIb) enhances grape resistance to powdery mildew by promoting SA signaling and related metabolite synthesis [41].VqWRKY53 (WRKY IIc) can promote stilbene biosynthesis and increase resistance to Pst DC3000 [42].Knockout of VvWRKY52 (WRKY III) can enhance the resistance of transgenic grape to gray mold [43].Overexpression of VqWRKY56 (WRKY IIb) in grapevine increased PA content and reduced susceptibility to powdery mildew [44].Although the functions of several WRKY IIa-IIc and WRKY III members in plant defense have been previously studied, the roles of other subgroup members remain unclear.
Herein, we demonstrate that the WRKY IIe subfamily member VvWRKY5 positively regulates grape white rot resistance by regulating the JA pathway.VvWRKY5 represses the expression of VvJAZ2 but activates the expression of VvMYC2 by directly binding to their promoters.Furthermore, the interaction between VvJAZ2 and VvWRKY5 enhances the suppression and promotion of VvJAZ2 and VvMYC2 activities by VvWRKY5, respectively.When VvWRKY5 was overexpressed in grape, the content of JA was also increased.Our findings provide a strong theoretical background for future research aimed at developing elite grape cultivars with enhanced pathogen resistance.

Cd resistance in grape berries
Exogenous application of MeJA (a stable derivative of JA) and SA can both activate the defense mechanisms of certain plant against pathogenic organisms [45,46].We cultured Cd mycelia in the same growth period and state on PDA plates containing different concentrations of MeJA and SA.Both MeJA (Fig. 1a and b) and SA (Supplementary Data Fig.S1) treatments were found to have inhibited Cd growth.However, compared with SA, MeJA had a stronger bacteriostatic effect at the same concentration (Fig. 1a and b; Supplementary Data Fig.S1).Furthermore, the higher the concentration of MeJA, the stronger its inhibitory effect on Cd megaspore formation (Fig. 1a and b).These findings demonstrated that MeJA treatment inhibited the growth of Cd more effectively.
Because 0.1 mM of MeJA appears to be a relatively low concentration but effectively inhibited the growth of Cd (Fig. 1a and b), we tested the effect of exogenous 0.1 mM MeJA treatment on Cd resistance in grape.After treating the 'Red Globe' grape fruits with or without 0.1 mM MeJA, we then inoculated them with Cd.We subsequently found that exogenous MeJA treatment effectively slowed Cd diffusion in grape fruits (Fig. 1c and d).Specifically, at 3 days post infection (dpi), non-MeJA-treated grape fruits began to rot and die from the top down, which was accompanied by the spread of white mycelia, whereas MeJA-treated fruits showed no evident necrosis or decomposition (Fig. 1c).Meanwhile, the fungal plaque area in MeJA-treated grape fruits was found to be ∼80% smaller than that of untreated fruits (Fig. 1d).These results indicated that MeJA treatment enhanced Cd resistance in grape.

VvWRKY5 was highly induced by Cd and MeJA
WRKY TFs play a key role in plant disease resistance [47,48].To identify the WRKY genes linked to grape white rot, the expression of 59 VvWRKY genes [49] after Cd infection in grape fruits was examined.RT-qPCR results showed that VvWRKY5 was highly induced after Cd inoculation (Supplementary Data Fig.S2).We also monitored the expression of VvWRKY5 in 'Red Globe' fruits during Cd infection.At 1 dpi, the expression of VvWRKY5 was induced almost 12-fold (Fig. 2a).Furthermore, we found that after 0.5 h of MeJA treatment VvWRKY5 was induced ∼17-fold in 'Red Globe' (Fig. 2b).These results indicated that VvWRKY5 was strongly induced by Cd and MeJA treatments in grape.
Phylogenetic analysis revealed that VvWRKY5 belongs to clade IIe of the WRKY TF family and was most closely related to AtWRKY22 and AtWRKY29 in Arabidopsis (Fig. 2c).AtWRKY22 and AtWRKY29 are required for resistance to fungi and bacteria [50,51].Amino acid homology analysis showed that VvWRKY5 contains a WRKY domain and a C2H2 zinc-finger motif (Fig. 2d).Three exons and two introns constitute the genomic sequence of VvWRKY5, similar to the exon-intron structure of AtWRKY22 and AtWRKY29 (Fig. 2e).Transient expression of the VvWRKY5-GFP protein in onion epidermal cells revealed that VvWRKY5 was localized within the nucleus (Fig. 2f), indicating the potential role of VvWRKY5 in transcriptional regulation.

VvWRKY5 played a positive role in resistance to Cd
To explore whether VvWRKY5 contributed to Cd resistance in grape, we conducted transient transfection for silencing and overexpressing VvWRKY5 in 'Red Globe' fruit mediated by Agrobacterium infection (Fig. 3a and b).Compared with the control, transiently silencing VvWRKY5 significantly reduced Cd resistance in grape, whereas, in contrast, overexpressing VvWRKY5 enhanced Cd resistance (Fig. 3a and c).Plants can limit, or even kill, pathogens by inducing ROS production [52].Therefore, we also measured the H 2 O 2 content, superoxide dismutase (SOD) activity, and peroxidase (POD) activity near the injection site of grape fruits on the third day after Cd infection.The results showed that VvWRKY5-pRI fruits had higher H 2 O 2 content, SOD activity, and POD activity than those of the control, whilst the VvWRKY5-TRV fruits were lower in these aspects (Fig. 3d-f).These findings indicated that VvWRKY5 improved grape resistance to Cd.
To further confirm these results, we obtained three stable VvWRKY5-overexpressing 'Gamay' grape calli (VvWRKY5-OE#1/2/5) (Fig. 3g-i).We subsequently found that the content of endogenous JA and JA-isoleucine (JA-ILE) in VvWRKY5-OE#2 was higher than that in the wild type (WT) (Fig. 3j and k), indicating that VvWRKY5 may can increase JA content.The severity of Cd infection in transgenic grape calli was assessed by spore infection.At 2 dpi, the fungal plaque areas of VvWRKY5-OE grape calli were smaller than those of the WT (Fig. 3l and m), which was consistent with observations in grape fruits (Fig. 3a and c).Values are means ± standard deviation of three replicates.Tukey's test was used for detecting significant differences using DPS software ( * * P < .01).
Staining with 3,3 -diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) showed that VvWRKY5-OE grape calli accumulated more ROS (Fig. 3n).The H 2 O 2 content, SOD activity, and POD activity were also all higher in VvWRKY5-OE grape calli than in the WT (Fig. 3o-q).Similarly, three VvWRKY5-overexpressing Arabidopsis lines (VvWRKY5-OE#1/2/8) were obtained (Supplementary Data Fig.S4a and b) and their Cd resistance was tested.We found that the overexpression of VvWRKY5 in Arabidopsis considerably boosted Cd resistance (Supplementary Data Fig.S4c-f).Furthermore, by identifying the resistance to Cd of 11 different grape varieties, we found that the expression level of VvWRKY5 in resistant varieties was significantly higher than that in susceptible varieties, indicating that the high Cd resistance of resistant grapevine varieties may be closely related to the high expression of VvWRKY5 after infection (Supplementary Data Fig.S5).Overall, these results indicated that VvWRKY5 positively regulated Cd resistance in grape and Arabidopsis.

VvWRKY5 regulated the transcription of jasmonic acid pathway-related genes
To further explore how VvWRKY5 regulated Cd resistance in grape, RNA-seq was performed on VvWRKY5-OE-Cd (VvWRKY5-OE#2 grape calli after infection with Cd) and WT-Cd (WT grape calli after infection with Cd) at 2 dpi.A total of 277 878 400 clean readings were obtained after low-quality reads were removed.The percentages of Q30 and GC were respectively 92.47-93.35and 44.33-44.99%(Supplementary Data Table S1), demonstrating the high credibility of the transcriptome sequencing data.
We examined the expression of 31 963 genes in VvWRKY5-OE-Cd and WT-Cd plants at 2 dpi using RNA-seq and obtained 1706 differentially expressed genes (DEGs).Compared with WT-Cd, 578 genes were upregulated whilst 1128 genes were downregulated in VvWRKY5-OE-Cd (Fig. 4a and b; Supplementary Data Table S2).Using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, we functionally annotated and categorized these 1706 genes.The significantly enriched GO terms of DEGs in VvWRKY5-OE-Cd vs WT-Cd included 'phenylpropanoid metabolic process', 'jasmonic acid mediated signaling pathway', 'monocarboxylic acid biosynthetic process', and 'regulation of jasmonic acid mediated signaling pathway' (Fig. 4c).KEGG enrichment analysis also showed that these DEGs were enriched in 'metabolic pathways', 'biosynthesis of secondary metabolites', 'plant hormone signal transduction', 'MAPK signaling pathway-plant', and 'plant-pathogen interaction' (Supplementary Data Fig.S6).These findings overall suggested that VvWRKY5 regulated complex biological pathways in grape following Cd infection.Moreover, RT-qPCR showed that after Cd infection the expression levels of VvJAZ6 were upregulated, whereas VvJAZ2, VvJAZ4, and VvJAZ5 were all downregulated in VvWRKY5-OE#2 calli compared with the WT (Fig. 4d), which was consistent with the RNA-seq results.

VvWRKY5 participated in jasmonic acid-mediated disease resistance by binding to the VvJAZ2 and VvMYC2 promoters
The expression levels of VvJAZs were considerably altered in the VvWRKY5 transgenic calli after Cd infection (Fig. 4d).To A tW R KY 1 investigate whether VvWRKY5 enhanced Cd resistance by directly binding to these genes, we selected VvJAZ2, VvJAZ4, VvJAZ5, and VvJAZ6, which were significantly inhibited or induced, as candidate genes.Considering that MYC2 is an important part of the JA signaling pathway [53][54][55], we also selected VvMYC2 as a candidate gene.
Interactions between VvWRKY5 and their promoters were examined using yeast one-hybrid (Y1H) assays.As shown in Fig. 5a, VvWRKY5 could bind to the promoters of VvJAZ2 and VvMYC2, but not VvJAZ4-6.WRKY TFs recognize the W-box (TTGACC) in the promoters of their target genes [56].The promoters of VvJAZ2 (Supplementary Data Fig.S7), VvJAZ4 (Supplementary Data Fig.S8), and VvMYC2 (Supplementary Data Fig.S11) contained three, one, and one W-box elements, respectively.However, no W-box elements were detected in the VvJAZ5 (Supplementary Data Fig.S9) and VvJAZ6 (Supplementary Data Fig.S10) promoters.To determine the VvJAZ2 promoter region interacting with VvWRKY5, we divided the VvJAZ2 promoter sequence into two segments (VvJAZ2pro1, −748 to −1 bp; VvJAZ2pro2, −1992 to −749 bp), with these fragments being used for Y1H experiments.As shown in Supplementary Data Fig.S12, the region that interacted with VvWRKY5 was VvJAZ2pro1, which contained one W-box element.Next, we designed electrophoretic mobility shift assay (EMSA) probes using W-box elements.The EMSAs showed that VvWRKY5 could bind to the W-box motifs of the VvJAZ2 (Fig. 5b) and VvMYC2 (Fig. 5c) promoters, respectively.To determine whether VvWRKY5 binds to the VvJAZ2 and VvMYC2 promoters in vivo, chromatin immunoprecipitation PCR (ChIP-PCR) analysis was conducted.The promoter fragments of VvJAZ2 (Fig. 5d) and VvMYC2 (Fig. 5e) were significantly enriched in VvWRKY5-OE grape calli.These results indicated that VvWRKY5 bound to the VvJAZ2 and VvMYC2 promoters in vitro and in vivo.

Physical interaction between VvWRKY5 and VvJAZ2
Previous research has demonstrated that WRKY40 can interact with JAZ proteins (JAZ1-8) to form protein complexes, thereby    C-terminal sequence (1-201 aa), but not in the N-terminal sequence (202-331 aa) (Fig. 6a).Y2H assays also showed that yeast cells co-transformed with VvWRKY5-N-BD and VvJAZ2-AD could grow in SD/−T/−L/−H/−A medium (Fig. 6b), indicating an interaction between VvWRKY5 and VvJAZ2.In contrast, VvWRKY5 did not interact with VvJAZ1, VvJAZ3, VvJAZ4, VvJAZ5, or VvJAZ6 (Supplementary Data Fig.S13).Furthermore, pull-down assays showed that VvJAZ2-GST was pulled down by VvWRKY5-HIS (Fig. 6c), indicating an interaction between VvWRKY5 and VvJAZ2 in vitro.Subsequent luciferase complementation imaging (LCI) experiments showed that the f luorescence signal generated by the co-expression of VvWRKY5-cLUC and VvJAZ2-nLUC in tobacco leaves was stronger than that generated in the control (Fig. 6d).Additionally, coimmunoprecipitation (Co-IP) assays using VvWRKY5-HA-and VvJAZ2-FLAG-transfected 'Garmay' grape calli were performed in vivo.The results of this showed that VvWRKY5-HA coprecipitated with VvJAZ2-FLAG (Fig. 6e).Overall, these observations indicated that VvWRKY5 interacted with VvJAZ2 both in vitro and in vivo.

Synergistic effects of VvWRKY5 and VvJAZ2 on VvJAZ2 and VvMYC2 expression
Based on the observed interactions between VvWRKY5 and VvJAZ2, we hypothesized that VvJAZ2 affects the transcriptional activity of VvWRKY5.We conducted transient dual-luciferase experiments in Nicotiana benthamiana using a luciferase (LUC) reporter gene driven by VvJAZ2 or VvMYC2 promoters.The LUC results revealed that VvWRKY5 directly inhibited and activated the VvJAZ2 and VvMYC2 promoters, respectively.When VvJAZ2 was coexpressed with VvWRKY5, the inhibitory effect of VvWRKY5 on the VvJAZ2 promoter and the activation effect of VvWRKY5 on the VvMYC2 promoter were significantly enhanced (Fig. 7a and b), indicating that VvJAZ2 enhanced the transcriptional regulatory activity of VvWRKY5.

Discussion
The JAZ-MYC module is a core signaling module involved in the transmission of JA signaling [58].Herein, we demonstrated that VvWRKY5, which belongs to the WRKY IIe subfamily, positively  after exogenous JA treatment (Fig. 2b), indicating that VvWRKY5 responded to JA signaling.In addition, JA and JA-ILE accumulated in VvWRKY5-overexpressing grape calli (Fig. 3j and k).Although we identified the molecular mechanisms of VvWRKY5-mediated JA signal transduction, the mechanism by which VvWRKY5 regulated JA biosynthesis in grape remains worth studying in the future.Notably, unlike the conditions prior to infection, the contents of JA and JA-ILE in VvWRKY5-OE#2 transgenic grape calli after Cd infection were lower than those in the WT calli (Supplementary Data Fig.S3a and b).Previous studies have reported that JA and SA pathways have antagonistic effects on plant defense responses [63].Considering this phenomenon, we examined the SA content in WT and VvWRKY5-OE#2 grape calli after Cd infection.As hypothesized, the SA content in the VvWRKY5-OE#2 grape calli was found to be higher than that in the WT calli after Cd infection (Supplementary Data Fig.S3c).Although JA-mediated defense responses can help plants adapt to various biotic and abiotic stresses, they may also have adverse effects on plant growth if these reactions continue to be excessive and are not terminated in time.Based on these results, we believe that this might be a mechanism of homeostatic regulation in plants.Moreover, our results confirmed that VvWRKY5 plays an essential role in the synergistic crosstalk between JA and SA.Many WRKY TFs have previously been reported to play significant regulatory functions in both SA and JA signaling pathways.For example, CaWRKY27 positively regulates tobacco resistance to R. solanacearum by regulating SA-, JA-, and ethephon-mediated pathways [17].
In addition to positively regulating resistance to Cd, we also found that VvWRKY5 had a positive defense function against the hemibiotrophic pathogen Pst DC3000 (Supplementary Data Fig.S14), indicating that VvWRKY5 may be a broadspectrum resistance gene.Crosstalk between hormonal pathways offers enormous regulatory potential for plants and may also allow them to adjust their defense responses to different invaders [64].VvWRKY5 plays an important role in the synergistic crosstalk between JA and SA, which may result in a synergistic role for VvWRKY5 in plant defense against necrotrophic and hemibiotrophic pathogens.
ROS networks also play an important role in signaling plant disease resistance [52].When plants are infected with a pathogen, ROS are quickly produced, thereby preventing the pathogen from entering cells or inducing resistant genes that inhibit pathogen growth [65].DAB and NBT staining also showed that VvWRKY5-overexpressing grape calli and Arabidopsis accumulated more ROS than the controls after Cd infection (Fig. 3n; Supplementary Data Fig.S4d).Moreover, GO enrichment analysis demonstrated that, after Cd infection, the DEGs in VvWRKY5-OE-Cd vs WT-Cd were related to 'oxidoreductase activity' (Fig. 4c), indicating that VvWRKY5 may have promoted antioxidant enzyme activity by regulating these DEGs.Furthermore, KEGG analysis revealed that some DEGs were related to 'cysteine and methionine metabolism' (Supplementary Data Fig.S6).Thiols of cysteine and methionine residues are vulnerable to nucleophilic attack by ROS.These data overall indicated that VvWRKY5 could also affect the expression of genes associated with the ROS-mediated defense pathway in grape.Interestingly, we found that the SOD and POD activities were also higher in VvWRKY5-overexpressing grape calli than the WT after Cd infection (Fig. 3p and q).Although SOD and POD are generally considered to be ROS-scavenging enzymes, different types of pathogens, different pathogenesis, different sampling time, and other complex factors may cause the relationship between SOD/POD and ROS to be more complex.Plant defense responses are complicated processes involving a variety of physiological, pathological, and molecular mechanisms, with transcriptional regulation being an important part of plant pathogen defense.In this study, we identified the role of VvWRKY5 in regulating the JA pathway to improve the grape defense response against Cd.Since our knowledge of the interaction between JA signaling and Cd primarily comes from studies in Arabidopsis and grape, and different Cd accessions may adopt different infection mechanisms for invading plants, our results provide theoretical guidance for the development of elite grape cultivars with improved pathogen resistance.
Arabidopsis (Col-0) and N. benthamiana plants were grown in an incubator with 16 h light/8 h dark periods at 25 • C.

RT-qPCR and RNA-seq
Total RNA was isolated using the RNA Plant Plus Reagent Kit (Tiangen, Beijing, China), and cDNA was synthesized using a Prime-Script™ RT Reagent Kit (TaKaRa, Dalian, China).Furthermore, a 7500 Real-Time PCR System (Applied Biosystems) was used for RT-qPCR.VvActin (GenBank number XM_002278316.4) was used as the internal control, and the expression levels were calculated using the 2 -Ct method [67].Supplementary Data Table S3 shows the details of all primers used.
WT and VvWRKY5-OE#2 grape calli after infection with Cd at 2 dpi were sampled to extract total RNA.The Illumina HiSeq platform (Metware Biotechnology, Wuhan, China) was used for sequencing.The Vitis genome sequences (https://urgi.versailles.inra.fr/Species/Vitis/Data-Sequences/Genome-sequences) were used to align the RNA-seq reads.

Transformation of grape calli with VvWRKY5
The coding sequence (CDS) of VvWRKY5 was inserted into the pRI101-GFP vector.Grape calli were transformed as previously described by Jia et al. [68].In brief, grape calli were incubated with Agrobacterium tumefaciens carrying recombinant plasmids for 30 min, then co-cultured in the dark in B5 medium without antibiotic at 24 • C for 2 days.The transformed calli with the VvWRKY5-pRI vector were cultured in B5 medium containing 40mg/l paromomycin sulfate and 300-mg/l cefotaxime sodium in the light at 24 • C.

Transient expression of VvWRKY5 in grapes
The VvWRKY5 CDS was inserted into the pRI101 vector for overexpression, whilst a 388-bp fragment of VvWRKY5 was inserted into the pTRV2 vector for silencing.The recombinant plasmids VvWRKY5-pRI and VvWRKY5-pTRV were then integrated into A. tumefaciens GV3101, respectively [69].Then, each transformed A. tumefaciens was injected into 'Red Globe' fruits [70].The injected 'Red Globe' fruits were incubated in the dark for 2 days and then used for pathogen infection experiments.The pericarp near the injection site was used for physiological index determination.Ten injected fruits were used as a biological replicate.Each fruit injection assay was performed using three biological replicates.

Pathogen infection assay
The Coniella diplodiella strain WR01 was grown on PDA medium in a 28 • C incubator for 7 days before use.'Gamay' grape calli and 'Red Globe' fruits were inoculated with a 1 × 10 7 /ml white rot spore suspension.All infected fruits were incubated in an incubator at 28 • C with 95% RH.The infected grape calli and fruit samples were incubated for 2 and 5 days, respectively.

Arabidopsis transformation and disease resistance analysis
Arabidopsis was transformed with A. tumefaciens GV3101 carrying the VvWRKY5-pRI plasmid with T 3 transgenic lines being used for pathogen infection analysis.Four-week-old Arabidopsis leaves were injected with a Pst DC3000 spore suspension as previously described [71].

Figure 1 .
Figure 1.Effect of MeJA treatment on Cd resistance in grapes.a MeJA affects Cd growth.Cd mycelial disks (1 cm diameter) were cultured on PDA medium supplemented with 0, 0.1, 0.5, 1.0, and 5.0 mM MeJA for 7 days.b Statistical analysis of the Cd mycelium growth rate shown in (a).Values are means ± standard deviation of three replicates.Statistical significance is indicated by different lowercase letters (P < .05).c Phenotype of 'Red Globe' fruit (with or without 0.1 mM MeJA treatment) inoculated with Cd for 3 days.d Determination of plaque area of 'Red Globe' fruit after being inoculated with Cd.Values are means ± standard deviation of three replicates.Tukey's test was used for detecting significant differences using DPS software ( * * P < .01).

Figure 2 .
Figure 2. Responses of VvWRKY5 to Cd and MeJA treatments.a Expression of VvWRKY5 in 'Red Globe' fruit after being inoculated with Cd.The density of Cd spore suspension used to inoculate the fruits was 1 × 10 7 /ml.b Expression of VvWRKY5 in 'Red Globe' fruit under 0.1 mM MeJA treatment.Values are means ± standard deviation of three replicates.Tukey's test was used for detecting significant differences using DPS software ( * * P < .01).c Phylogenetic analysis of VvWRKY5 and 75 members of the Arabidopsis WRKY family.Accession numbers are listed in Supplementary Data Table S4.d Multiple sequence alignment of VvWRKY5 with eight IIe subfamily members of Arabidopsis.e Genomic structures of VvWRKY5, AtWRKY22, and AtWRKY29.Boxes represent exons and lines represent introns.f Subcellular localization of VvWRKY5.Onion epidermal cells were transformed with the VvWRKY5-GFP plasmid.Scale bars, 25 μm.

Figure 3 .
Figure 3. VvWRKY5 enhances grape resistance to white rot. a Phenotype of 'Red Globe' fruit (VvWRKY5 transiently silenced or overexpressed) inoculated with Cd for 3 days.The density of Cd spore suspension used to inoculate 'Red Globe' fruits was 1 × 10 7 /ml.b Expression of VvWRKY5 in transiently transformed grape fruits.c Determination of plaque area of 'Red Globe' fruit after inoculation with Cd. d-f H 2 O 2 content (d), SOD activity (e), and POD activity (f) of 'Red Globe" fruit after being inoculated with Cd. g Phenotype of WT and VvWRKY5-overexpressing grape calli (VvWRKY5-OE#1/2/5).h, i Expression levels of DNA (h) and RNA (i) to detect stable VvWRKY5-overexpressing grape calli.j, k Quantification of JA (j) and JA-ILE (k) concentration in the WT and VvWRKY5-OE#2 grape calli.l Phenotype of WT and VvWRKY5-overexpressing grape calli after being infected with Cd for 2 days.The density of Cd spore suspension used to inoculate the grape calli was 1 × 10 7 /ml.m Determination of plaque area of WT and VvWRKY5-overexpressing calli after being inoculated with Cd. n DAB and NBT staining of WT and VvWRKY5-overexpressing calli after being infected with Cd. o-q H 2 O 2 content (o), SOD activity (p), and POD activity (q) of WT and VvWRKY5-overexpressing calli infected with Cd.Values are means ± standard deviation of three replicates.Tukey's test was used for detecting significant differences by DPS software ( * * P < .01).

Figure 4 .
Figure 4. RNA-seq analysis of the role of the VvWRKY5 regulator in Cd response.a Number of DEGs among VvWRKY5-OE-Cd vs WT-Cd at 2 dpi by RNA-seq.VvWRKY5-OE-Cd, VvWRKY5-OE#2 grape calli after infection with Cd; WT-Cd, wild-type grape calli after infection with Cd. b Heat map of DEGs based on RNA-seq data of VvWRKY5-OE-Cd vs WT-Cd at 2 dpi.c GO enrichment analysis of DEGs in VvWRKY5-OE-Cd vs WT-Cd at 2 dpi.d Expression of VvMYC2, VvJAZ2, VvJAZ4, and VvJAZ6 in WT and VvWRKY5-OE#2 grape calli at 0 and 2 dpi with Cd.Values are means ± standard deviation of three replicates.Tukey's test was used for detecting significant differences using DPS software ( * P < .05,* * P < .01).

Figure 5 .Figure 7 .
Figure 5. VvWRKY5 binds to the promoters of VvJAZ2 and VvMYC2.a Y1H assays.The AD empty vector and the VvJAZ2, VvJAZ4, VvJAZ5, VvJAZ6, and VvMYC2 promoters were used as negative controls.b, c EMSA assays showed the binding of VvWRKY5 to the VvJAZ2 (b) and VvMYC2 (c) promoters.Hot probes were biotin-conjugated promoter fragments with specific W-box motifs.Cold probe was an unlabeled competitive probe.Mutant probes were unlabeled fragments containing mutated nucleotides of the W-box motifs of VvJAZ2 and VvMYC2.Cold and mutant probes were added in increasing amounts (100× or 200×).d, e ChIP-PCR assays confirmed the binding of VvWRKY5 to the VvJAZ2 (d) and VvMYC2 (e) promoters.The DNA fragments enriched in each ChIP served as the biological replicate in PCR.

Figure 8 .
Figure 8. Proposed model showing the role of VvWRKY5 in JA-mediated white rot resistance in grape.VvWRKY5 binds to the promoters of VvJAZ2 and VvMYC2, repressing VvJAZ2 transcription and activating VvMYC2 transcription, thereby enhancing the resistance of grape to Cd.In addition, VvJAZ2 interacts with VvWRKY5 and enhances the transcriptional activities of VvWRKY5 to VvJAZ2 and VvMYC2.→, promotion; ⊥, suppression.