VqMAPKKK38 is essential for stilbene accumulation in grapevine

Vitis species, including grapevine, produce a class of secondary metabolites called stilbenes that are important for plant disease resistance and can have positive effects on human health. Mitogen-activated protein kinase (MAPK) signaling cascades not only play key roles in plant defense responses but also contribute to stilbene biosynthesis in grapevine. MAPKKKs function at the upper level of the MAPK network and initiate signaling through this pathway. In this study, a Raf-like MAPKKK gene, VqMAPKKK38, was identified and functionally characterized from the Chinese wild grapevine V. quinquangularis accession ‘Danfeng-2’. We observed that VqMAPKKK38 transcript levels were elevated by powdery mildew infection, high salinity conditions and chilling stresses, as well as in response to treatments by the hormones salicylic acid (SA), methyl jasmonate (MeJA), ethylene (Eth) and abscisic acid (ABA). In addition, based on both transient overexpression and gene suppression of VqMAPKKK38 in grapevine leaves, we found that VqMAPKKK38 positively regulates stilbene synthase transcription and stilbene accumulation probably by mediating the activation of the transcription factor MYB14. In addition, both hydrogen peroxide (H2O2) and calcium influx activated VqMAPKKK38 expression and stilbene biosynthesis, which suggests that VqMAPKKK38 may be involved in the calcium signaling and ROS signaling pathways.


INTRODUCTION
As sessile organisms, plants are constantly exposed to a wide range of biotic and abiotic stresses and have evolved a large number of sophisticated signal transduction mechanisms to both regulate their development and enhance their resistance to these stressors. For example, the mitogen-activated protein kinase (MAPK) cascade is commonly used by eukaryotes to transduce extracellular stimuli into intracellular responses. 1 The basic components of an MAPK cascade comprise three interconnected kinase modules: MAPKKK/MEKK, MAPKK/MKK and MAPK/MPK. 2 MAPKKK proteins function in the beginning of the cascade, receiving signals from upstream sensors to initiate the pathway, and activate the MAPKK proteins by phosphorylating the serine/ threonine residues in a conserved motif (Ser/Thr-X 3-5 -Ser/Thr, X indicating any amino acid) of the activation loop. 3,4 The activated MAPKK proteins in turn mediate the activation of downstream MAPK proteins through the phosphorylation of threonine and/or tyrosine residues in the T-X-Y motif. 3,4 The phosphorylated MAPK proteins then act as regulators of multiple effector proteins in the nucleus or cytoplasm that can be transcription factors, cytoskeletal components and protein kinases. 3,4 MAPK cascades thus connect upstream signals to downstream targets and participate in the adaptation to a broad range of pathogenic and environmental threats such as bacterial or fungal attack, viral infection, wounding, high salinity, drought, osmotic stress, ultraviolet (UV) irradiation and temperature extremes. 5 Several three-kinase modules have been functionally characterized in plants. Recent studies in Arabidopsis thaliana showed that the MAPKKK17/18-MKK3-MPK1/2/7/14 cascade operates downstream from abscisic acid (ABA)-induced stress signaling 6 and plays an important role in ABA-modulated leaf senescence. 7 The AIK1-MKK5-MPK6 module was shown to be activated by ABA and to regulate ABA responses, including root development and stomatal behavior. 8 Furthermore, two MAPK cascades, MEKK1-MKK1/2-MPK4 and MEKK1-MKK4/5-MPK3/6, have been shown to play a role in flagellin-induced signal transmission, 9 while the CTR1-MKK9-MPK3/6 cascade is involved in ethylene-regulated signaling. 10 In addition, in tobacco, the two modules, MAPKKKα-MEK2-SIPK and NPK1-MEK1/NQK1-NTF6/ NRK1, contribute to pathogen defense and plant cytokinesis, [11][12][13] and tomato (Solanum lycopersicum) MAPKKKα-MKK2/MKK4-MPK2/MPK3 is a component of the Pto-mediated effector triggered immunity (ETI) pathway. 14 Whole-genome sequencing of numerous plant species has generated a valuable resource in the form of an inventory of MAPK families from those species. For instance, there are 20 MAPKs, 10 MAPKKs and at least 80 MAPKKKs in A. thaliana, while the grapevine (Vitis vinifera) genome contains 14 MAPKs, five MAPKKs and 62 MAPKKKs. 10,[15][16][17] The MAPKKKs of a given species show enhanced sequence diversity compared to members of the MAPKK or MAPK families. 18 On the basis of the sequence in the conserved kinase domain, the MAPKKK family in higher plants has been classified into three clades: the MEKK-like subfamily has a conserved signature (G(T/S)Px(W/Y/F)MAPEV); the ZIK subfamily contains a GTPEFMAPE(L/V)Y sequence, and the Raf-like subfamily has a specific GTxx(W/Y)MAPE signature. 18,19 MEKK1 is the most thoroughly studied MAPKKK in A. thaliana and is known to be involved in multiple stress responses, including flagellin signaling, 9 wounding, 20 and cold and salt stimuli. 21 Two well-known Raf-like MAPKKKs, CTR1 (Constitutive Triple Response 1) and EDR1 (Enhanced Disease Resistance 1), function as negative regulators of the ethylene 22 and pathogen defense responses, 23 respectively. In cotton (Gossypium hirsutum), GhRaf19 has been shown to enhance the tolerance to chilling stress but to decrease drought and salt stress resistance, 24 while GhMKK5 is significantly triggered by salicylic acid (SA) and induces the transcription of pathogenesisrelated (PR) genes. 25 Recently, A. thaliana AIK1 and MAPKKK17/18 were identified as key regulators of ABA signal transduction, 7,8 but relatively little information is available regarding MAPKKKs in grapevine. A genome-wide analysis of MAPK cascades in V. vinifera revealed 21 MEKKs, 12 ZIKs and 29 Rafs among the 62 MAPKKKs. 16 Expression profiles of 45 grapevine MAPKKK genes following exposure to various stress conditions suggested that these candidate MAPKKK genes may participate in responses to powdery mildew, drought, SA, ethylene (Eth) and hydrogen peroxide (H 2 O 2 ). 26 However, a more detailed characterization of individual MAPKKKs is required to better define their biological and physiological roles.
China is a major biodiversity center for Vitis, 27 and Chinese wild grapevines provide valuable gene pools with a number of resistance factors that are thought to be important for pathogen immunity. The Chinese wild grapevine species V. quinquangularis, particularly the accession 'Danfeng-2', has attracted attention because of its high level of resistance to pathogen infection and its high content of stilbene-type phytoalexins. [28][29][30] Stilbenes are secondary metabolites that help promote resistance to a diverse range of pathogens, and they also have pharmacological value. [31][32][33] The accumulation of stilbenes can be induced by factors such as pathogen infection, 32 ozone damage, 34 wounding, 35 salt stress 36 and UV irradiation. 34,37 Perhaps the most widely studied stilbene, resveratrol, is synthesized by a side branch of the well-characterized phenylalanine/polymalonate pathway where the final step is catalyzed by stilbene synthase (STS). 38 The R2R3-MYB-type transcription factors MYB14 and MYB15 are thought to regulate the biosynthesis of stilbenes by up-regulating STS transcription. 39 It has also been suggested that MAPK cascades are required for stilbene biosynthesis, and a specific MAPK cascade inhibitor (PD98059) can efficiently suppress the activation of STS by flagellin 22 (flg22), a bacterial elicitor harpin, 40 or by SA. 41 PD98059 can also block the induction of MYB14 by flg22 in grapevine cell suspension cultures. 42 In this study, we sought to identify genes in the MAPK pathway that are involved in the regulation of stilbene accumulation in V. quinquangularis. We focused on VqMAPKKK38, a Raf-like subfamily member that is responsive to various stressors and examined its role in signal transduction and stilbene biosynthesis. We present functional studies involving transient overexpression and suppression using RNAi in grapevine leaves and propose a mechanism by which VqMAPKKK38 promotes stilbene biosynthesis, as well as how its involvement in signaling is triggered by calcium and reactive oxygen species (ROS).

Plant materials and stress treatments
Chinese wild V. quinquangularis accession Danfeng-2 was cultivated in the grape germplasm resources garden at the Northwest A&F University, Yangling, Shaanxi, China. Samples of young leaves (the second to fourth leaf from the tip), mature leaves (dark-green leaves collected when berries were enlarging), stems (the woody stem), inflorescences (with single flowers in dense groups), young berries (berries were enlarging, 25 days after anthesis) and mature berries (berries were harvest-ripe, 80 days after anthesis) were collected for expression analyses.
Fresh young leaves were collected and subjected to different forms of stress. Powdery mildew (Erysiphe necator) inoculation was carried out as described previously, 43 and inoculated leaves were collected at 0, 12, 24, 48, 72, 96 and 120 h post-inoculation. For abiotic stress treatments, young leaves were either wounded with sterile scissors, sprayed with aqueous 250 mM NaCl, or exposed to low (4°C) or high temperature (37°C) for 0, 0.5, 1, 2, 6 or 10 h. Treatment with the signaling molecules was carried out by spraying the young leaves with one of the following solutions: 100 μM Figure 1. Chromosome location (a), sequence alignment analysis (b) and phylogenetic analysis (c) of VqMAPKKK38. The Raf proteins used for the alignment were VviRaf23 (Gene ID: VIT05s0094g01080), AtRaf23 (Gene ID: At2G31800), and BnaRaf23 (GenBank: AHL77715.1). Identical residues are shaded gray. The Raf motif is marked with triangles. The active site (VIHCDLKPKNILL) is boxed, and the kinase catalytic domain is located between the two arrows. The phylogenetic analysis of VqMAPKKK38 and MAPKKK proteins from Vitis vinifera, Arabidopsis thaliana, Brassica napus and Gossypium hirsutum was carried out using MEGA 7 software.
VqMAPKKK38 is essential for stilbene accumulation in grapevines Y Jiao et al.
SA, 100 μM MeJA, 100 μM Eth, 100 μM ABA, 5 mM CaCl 2 or 1% H 2 O 2 (w/v). The leaves were harvested at 0, 0.5, 1, 2, 6 and 10 h post-treatment. The leaves were pretreated with 20 μM GdCl 3 for 30 min before 5 mM CaCl 2 was administered to study the roles of Ca 2+ and ROS. Dimethylthiourea (DMTU), an H 2 O 2 scavenger, was used to pretreat the leaves for 30 min before 1% H 2 O 2 was administered. GdCl 3 or DMTU was also added without a subsequent treatment to assess the effect of the inhibitors on the leaves. Leaves treated with the solvent in which the elicitors were dissolved served as negative controls. Three samples were treated in each treatment, and each treatment was repeated three times.

Expression analysis
Total RNA was extracted from leaves with the EZNA Total RNA kit II (Omega Bio-Tek) and reverse-transcribed into complementary DNA (cDNA) using Prime Script Reverse Transcriptase (TaKaRa) following the manufacturer's instructions. Semi-quantitative reverse transcription-PCR was performed with the following parameters: 94°C for 3 min followed by 30 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 1 min. The amplified products were separated on a 1% agarose gel and visualized with ethidium bromide. Quantitative real-time PCR (qRT-PCR) was carried out as described previously. 44 The specific primers used for gene expression analysis are shown in Supplementary Table S1. Gene transcript levels were quantified with normalization to grapevine GAPDH (GenBank accession no. GR883080) and EF1γ (GenBank accession no. AF176496) as internal standards. Each experiment was carried out with three biological replicates, and each biological sample was analyzed in three technical replicates.
Cloning and VqMAPKKK38 sequence analysis The specific primers used to isolate the full-length VqMAPKKK38 cDNA (see Supplementary Table S1) were designed according to the homologous sequences from the reference genome of V. vinifera cv. 'Pinot Noir' clone P40024. 45 The VqMAPKKK38 gene is located on the fifth chromosome according to a BLAST search in the Genoscope Genome Browser (http:// www.genoscope.cns.fr/blat-server/cgi-bin/vitis/webBlat). DNAMAN software was used to carry out amino-acid sequence alignment analyses of four Raf proteins, including VqMAPKKK38, VviRaf23 (Gene ID: VIT05s0094g01080), AtRaf23 (Gene ID: At2G31800) and BnaRaf23 (Gen-Bank: AHL77715.1). The phylogenetic tree was constructed using MEGA 7 software to analyze the evolutionary relationship between these proteins.

Plasmid construction and transient expression assays in grapevine
To generate the over-expression construct, the amplified product (open reading frame of VqMAPKKK38) was inserted into the pART-CAM-S vector 46 after digestion with SacI and ClaI. To create the silencing construct, a fragment from 545 to 1405 bp of VqMAPKKK38 was isolated as sense and antisense sequences. Both sense and antisense sequences were cloned into pKANNIBAL, 47 and then into pART27 48 after restriction digestion by NotI. Each sequenced plasmid was transformed separately into Agrobacterium tumefaciens strain GV3101 using electroporation and then introduced into 8-week-old V. quinquangularis leaves by Agrobacterium-mediated transient expression as described previously. 44 Stilbene quantification Stilbene levels in the transgenic leaves were measured as described previously 44 with minor modifications. The leaf samples were ground in   (Figures 1b and c).
VqMAPKKK38 expression in different organs and in response to environmental stimuli and hormone treatments qRT-PCR revealed that VqMAPKKK38 transcripts were expressed in all of the organs tested, including young leaves, mature leaves, stems, inflorescences, young berries and mature berries. Since the highest levels were observed in young leaves (Figure 2a), these were used for subsequent expression studies. It has been established that MAPK cascades function in basal immunity 49 and are activated in response to various abiotic and biotic stresses. 5 Therefore, we analyzed the expression profiles of VqMAPKKK38 during powdery mildew infection, as well as following salt, chilling, heat and wounding treatments. After inoculation with grapevine powdery mildew (Uncinula necator), the expression of VqMAPKKK38 remained at basal levels until 72hour post inoculation (hpi), and then increased at 96 and 120 hpi. (Figure 2b). Following both salt and chilling treatments, the abundance of the VqMAPKKK38 transcripts was significantly elevated at 0.5 h, and then remained at high levels throughout the experiment (Figures 2c and d). In contrast, neither the heat or the wounding treatments stimulated VqMAPKKK38 gene  Hormones play central roles in plant growth and stress responses, and a number of hormone signals are known to be active via MAPK cascades in plant cells. 50 We examined VqMAPKKK38 expression in response to phytohormones and found that SA induced expression within 30 min of treatment, and this persisted over the entire 10 h duration of the experiment (Figure 2g). After MeJA application, the VqMAPKKK38 transcript levels were strongly up-regulated after 1 h and peaked at 6 h, followed by a sharp decline at 10 h (Figure 2h). Treatment with Eth or ABA led to an upregulation of VqMAPKKK38 expression at 6 and 2 h, respectively (Figures 2i and j).
VqMAPKKK38 promotes stilbene biosynthesis in grapevine Our previous studies of grapevine suspension culture cells showed that MAPK signaling is necessary for the activation of STS promoters by SA. 41 As shown in Figure 2, VqMAPKKK38 expression was strongly induced by SA, so we investigated whether VqMAPKKK38 plays a role in SA-triggered stilbene biosynthesis. An Agrobacterium-mediated transient expression system was used to overexpress (OE) or suppress via RNA interference (RNAi) VqMAPKKK38 expression in grapevine leaves. pART-CAM-S and pART27 without a target gene served as negative controls. Both semi-quantitative reverse transcription-PCR and qRT-PCR analysis confirmed that VqMAPKKK38 was successfully overexpressed or silenced (Figure 3a). We measured the stilbene levels in the transformed grapevine leaves before and after 100 μM SA treatment by high-performance liquid chromatography (HPLC). Figure 3b, both trans-resveratrol and trans-piceid levels increased after VqMAPKKK38 was overexpressed. In addition, after SA treatment, the VqMAPKKK38 overexpressor accumulated significantly higher levels of these compounds than the control. This suggested that VqMAPKKK38 promotes stilbene accumulation in grapevine. A transient silencing assay further confirmed this hypothesis, since RNAi-VqMAPKKK38 leaves exposed to the SA treatment contained much less trans-resveratrol and trans-piceid than the control leaves. Glucoside cis-piceid was also detected in all overexpressing/silenced leaves but showed the same levels as those observed in the controls.

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To investigate whether the upregulation of stilbene biosynthesis caused by VqMAPKKK38 overexpression was linked to the activation of STS, the expression of several VqSTS genes was determined. qRT-PCR data showed that the expression of the VqSTS genes was significantly induced in OE-VqMAPKKK38 leaves, especially after SA treatment (Figure 4a). In addition, the induction of the VqSTS genes by SA was markedly suppressed in RNAi-VqMAPKKK38 leaves (Figure 4b). These results are consistent with VqMAPKKK38 up-regulating the expression of VqSTSs.
It has been shown that the R2R3-MYB-type transcription factors MYB14 and MYB15 are responsible for the regulation of STS in grapevine 39 and that the activation of MYB14 by flg22 is dependent on MAPK signaling. 42 To investigate whether the accumulation of stilbenes and the activation of STS genes regulated by VqMAPKKK38 correlated with induction of these transcription factors, we examined their transcript levels in the leaves of the OE-and RNAi-VqMAPKKK38 lines. In response to SA treatment, the expression of MYB14 was significantly upregulated in the OE-VqMAPKKK38 leaves and downregulated in the  RNAi-VqMAPKKK38 leaves compared with the control (Figure 5a). In contrast, the induction of MYB15 by SA seemed to be marginal, and we concluded that it was not regulated by VqMAPKKK38 (Figure 5b).
VqMAPKKK38 activation by the calcium ionophore and H 2 O 2 The influx of Ca 2+ , generation of ROS and activation of MAPK cascades are early signaling events associated with immune responses in plants. During unfavorable conditions, these factors are able to trigger and regulate one another, 49,51-54 leading us to hypothesize that VqMAPKKK38 expression could be induced by changing the cytoplasmic Ca 2+ or ROS levels. Young V. quinquangularis leaves were treated with 5 mM CaCl 2 or 1% H 2 O 2 (w/v) and sampled at 0.5, 1, 2, 6 and 10 h post-treatment. qRT-PCR analysis revealed that VqMAPKKK38 expression was significantly induced by exposure to either CaCl 2 or H 2 O 2 (Figures 6a and b).
To further investigate the role of Ca 2+ and H 2 O 2 in VqMAPKKK38 transcription, we tested the effect of GdCl 3 , an inhibitor of mechanosensitive calcium channels, on the induction of VqMAPKKK38 by CaCl 2 , as well as the effect of the H 2 O 2 scavenger dimethylthiourea (DMTU) on the activation of VqMAPKKK38 by H 2 O 2 . Compared to the solvent controls, the accumulation of VqMAPKKK38 transcripts significantly increased after adding either CaCl 2 or H 2 O 2 for 2 h (Figures 6c and d). When the young leaves were pre-treated with 20 μM GdCl 3 for 30 min before the CaCl 2 was administered, the VqMAPKKK38 expression was higher than that in the solvent control, but significantly lower than in the group treated with CaCl 2 alone (Figure 6c). The pretreatment of leaves with 5 mM DMTU for 30 min significantly decreased the H 2 O 2 induction of VqMAPKKK38 in a manner similar to that of the GdCl 3 treatment (Figure 6d). Neither GdCl 3 nor DMTU themselves affected the expression of VqMAPKKK38. These results indicated that VqMAPKKK38 acts downstream of both the calcium and ROS signaling, and so either or both might induce VqMAPKKK38 expression.
Stilbene accumulation can be triggered by calcium and H 2 O 2 Since VqMAPKKK38 expression was induced in response to either calcium influx or H 2 O 2 treatment, we examined the potential correlation between stilbene induction and exogenous calcium and H 2 O 2 . We found that the accumulation of trans-resveratrol, trans-piceid and cis-piceid markedly increased after treatment with either exogenous CaCl 2 or H 2 O 2 . In addition, the accumulation of the three types of stilbenes induced by CaCl 2 was significantly limited by the calcium channel inhibitor GdCl 3 , while the H 2 O 2 scavenger DMTU effectively suppressed the H 2 O 2induced accumulation of trans-resveratrol (Figures 7a and b). These findings suggest that Ca 2+ and ROS signaling are involved in stilbene accumulation. qRT-PCR analysis further confirmed that the expression of the STS genes was induced in response to calcium influx and ROS signaling. We found that the induction of VqSTS6, VqSTS19, VqSTS24 and VqSTS32 by the calcium ionophore was strongly inhibited by GdCl 3 (Supplementary Figure 1a). Moreover, the activation of VqSTS6, VqSTS19, VqSTS26 and VqSTS32 by H 2 O 2 was suppressed when leaves were pretreated with DMTU before the application of H 2 O 2 (Supplementary Figure 1b).

DISCUSSION
The resistance to pathogen attack of Chinese wild Vitis species such as V. quinquangularis accession 'Danfeng-2', is correlated with high concentrations of trans-resveratrol. 28 Our previous study of grapevine cell cultures documented the responsiveness of STS to SA depended on MAPK signaling. 41 However, the specific elements of this response pathway were unknown. Recent transcriptome data from four developmental stages of berry material from V. quinquangularis accession 'Danfeng-2' were analyzed by our colleagues (SRA; SRP067690), and VqMAPKKK38 was predicted to play a role in the regulation of stilbene accumulation (unpublished). In this study, we investigated the involvement of VqMAPKKK38 in stilbene biosynthesis and signal transduction.
In grapevine, the biosynthesis of resveratrol is catalyzed by the key enzyme STS that is specifically activated by MYB14. 39 As previously reported, MAPK signaling can mediate the activation of STS transcription. 40,41 Consistent with this, Duan et al. 42 confirmed that MAPK cascades are essential in the activation of grapevine MYB14. 42 In this study, we show that VqMAPKKK38 overexpression in grapevine leaves can significantly enhance SA-induced stilbene accumulation, accompanied by the strong induction of STS and MYB14 expression. We also observed that the accumulation of stilbenes was almost abolished in RNAi-VqMAPKKK38 transgenic leaves, showing that VqMAPKKK38 is required for stilbene biosynthesis and that a MAPKKK38-based cascade is likely to be involved in this process.
The rapid influx of calcium and the generation of ROS are among the earliest cellular responses to biotic and abiotic stresses. 55,56 The levels of Ca 2+ and ROS (O 2 − , H 2 O 2 , HO· and NO·) are maintained at low levels in plant cells under normal physiological conditions. However, environmental signals can trigger rapid calcium fluxes and increases in the levels of ROS. 57,58 Either of these responses can activate a number of molecular  The regulation of stilbene accumulation by signaling events has been widely studied in grapevine suspension cell lines. A number of pathogen elicitors can induce stilbene accumulation, including flg22 and harpin, as can hormones such as SA and JA. This inducibility requires an influx of calcium, an oxidative burst and MAPK cascades. 40,41,59,60 The signaling events are often shared among different induction processes, but different elicitors can generate different types of stilbene output, mainly due to the relative sequence of calcium influx and an apoplastic burst. 40 Since we found that both calcium and the ROS signaling operate upstream of VqMAPKKK38, we examined the effects of calcium and ROS on STSs transcription and stilbene biosynthesis. The observation that both the expression of STS genes and the accumulation of stilbenes were elevated by Ca 2+ /H 2 O 2 and could also be inhibited by Ca 2+ /H 2 O 2 blockers suggested the involvement of Ca 2+ and ROS signaling in the regulation of stilbene biosynthesis.
It has been shown that exogenous resveratrol can act as a regulator of the hypersensitive reaction accompanied by a stimulation of an oxidative burst in V. rupestris suspension cells. 60 In this study, we found that VqMAPKKK38 is involved in the ROS signaling pathway, raising the possibility that endogenous stilbenes may in turn regulate the upstream acting VqMAPKKK38. We therefore measured the accumulation of VqMAPKKK38 transcripts in VqSTS6-, VqSTS23-, or VqSTS32-overexpressing transgenic grapevine plants both before and after powdery mildew treatment. However, since we observed no difference in the expression of VqMAPKKK38 between transgenic and non-transgenic plants (Supplementary Figure S2), we conclude that there is likely no direct feedback regulation between VqMAPKKK38 and downstream stilbene accumulation in grapevine.
In conclusion, this study provides new insights into the biological roles of a grapevine MAPKKK gene, VqMAPKKK38, that has the same coding sequence as VviMAPKKK38 from V. vinifera (ID: VIT05s0094g01080). qRT-PCR analysis revealed that Vvi-MAPKKK38 expression is strongly induced by Erysiphe necator, SA, ethylene and H 2 O 2 , 26 which is consistent with our report of the expression of VqMAPKKK38 being induced in response to biotic (E. necator) and abiotic (salt, chilling) stresses, as well as defenserelated hormone (SA, MeJA, ABA, Eth) treatments. Since the expression profile may be an indicator of gene function, we hypothesize that VqMAPKKK38 is a stress-inducible gene that is recruited for effective defense against a range of stressors. The evidence from the over-and RNAi-expression experiments with grapevine leaves indicates that VqMAPKKK38 is involved in a stilbene-type phytoalexin biosynthesis by mediating the transcription of STS genes and MYB14. Future studies will focus on   VqMAPKKK38 is essential for stilbene accumulation in grapevines Y Jiao et al.