Chinese Wild Grape Research Articles

Transcriptomic and metabolomic analyses reveal mechanisms underpinning resistance of Chinese wild grape to Colletotrichum viniferum

Grape ripe rot is one of the most important diseases caused by Colletotrichum spp. Chinese wild grape (Vitis davidii) is highly resistant to Colletotrichum viniferum infection. But mechanisms underlying the resistance remain largely unclear. In this study, transcriptomic and metabolomic responses of V. davidii to C. viniferum were studied before and after 1, 2, 4, and 6 days of inoculation. C. viniferum infection induced the expression of a large number of defense-related genes. KEGG analysis indicated that the differentially expressed genes (DEGs) were largely those involved in alpha-linolenic acid metabolism, flavonoid biosynthesis, phenylpropanoid biosynthesis, stilbenoid biosynthesis, and other defense-related metabolic pathways. Based on transcriptome data and experimental analysis, we found that jasmonic acid (JA) biosynthesis was closely related to V. davidii resistance to C. viniferum. In addition, many genes related to the synthesis of lignin and phytoalexin resveratrol are upregulated by pathogen infection, and metabolomic analysis showed that there was an increasing accumulation of resveratrol on day 6 of C. viniferum inoculation. Further analysis indicated that transcription factors, such as VdWRKY75 regulated the biosynthesis of lignin and stilbenes. A working model for V. davidii against C. viniferum infection was proposed. The infection of C. viniferum induced JA production, JA along with transcription factors regulated the biosynthesis of secondary metabolites, such as lignin and resveratrol that enhanced plant resistance to C. viniferum. This study elucidated molecular mechanisms underlying the resistance of Chinese wild V. davidii to C. viniferum which can provide a theoretical basis for grape disease resistance breeding.

VqNAC44 enhances stilbene synthesis and disease resistance in Chinese wild grape by interacting with VqMYB15

As significant phytoalexins, stilbene compounds can improve the stress resistance of grapes under biotic and abiotic stress conditions and have biological effects such as antitumour, antioxidant, immune regulation and cardiovascular protection activities in humans. RESVERATROL SYNTHASE (RS), also known as STILBENE SYNTHASE (STS), is the critical enzyme regulating stilbene synthesis and has been identified in a few plant species. However, the regulatory mechanisms of stilbene synthesis are uncertain. In this study, an NAC family transcription factor from Vitis quinquangularis, named VqNAC44, was characterized as an indirect regulator of stilbene synthesis. It is worth noting that VqNAC44 did not bind to the STS promoter nor did it interact with the STS protein but interacted with the MYB transcription factor VqMYB15. This interaction between VqMYB15 and VqNAC44 was validated by a yeast two-hybrid assay and bimolecular fluorescence complementation. Overexpressing VqNAC44 in Arabidopsis thaliana significantly increased its tolerance to biotic and abiotic stresses. Transient overexpression of VqNAC44 and VqMYB15 in grape leaves resulted in increased expression of the STS gene and increased production of stilbene compounds. The experimental results confirmed that VqNAC44 regulated stilbene synthesis by interacting with VqMYB15, thereby enhancing the plant stress resistance.

Overexpression of the VyP5CR gene increases drought tolerance in transgenic grapevine (V. vinifera L.)

Proline is an important osmotic regulatory substance that can rapidly accumulate in plants under drought stress. A key gene in the proline biosynthesis pathway is P5CR, which responds to abiotic stresses and hormones. When the VyP5CR gene cloned from extremely drought-resistant Chinese wild grape Vitis yeshanensis accession 'Yanshan-1′ is overexpressed in Arabidopsis thaliana, drought tolerance improves. In this study, the effect of VyP5CR on drought resistance was examined in transgenic grapevine, and its upstream regulatory factors were determined. The results showed that when VyP5CR was overexpressed in V. vinifera cultivar 'Thompson Seedless', transgenic vines were more drought-tolerant than the wild type under drought stress, with much less wilting and significantly smaller stomatal apertures and lower transpiration rate. Malondialdehyde content, electrolyte leakage, and H2O2 and O2− accumulations were lower, but the contents of chlorophyll and proline, the net photosynthetic rate and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were higher. The expression of drought stress-related genes was also significantly up-regulated in transgenic plants. Thus, VyP5CR increased drought tolerance of transgenic grapevine. Yeast one-hybrid and luciferase activity assays indicated that MADS-box transcription factors VyAGL42 and VyMADS23 directly bound to the VyP5CR gene promoter and activated its transcription. Therefore, VyAGL42 and VyMADS23 might synergistically regulate drought tolerance of grapes with VyP5CR.

VyUSPA3, a universal stress protein from the Chinese wild grape Vitis yeshanensis, confers drought tolerance to transgenic V. vinifera.

VyUSPA3 from the Chinese wild grape Vitis yeshanensis interacts with ERF105, PUB24 and NF-YB3, and overexpression of the VyUSPA3 gene in V. vinifera cv. 'Thompson Seedless' confers drought tolerance. Drought is a major abiotic stress factor that seriously affects the growth and yield of grapevine. Although many drought-related genes have been identified in Arabidopsis and other plants, the functions of only a few of their counterparts have been revealed in grape. Here, a universal stress protein (USP) A from the Chinese wild grape Vitis yeshanensis, VyUSPA3, was identified and its function was subsequently characterized by overexpressing or silencing the VyUSPA3 gene in V. vinifera cv. 'Thompson Seedless' via Agrobacterium-mediated genetic transformation. After 21 d of the drought treatment, most leaves of the untransformed (UT) 'Thompson Seedless' lines wilted, yet UT lines were less damaged compared to the RNAi-VyUSPA3 lines, nonetheless, the OE-VyUSPA3 lines were mostly unaffected. Meanwhile, OE-VyUSPA3 lines showed smaller stomatal aperture, more developed roots, higher leaf relative water content, proline content, and antioxidant enzyme activities, as well as lower malondialdehyde, H2O2 and O2•- accumulation than UT lines, but this response pattern was reversed in the RNAi-VyUSPA3 lines. Besides, the transcript levels of four drought-related genes (RD22, RD29B, DREB2A, and NCED1) in OE-VyUSPA3 lines were greater than those in the RNAi-VyUSPA3 and UT lines. In addition, a yeast two-hybrid assay and a bimolecular fluorescence complementation assay confirmed that VyUSPA3 interacted with ERF105, PUB24, and NF-YB3, respectively. This study revealed that VyUSPA3 improved drought tolerance in transgenic grapevines possibly through interaction with the hormone signaling, ubiquitination system, ethylene-responsive element binding factor and nuclear factors.

The transcription factor VaMYC2 from Chinese wild Vitis amurensis enhances cold tolerance of grape (V. vinifera) by up-regulating VaCBF1 and VaP5CS

Cultivated grapes, one of the most important fruit crops in the world, are sensitive to low temperature. Since Chinese wild grape Vitis amurensis is highly tolerant to cold, it is imperative to study and utilize its cold-tolerance genes for molecular breeding. Here, a VaMYC2 gene from V. amurensis was cloned, and its function was investigated by expressing VaMYC2 in the cold-sensitive V. vinifera cultivar ‘Thompson Seedless’. The expression of VaMYC2 could be induced by cold stress, methyl jasmonate and ethylene treatment, but was inhibited by abscisic acid in leaves of V. amurensis. When transgenic grape lines expressing VaMYC2 were subjected to cold stress (−1 °C) for 41 h, the transgenic lines showed less freezing injury and lower electrolyte leakage and malondialdehyde content, but higher contents of soluble sugars, soluble proteins and proline, and antioxidant enzyme activities compared with wild-type. Moreover, the expression of some cold-tolerance related genes increased in transgenic lines. Besides, the interactions of VaMYC2 with VaJAZ1 and VaJAZ7B were confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays. Yeast one-hybrid and dual luciferase assays showed that VaMYC2 can bind to the promoters of VaCBF1 and VaP5CS and activate their expressions. In conclusion, expression of VaMYC2 in V. vinifera enhances cold tolerance of transgenic grapes which is attributed to enhanced accumulation of osmotic regulatory substances, cell membrane stability, antioxidant enzyme activity, and expression of cold tolerance-related genes. Also, VaMYC2 interacts with VaJAZ1 and VaJAZ7, and activates the expression of VaCBF1 and VaP5CS to mediate cold tolerance in grapes.

Ectopic expression of VyCIPK1 gene, isolated from wild grape Vitis yanshanesis J, X. Chen., confers the tolerance to salt in transgenic tobacco

ABSTRACT Calmodulin-like interacting protein kinases play an important role in plant response to abiotic stresses and development. But the role of the CIPK gene in grapevine is unknown. In this study, VyCIPK1, isolated from the Chinese wild grape V. Yanshanesis, was strongly induced by salt stress. Overexpressing VyCIPK1 could induce AOC and AOS, and result in notably increased jamonate levels in tobacco. Under salt stress, transgenic plants showed higher germination rate, leaf number, and fresh weight than wild-type plants. Moreover, transgenic plants displayed higher chlorophyll content, catalase activity, peroxidase activity, superoxide dismutase activity, and lower malondialdehyde content, H2O2, and O2- content than that of wild type under salt stress conditions. And the stress-related genes, including ERD10C, ERD10D, LEA5, POD, SOD, and CAT, were up-regulated in transgenic plants. Our founding demonstrated that the VyCIPK1 has the potential for grape molecular breeding of salt tolerance as a candidate gene.

The transcription factors VaERF16 and VaMYB306 interact to enhance resistance of grapevine to Botrytis cinerea infection.

Botrytis cinerea is a fungus that infects cultivated grape (Vitis vinifera); the identification and characterization of resistance mechanisms in the host is of great importance for the grape industry. Here, we report that a transcription factor in the ethylene‐responsive factor (ERF) family (VaERF16) from Chinese wild grape (Vitis amurensis ‘Shuang You’) is expressed during B. cinerea infection and in response to treatments with the hormones ethylene and methyl jasmonate. Heterologous overexpression of VaERF16 in Arabidopsis thaliana substantially enhanced resistance to B. cinerea and the bacterium Pseudomonas syringae DC3000 via the salicylic acid and jasmonate/ethylene signalling pathways. Yeast two‐hybrid, bimolecular fluorescence complementation, and co‐immunoprecipitation assays indicated that VaERF16 interacts with the MYB family transcription factor VaMYB306. Overexpression of VaERF16 or VaMYB306 in grape leaves increased resistance to B. cinerea and caused an up‐regulation of the defence‐related gene PDF1.2, which encodes a defensin‐like protein. Conversely, silencing of either gene resulted in increased susceptibility to B. cinerea. Yeast one‐hybrid and dual‐luciferase assays indicated that VaERF16 increased the transcript levels of VaPDF1.2 by binding directly to the GCC box in its promoter. Notably, VaMYB306 alone did not bind to the VaPDF1.2 promoter, but the VaERF16–VaMYB306 transcriptional complex resulted in higher transcript levels of VaPDF1.2, suggesting that the proteins function through their mutual interaction. Elucidation of this regulatory module may be of value in enhancing resistance of grapevine to B. cinerea infection.

Evaluation of volatile aroma compounds from Chinese wild grape berries by headspace-SPME with GC-MS

Evaluation of volatile aroma compounds from Chinese wild grape species aimed to explore new grape species via fruit aroma. In this study, the volatile organic compounds (VOCs) from ten grape cultivars belonging to nine different grape species were detected, by using headspace solid-phase microextraction with GC-MS. Chinese wild grape species were studied along with table grape (Muscat Hamburg, Concord) and wine grape (Cabernet Sauvignon) cultivars belonging to different origins. Total forty-five volatile compounds were detected, upon which several VOCs including pentanoic acid, cyclobutyl ester, maltol, (+)-neoisomenthol, and 8-methyloctahydrocoumarin were detected in Chinese wild grapes that have not been detected in other Vitis species. The results revealed that C6 volatile compounds were pre-dominant VOCs. Esters were found abundant in V. labrusca (Concord) and terpenoids were dominant in V. vinafera (Hamburg Muscat). The principal component analysis showed that Chinese wild grape cultivars V. yeshanensis, V. facifolia, V. davidii, V. betulifolia V. heyneana, V. amurensis and V. adenoclada are correlated with wine grape cultivar ‘Cabernet Sauvignon’, while table grape cultivars are distributed separately. These results can provide a reference for the utilization of new germplasm for specific purposes.

Grape Transcriptome Response to Powdery Mildew Infection: Comparative Transcriptome Profiling of Chinese Wild Grapes Provides Insights Into Powdery Mildew Resistance.

Erysiphe necator, the fungal pathogen of grape powdery mildew disease, poses a great threat to the grape market and the wine industry. To better understand the molecular basis of grape responses to E. necator, we performed comparative transcriptome profiling on two Chinese wild grape accessions with varying degrees of resistance to E. necator. At 6-, 24-, and 96-h postinoculation of E. necator, 2,856, 2,678, and 1,542 differentially expressed genes (DEGs) were identified in the susceptible accession Vitis pseudoreticulata 'Hunan-1', and at those same time points, 1,921, 2,498, and 3,249 DEGs, respectively, were identified in the resistant accession V. quinquangularis 'Shang-24'. 'Hunan-1' had a substantially larger fraction of down-regulated genes than 'Shang-24' at every infection stage. Analysis of DEGs revealed that up-regulated genes were mostly associated with defense response and disease resistance-related metabolite biosynthesis, and such signaling genes were significantly suppressed in 'Hunan-1'. Interestingly, fatty acid biosynthesis- and elongation-related genes were suppressed by the fungus in the 'Shang-24' accession but somehow induced in the 'Hunan-1' accession, consistent with the concept that E. necator is likely to be a fatty acid auxotroph that requires lipids from the host. Moreover, genes involved in biosynthesis and signaling of phytohormones, such as jasmonic acid and cytokinin, as well as genes encoding protein kinases and nucleotide-binding domain leucine-rich repeat proteins, differentially responded to E. necator in the two wild grapes. The variation of gene regulation associated with nutrient uptake by the fungus and with signaling transduction and pathogen recognition suggests a multilayered regulatory network that works in concert to assist in the establishment of fungal pathogen infections.

Genetic Diversity and Population Structure Analysis of Chinese Wild Grape Using Simple Sequence Repeat Markers

Chinese wild Vitis is a useful gene source for resistance to biotic and abiotic stresses, although there is little research on its genetic diversity and structure. In this study, nine simple sequence repeat (SSR) markers were used to assess the genetic diversity and genetic structure among 100 Vitis materials. These materials included 77 indigenous accessions representing 23 of 38 wild Vitis species/cultivars in China, 18 V. vinifera cultivars, and the five North American species V. aestivalis, V. girdiana, V. monticola, V. acerifolia, and V. riparia. The SSR loci used in this study for establishing an international database (Vitis International Variety Catalogue) revealed a total of 186 alleles in 100 Vitis accessions. The mean values for the gene diversity (GD) and polymorphism information content (PIC) per locus were 0.91 and 0.90, respectively, which indicates that the discriminatory power of the markers is high. Based on the genetic distance data, the 100 Vitis accessions were divided into five primary clusters by cluster analysis, and five populations by structure analysis; these results indicate these Chinese wild grapes were more genetically close to European grapes than to North American species. In addition, the clustering patterns of most accessions correlated with the geographic distribution. An analysis of molecular variance (AMOVA) revealed that 3.28%, 3.27%, and 93.46% of the variance occurred between populations, between individuals within populations, and between individuals within the entire population, respectively. In addition, we identified three previously undescribed accessions (Wuzhi-1, MZL-5, and MZL-6) by cluster analysis. Our results reveal a high level of genetic diversity and variability in Vitis from China, which will be helpful in the use of genetic resources in future breeding programs. In addition, our study demonstrates that SSR markers are highly suitable for further genetic diversity analyses of Chinese wild grapes.

Pathogenesis and Immune Response in Resistant and Susceptible Cultivars of Grapevine (Vitis spp.) Against Elsinoë ampelina Infection.

Elsinoë ampelina is the main cause of grape anthracnose, and the majority of grapevine cultivars are susceptible to this fungus. Some Chinese wild grape cultivars are resistant, however. It is therefore apt to compare the pathogenesis and immune responses in susceptible and resistant cultivars of grapevine to explore the detailed molecular and biochemical mechanisms of resistance to this fungus. In this study, ultrastructural and histopathological observations were used to demonstrate the resistance responses to E. ampelina in the resistant Chinese wild cultivar Vitis quinquangularis clone 'Shang-24' and the susceptible cultivars V. davidii 'Tangwei' and V. vinifera 'Thompson Seedless'. Seventy-two hours postinoculation (hpi) with E. ampelina, brown necrotic spots were clearly visible on the leaves of the susceptible 'Tangwei' and 'Thompson Seedless'. The infection was characterized by rapid colonization of the host cells by hyphae and massive spread of the pathogen in the intercellular spaces, ultimately leading to host cell collapse, cuticle dissolution, and extensive hyphal growth. In the resistant clone 'Shang-24', the conidia were lysed, a large quantity of electronically dense matter appeared, the hyphal growth was suppressed, and the host cells remained intact. In addition, six genes associated with disease resistance were differentially expressed in the susceptible and resistant cultivars. These disease-related genes were significantly up-regulated following infection with E. ampelina. This study illustrates the differences in infection and colonization of E. ampelina in resistant and susceptible grape leaves.

Cloning and Functional Analysis of the CDS and Promoter of VpPR4b Gene Response to Downy Mildew in Chinese Wild Grape

Cloning and Functional Analysis of the CDS and Promoter of VpPR4b Gene Response to Downy Mildew in Chinese Wild Grape

Polymorphism of anthocyanin concentration and composition in Chinese wild grapes

Background and Aims Abundant wild grape resources are distributed in China and are rich in anthocyanins, but the systematic analysis and evaluation of the anthocyanin composition in different species of Chinese wild grapes have been little studied. We investigated the interspecific polymorphism of anthocyanins in Chinese wild grapes. Methods and Results The anthocyanin concentration and individual anthocyanins in 40 grape genotypes from 11 Vitis species were detected and analysed by ultra-performance liquid chromatography (UPLC)–MS. Vitis bryoniaefolia had the highest anthocyanin concentration, whereas V. betulifolia had the lowest. Twenty-eight anthocyanins were identified and V. bryoniaefolia had the maximum number of anthocyanins, whereas V. davidii had the minimum. Mono- and diglucoside anthocyanins were detected, among which malvidin 3,5-O-diglucoside was dominant. Among the acylated anthocyanins detected in Chinese wild grapes, coumaroyl derivatives were dominant, except for V. betulifolia, in which acetylated anthocyanins accounted for >95% of the acylated anthocyanins. These were not detected in V. amurensis and in some genotypes of V. heyneana s. ficifolia, V. piasezkii and V. heyneana. Conclusions Chinese wild grape species show polymorphism in anthocyanin concentration and composition. Significance of the Study This study is important for the systematic evaluation and utilisation of Chinese wild grape resources.

Targeted Metabolomic and Transcript Level Analysis Reveals Quality Characteristic of Chinese Wild Grapes (Vitis davidii Foex).

Native to China, spine grapes (Vitis davidii Foex) are an important wild grape species. Here, the quality characteristics of one white and three red spine grape clones were evaluated via targeted metabolomic and transcription level analysis. Xiangzhenzhu (XZZ) had the highest soluble sugar and organic acid content. Malvidin-3-acetyl-glucoside and cyanidin-3-glucoside were the characteristic anthocyanins in spine grapes, and significant differences in anthocyanin composition between different clones were detected. Anthocyanins were not detected in Baiyu (BY) grapes. The transcript levels of VdGST, VdF3′H, VdOMT, VdLDOX, and VdUFGT were significantly related to the anthocyanin biosynthesis and proportions. A total of 27 kinds of glycosidically bound volatiles (including alcohols, monoterpenes, esters, aldehydes, ketones, and phenolic acid) were identified in spine grapes, with Gaoshan #4 (G4) and BY grapes having the highest concentrations. The VdGT expression levels were closely related to glycosidically bound volatile concentrations. These results increase our understanding of the quality of wild spine grapes and further promote the development and use of wild grape resources.

The process of embryo abortion of stenospermocarpic grape and it develops into plantlet in vitro using embryo rescue

Using stenospermocarpic grapes as female parents could improve the seedless ratio of offspring via embryo rescue techniques, so they are widely used for breeding seedless grapes. Here, we report the variation in phenotype, endogenous hormones, and internal cellular structure of ovules during embryo abortion in ‘Thompson Seedless’. Moreover, we used the stenospermocarpic grapes as female parents to breed new seedless grapes. Our result indicated that the seed coat of abnormal ovules turned brown from 38 to 42 days after flowering (DAF). In hormones level, two ratios of endogenous hormones’ content [gibberellic acid (GA3) + indole-3-acetic acid (IAA)]/abscisic acid (ABA) and zeatin riboside (ZR) /abscisic acid (ABA) had decreased by 36 DAF. In paraffin sections of embryo sac, indicated that there were few or no embryos in ovules were observed from 37 to 42 DAF. Importantly, the globular embryos in ‘Thompson Seedless’ were significantly smaller than those in ‘Ruby Seedless’ at 40 DAF, indicating the former had a lower embryo rescue efficiency as a female parent; accordingly, the most suitable time for embryo cultured in vitro was 39 DAF for ‘Thompson Seedless’. Result from embryo rescue, demonstrated that 5 mg/L exogenous brassinolide (BR) promoted embryo recovery, embryo germination, and polyembryony formation of ‘Flame Seedless’. Concurrently, WPM medium containing 1 µM of 6-BA helped embryos germinate into normal plantlets and reduced the abnormal plantlet development percentage. A total of 3573 hybrid ovules and 402 new progeny plants were obtained using the stenospermocarpic grapes as female parents crossed with disease-resistant Chinese wild grapes, among which 212 progeny with seedless trait and 23 with disease-resistant were detected by molecular markers, respectively. By studying phenotype, hormone, and anatomy, the optimal time for embryo rescue in Vitis vinifera cv. ‘Thompson Seedless’ was found. In all, 402 new seedless and disease-resistant hybrids were obtained via embryo rescue when using stenospermocarpic grapevine cultivars as female parents.

First study on the fatty acids and their derived volatile profiles from six Chinese wild spine grape clones (Vitis davidii Foex)

Spine grapes (Vitis davidii Foex) native to China are an important wild grape species. To evaluate the differences in the fatty acids which may contribute to the aroma of spine grapes, four red and two white spine grape clones were used as materials. GC-MS analysis of spine grapes revealed ten fatty acids, and 9,12-octadecadieneoic acid and linolenic acid were the major UFAs in the six clones. The highest concentrations of C6 and C9 volatiles were detected in Gaoshan #4 and Baiputao #1, respectively. The concentrations of C6 compounds in spine grapes were closely related to linolenic acid and linoleic acid, and positively correlated with the expressions of VdLOXA, VdLOXC, VdLOXD and VdHPL1. These results would fill the gap knowledge of fatty acids in spine grapes and provide information for future research on the classification of spine grapes flavor type.

Endophytic fungal community in grape is correlated to foliar age and domestication

PurposeThe composition of endophytic communities has been shown to depend on grape genotypes and viticultural managements in leaves, stems, and berries of grape, but there have been relatively few reports exploring fungal endophytes associated with wild grape and foliar age.MethodsThe regions of internally transcribed spacer (ITS) were sequenced using the Illumina HiSeq to determine the diversity of fungal endophytes associated with European grape (Vitis vinifera cv. Red Globe) and Chinese wild grape (Vitis amurensis cv. Shuangyou) in young and mature leaves.ResultsA total of 3 phyla, 23 classes, 51 orders, 97 families, and 150 fungal genera were identified. Young leaves have significantly higher diversity and richness than that in mature leaves in both cultivars. Endophytic fungal diversity was greater in wild grapevines (119 genera) than in cultivated grapevines (81 genera) in both young and mature leaves. Endophytic fungal community structure was also significantly different between young leaves and mature leaves as well as in both cultivars based on statistical tests of ANOSIM and MRPP.ConclusionsOur results suggest that endophytic fungal communities were strongly affected by foliar age and domestication, which are crucial factors in establishing symbiotic associations with a selective enrichment for specific endophytes.

The WRKY53 transcription factor enhances stilbene synthesis and disease resistance by interacting with MYB14 and MYB15 in Chinese wild grape.

Resveratrol is notable not only for its functions in disease resistance in plants but also for its health benefits when it forms part of the human diet. Identification of new transcription factors helps to reveal the regulatory mechanisms of stilbene synthesis. Here, the WRKY53 transcription factor was isolated from the Chinese wild grape, Vitis quinquangularis. Vqwrky53 was expressed in a variety of tissues and responded to powdery mildew infection and to exogenous hormone application. VqWRKY53 was located in the nucleus and had transcriptional activation activity in yeast. A yeast two-hybrid assay and a bimolecular fluorescence complementation assay confirmed that VqWRKY53 interacted physically with VqMYB14 and VqMYB15, which have previously been reported to regulate stilbene synthesis. When Vqwrky53 was overexpressed in grape leaves, the expression of VqSTS32 and VqSTS41 and the content of stilbenes were increased. A yeast one-hybrid assay demonstrated that VqWRKY53 could bind directly to the promoters of STS genes. Overexpression of Vqwrky53 activated β-glucuronidase expression, driven by STS promoters, and co-expressing Vqwrky53 with VqMYB14 and VqMYB15 showed stronger regulatory functions. Heterologous overexpression of Vqwrky53 in Arabidopsis accelerated leaf senescence and disease resistance to PstDC3000.

The Effect of Transcription Factor MYB14 on Defense Mechanisms in Vitis quinquangularis-Pingyi.

In the current study, we identified a transcription factor, MYB14, from Chinese wild grape, Vitis quinquangularis-Pingyi (V. quinquangularis-PY), which could enhance the main stilbene contents and expression of stilbene biosynthesis genes (StSy/RS) by overexpression of VqMYB14. The promoter of VqMYB14 (pVqMYB14) was shown to be induced as part of both basal immunity (also called pathogen-associated molecular pattern (PAMP)-triggered immunity, PTI) and effector-triggered immunity (ETI), triggered by the elicitors flg22 and harpin, respectively. This was demonstrated by expression of pVqMYB14 in Nicotiana benthamiana and Vitis. We identified sequence differences, notably an 11 bp segment in pVqMYB14 that is important for the PTI/ETI, and particularly for the harpin-induced ETI response. In addition, we showed that activation of the MYB14 promoter correlates with differences in the expression of MYB14 and stilbene pattern induced by flg22 and harpin. An experimental model of upstream signaling in V. quinquangularis-PY is presented, where early defense responses triggered by flg22 and harpin partially overlap, but where the timing and levels differ. This translates into a qualitative difference with respect to patterns of stilbene accumulation.

The grapevine R2R3-type MYB transcription factor VdMYB1 positively regulates defense responses by activating the stilbene synthase gene 2 (VdSTS2)

BackgroundResveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of trans-resveratrol to human health and plant protection against fungal pathogens and abiotic stresses are well-established, yet little is known about the molecular mechanisms regulating stilbene biosynthesis in plant defense progress.ResultsHere, we cloned and identified the Chinese wild grape (Vitis davidii) R2R3-MYB transcription factor VdMYB1, which activates defense responses against invading pathogen. VdMYB1 transcripts were significantly upregulated after inoculation with the grapevine powdery mildew fungus Erysiphe necator (Schw.) Burr. Transient expression analysis using onion epidermal cells and Arabidopsis thaliana protoplasts showed that VdMYB1 was localized in the nucleus. Yeast one-hybrid assays revealed that VdMYB1 acts as a transcriptional activator. Grapevine leaves transiently overexpressing VdMYB1 showed a lower number of fungal conidiophores compared with wild-type leaves. Overexpression of VdMYB1 in grapevine leaves did not alter the expression of genes in salicylic acid- and jasmonate-dependent pathways, but affected the expression of stilbene synthase (STS) genes, key regulators of flavonoid metabolism. Results of electrophoretic mobility shift assays and in vivo transcriptional activation assays showed that VdMYB1 binds to the MYB binding site (MYBBS) in the STS2 gene promoter, thus activating STS2 transcription. In heterologous expression assays using tobacco leaves, VdMYB1 activated STS2 gene expression and increased the accumulation of resveratrol.ConclusionsOur study showed that VdMYB1 activates STS2 gene expression to positively regulate defense responses, and increases the content of resveratrol in leaves.