Chinese Wild Vitis Quinquangularis Research Articles

VqMAPK3/VqMAPK6, VqWRKY33, and VqNSTS3 constitute a regulatory node in enhancing resistance to powdery mildew in grapevine.

Grapevine powdery mildew is caused by Erysiphe necator, which seriously harms grape production in the world. Stilbene synthase makes phytoalexins that contribute to the resistance of grapevine against powdery mildew. A novel VqNSTS3 was identified and cloned from Chinese wild Vitis quinquangularis accession Danfeng-2. The novel VqNSTS3 was transferred into susceptible 'Thompson Seedless' by Agrobacterium-mediated transformation. The transgenic plants showed resistance to the disease and activated other resistance-related genes. VqNSTS3 expression in grapevine is regulated by VqWRKY33, and which binds to TTGACC in the VqNSTS3 promoter. Furthermore, VqWRKY33 was phosphorylated by VqMAPK3/VqMAPK6 and thus led to enhanced signal transduction and increased VqNSTS3 expression. ProVqNSTS3::VqNSTS3-GFP of transgenic VqNSTS3 in Arabidopsis thaliana was observed to move to and wrap the pathogen's haustoria and block invasion by Golovinomyces cichoracearum. These results demonstrate that stilbene accumulation of novel VqNSTS3 of the Chinese wild Vitis quinquangularis accession Danfeng-2 prevented pathogen invasion and enhanced resistance to powdery mildew. Therefore, VqNSTS3 can be used in generating powdery mildew-resistant grapevines.

Transcriptome and metabolome analysis of stress tolerance to aluminium in Vitis quinquangularis.

Transcriptional and metabolic regulation of aluminium tolerance of Chinese wild Vitis quinquangularis after Al treatment for 12h: genes and pathways related to stress resistance are activated to cope with Al stress. The phytotoxicity of aluminium (Al) has become a major issue in inhibiting plant growth in acidic soils. Chinese wild Vitis species have excellent stress resistance. In this study, to explore the mechanism underlying Al tolerance in Chinese wild Vitis quinquangularis, we conducted a transcriptome analysis to understand the changes in gene expression and pathways in V. quinquangularis leaves after Al treatment for 12h (Al_12h). Compared with the control (CK) treatment, 2266 upregulated differentially expressed genes (DEGs) and 2943 downregulated DEGs were identified after Al treatment. We analysed the top 60 upregulated DEGs and found that these genes were related mostly to cell wall organization or biogenesis, transition metal ion binding, etc. Another analysis of all the upregulated DEGs showed that genes related to the ABC transport pathway, salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) hormone signalling pathway were expressed. Transcriptome and metabolome analysis revealed that genes and metabolites (phenylalanine, cinnamate and quercetin) related to the phenylalanine metabolic pathway were expressed. In summary, the results provide a new contribution to a better understanding of the metabolic changes that occur in grapes after Al stress as well as to research on improving the resistance of grape cultivars.

Heterologous expression of Chinese wild grapevine VqERFs in Arabidopsis thaliana enhance resistance to Pseudomonas syringae pv. tomato DC3000 and to Botrytis cinerea

When a plant is attacked by a pathogen, an immune response is activated to help protect it from harm. ERF transcription factors have been reported to regulate immune responses in plants. Here, three ERF transcription factors from Chinese wild Vitis quinquangularis, VqERF112, VqERF114 and VqERF072, are shown to respond to pathogen inoculation by powdery mildew, Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea and to hormone treatments including with ET, SA, MeJA or ABA. Tissue specific expression analysis shows the highest expression levels of VqERF112 and VqERF114 were in mature berries and of VqERF072 was in tendrils. A GUS activity assay indicates that the promoters of VqERF112, VqERF114 and VqERF072 can be induced by powdery mildew inoculation and by hormone treatment, including with ET, SA and MeJA. Overexpression of VqERF112, VqERF114 and VqERF072 in transgenic Arabidopsis enhanced the resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and B. cinerea, and it increased the expression of the SA signaling-related genes AtNPR1 and AtPR1 and of the JA/ET signaling-related genes AtPDF1.2, AtLOX3, AtPR3 and AtPR4. Compared to Col-0 plants, the H2O2 accumulation in transgenic Arabidopsis increased after Pst DC3000 inoculation but decreased after B. cinerea inoculation. These results demonstrate that VqERF112, VqERF114 and VqERF072 positively regulate resistance to Pst DC3000 and B. cinerea.

Transcription factor VqERF114 regulates stilbene synthesis in Chinese wild Vitis quinquangularis by interacting with VqMYB35.

VqERF114 regulates stilbene synthesis by interacting with VqMYB35. Resveratrol is a stilbene, an important class of secondary metabolites that accumulates in some plant species, including grapevine. In the plant, these are involved in the response to attack by plant pathogens and, as a component of the human diet, they offer a range of significant health benefits. Stilbene synthase (STS), the key enzyme responsible for resveratrol synthesis, has been characterised in a small number of plant species. However, the regulatory mechanisms for stilbene synthesis are uncertain. Here, an ERF family transcription factor from Chinese wild Vitis quinquangularis, VqERF114, was characterised as an indirect regulator of stilbene synthesis. A transient overexpression assay of VqERF114 in grapevine leaves led to increased STS expression and stilbene accumulation. However, VqERF114 did not bind to the promoters of VqSTSs but the MYB transcription factor, VqMYB35, did interact with VqERF114. This interaction was confirmed by a yeast two-hybrid assay and bimolecular fluorescence complementation. Furthermore, VqMYB35 showed activation effects on the expressions of VqSTS15, VqSTS28, VqSTS42 and VqSTS46 by binding directly to the MBS elements in their promoters. Co-overexpression of VqERF114 and VqMYB35 resulted in higher VqSTSs expression and more stilbene synthesis. These results demonstrate that VqERF114 regulates stilbene synthesis by interacting with VqMYB35.

VqbZIP1 isolated from Chinese wild Vitis quinquangularis is involved in the ABA signaling pathway and regulates stilbene synthesis

Resveratrol is an important phytoalexin in grapevine. Not only does it confer increased disease resistance and but as a food component it offers significant benefits in human health. Abscisic acid (ABA) is an important phytohormone involved in many biological processes in plants and can also promote the accumulation of stilbenes. Stilbene synthase (STS) is an important enzyme which catalyzes the last step of resveratrol synthesis. Our study characterizes a basic leucine zipper (bZIP) transcription factor, VqbZIP1, isolated from Chinese wild Vitis quinquangularis accession Danfeng-2. The results show that VqbZIP1 encodes 299 amino acids and belongs to the Group A subfamily of the bZIP family. VqbZIP1 showed transcriptional activation activity in yeast and is predicted to be located in the nucleus. The yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) assay together show that VqbZIP1 interacts with VqSnRK2.4 and VqSnRK2.6. VqbZIP1, the STS genes, VqSnRK2.4 and VqSnRK2.6 can all be induced by ABA treatment. A GUS activity experiment indicates VqbZIP1 can activate the GUS reporter gene driven by STS promoters. Further studies show that co-expression of VqbZIP1 with VqSnRK2.4 or VqSnRK2.6 can confer higher efficiency than expression of VqbZIP1 alone in activating the STS promoters. Overexpression of VqbZIP1 in grape leaves promoted the transcript level of the STS genes and the accumulation of stilbenes. Overexpression of VqbZIP1 in Arabidopsis thaliana can confer ABA sensitivity. In summary, our results suggest VqbZIP1 participates in the ABA signaling pathway and regulates stilbene synthesis.

Ectopic Expression of the Wild Grape WRKY Transcription Factor VqWRKY52 in Arabidopsis thaliana Enhances Resistance to the Biotrophic Pathogen Powdery Mildew But Not to the Necrotrophic Pathogen Botrytis cinerea.

WRKY transcription factors are known to play important roles in plant responses to biotic stresses. We previously showed that the expression of the WRKY gene, VqWRKY52, from Chinese wild Vitis quinquangularis was strongly induced 24 h post inoculation with powdery mildew. In this study, we analyzed the expression levels of VqWRKY52 following treatment with the defense related hormones salicylic acid (SA) and methyl jasmonate, revealing that VqWRKY52 was strongly induced by SA but not JA. We characterized the VqWRKY52 gene, which encodes a WRKY III gene family member, and found that ectopic expression in Arabidopsis thaliana enhanced resistance to powdery mildew and Pseudomonas syringae pv. tomato DC3000, but increased susceptibility to Botrytis cinerea, compared with wild type (WT) plants. The transgenic A. thaliana lines displayed strong cell death induced by the biotrophic powdery mildew pathogen, the hemibiotrophic P. syringe pathogen and the necrotrophic pathogen B. cinerea. In addition, the relative expression levels of various defense-related genes were compared between the transgenic A. thaliana lines and WT plants following the infection by different pathogens. Collectively, the results indicated that VqWRKY52 plays essential roles in the SA dependent signal transduction pathway and that it can enhance the hypersensitive response cell death triggered by microbial pathogens.

Functional analysis of the fruit-specific promoter of VqSTS6 from the Chinese wild grape, Vitis quinquangularis

Stilbene synthase plays important roles in the secondary metabolic pathways of many plants as polyketide synthase. Increased consumption of its metabolite, resveratrol, is highly beneficial to our health. The stilbene synthase 6 gene (VqSTS6) has been obtained from the Chinese wild Vitis quinquangularis accession ‘Danfeng-2’. Our previous studies have shown that this stilbene synthase gene exhibits higher levels of expression than other VqSTSs in the all six grape organs. To characterize gene expression, we cloned the promoter of VqSTS6 by homologous cloning. Alignment between the VqSTS6 gene sequence and Vitis vinifera VvSTS48 showed 99.24% identity. The promoter of VqSTS6 fused with the β-glucuronidase (GUS) gene was transformed into tomato (Solanum lycopersicum L. ‘Micro-Tom’) and the activity expression in four organs (roots, stems, leaves and fruits) was analyzed. This revealed that expression was much higher in the fruit than in the other three organs. We also used promoter deletion to show that the –518 bp to –411 bp region contained skn-1_motifs are the core functional promoter region in fruit development. These results evidence the special role of VqSTS6 promoter in regulating its expression in a high-resveratrol grapevine.

Ectopic expression of a grape aspartic protease gene, AP13, in Arabidopsis thaliana improves resistance to powdery mildew but increases susceptibility to Botrytis cinerea

The grape aspartic protease gene, AP13 was previously reported to be responsive, in Chinese wild Vitis quinquangularis cv. ‘Shang-24’, to infection by Erysiphe necator, the causal agent of powdery mildew disease, as well as to treatment with salicylic acid in V. labrusca×V. vinifera cv. ‘Kyoho’. In the current study, we evaluated the expression levels of AP13 in ‘Shang-24’ in response to salicylic acid (SA), methyl jasmonate (MeJA) and ethylene (ET) treatments, as well as to infection by the necrotrophic fungus, Botrytis cinerea, and the transcript levels of VqAP13 decreased after B. cinerea infection and MeJA treatment, but increased following ET and SA treatments. Transgenic Arabidopsis thaliana lines over-expressing VqAP13 under the control of a constitutive promoter showed enhanced resistance to powdery mildew and to the bacterium Pseudomonas syringae pv. tomato DC3000, and accumulated more callose than wild type plants, while the resistance of transgenic A. thaliana lines to B. cinerea inoculation was reduced. In addition, the expression profiles of various disease resistance- related genes in the transgenic A. thaliana lines following infection by different pathogens were compared to the equivalent profiles in the wild type plants. The results suggest that VqAP13 action promotes the SA dependent signal transduction pathway, but suppresses the JA signal transduction pathway.

Genetic transformation of a fruit-specific, highly expressed stilbene synthase gene from Chinese wild Vitis quinquangularis.

The stilbene synthase gene VqSTS6, from Chinese wild type Vitis quinquangularis accession Danfeng-2, increases the resveratrol content and pathogen resistance of transgenic plants of V. vinifera Thompson Seedless. This study successfully created transgenic plants of V. vinifera Thompson Seedless which overexpressed VqSTS6, cloned from Chinese wild type V. quinquangularis accession Danfeng-2. Western blot and qRT-PCR showed a variable range in transcript levels among transgenic lines. The resistance to powdery mildew (Uncinula necator) was particularly enhanced in lines most highly expressing VqSTS6. Compared with the non-transformed controls, trans-resveratrol and other stilbene compounds were significantly increased in the transgenic lines. The correlation between high resveratrol content and high pathogen resistance in transgenic grapes is discussed. We hypothesize that the fruit-specific, highly expressed gene VqSTS6 from Chinese wild V. quinquangularis accession Danfeng-2, is directly involved in the resveratrol synthesis pathway in grapes, and plays an important role in the plant's defense against pathogens. Genetic transformation of VqSTS6explored the potential of the wild Chinese grape species for use in breeding, the results of which would raise both the disease resistance and the fruit quality of V. vinifera grapevines.

Characterization of Erysiphe necator-responsive genes in Chinese Wild Vitis quinquangularis.

Powdery mildew (PM), caused by fungus Erysiphe necator, is one of the most devastating diseases of grapevine. To better understand grapevine-PM interaction and provide candidate resources for grapevine breeding, a suppression subtractive hybridization (SSH) cDNA library was constructed from E. necator-infected leaves of a resistant Chinese wild Vitis quinquangularis clone “Shang-24”. A total of 492 high quality expressed sequence tags (ESTs) were obtained and assembled into 266 unigenes. Gene ontology (GO) analysis indicated that 188 unigenes could be assigned with at least one GO term in the biological process category, and 176 in the molecular function category. Sequence analysis showed that a large number of these genes were homologous to those involved in defense responses. Genes involved in metabolism, photosynthesis, transport and signal transduction were also enriched in the library. Expression analysis of 13 selected genes by qRT-PCR revealed that most were induced more quickly and intensely in the resistant material “Shang-24” than in the sensitive V. pseudoreticulata clone “Hunan-1” by E. necator infection. The ESTs reported here provide new clues to understand the disease-resistance mechanism in Chinese wild grapevine species and may enable us to investigate E. necator-responsive genes involved in PM resistance in grapevine germplasm.

Identification of genes differentially expressed in grapevine associated with resistance to Elsinoe ampelina through suppressive subtraction hybridization

Anthracnose, caused by the biotrophic fungus Elsinoe ampelina, is an economically devastating disease of grapevine (Vitis vinifera L.) prevalent in warm and humid regions of the world. In order to investigate the molecular resistance mechanisms and identify genes related to anthracnose resistance in grapevine, a Suppression Subtractive Hybridization (SSH) library was constructed using mixed cDNAs prepared from leaves of Chinese wild Vitis quinquangularis clone 'Shang-24', cDNA prepared from leaves infected with the pathogen E.ampelina served as tester and cDNA from mock-inoculated leaves as driver. A total of 670 high-quality ESTs were clustered and assembled into a collection of 461 unique genes comprising 85 contigs and 376 singletons. By Gene ontology (GO) analysis 310 unigenes were assigned to 22 GO slims within the molecular function category, while 317 unigenes could be sorted into 43 GO slims within the biological process category. The expression profiles of 20 selected genes, monitored by quantitative RT-PCR, indicated that expression of these genes in the E.ampelina-resistant 'Shang-24' was quicker and more intense, than in the susceptible 'Red Globe' where the reaction was delayed and limited. The results imply that these up-regulated genes could be involved in grapevine responses against E.ampelina infection.

Identification and expression analysis of a pathogen-responsive PR-1 gene from Chinese wild Vitis quinquangularis

In response to pathogen attacks, plant produces a wide range of pathogenesis-related (PR) proteins. PR-1 genes represent the first identified PR gene family. Most members of PR-1 gene family are not inducible by pathogen attacks. In this study, we identified a pathogen-responsive PR-1 gene designated as VqPR-1 (GenBank accession no. JN256202), in a subtractive suppression hybridization (SSH) cDNAlibrary from Elsinoe ampelina -inoculated young leaves of Chinese wild Vitis quinquangularis clone ‘shang-24’. VqPR-1 protein contained the requisite signal sequence at the N-terminus, a conserved three-dimensional structure called ‘PR-1 fold’ and a highly conserved six-cysteine motif. Expression level of VqPR-1 rose rapidly in response to E. ampelina infection. The three tested plant defence signaling molecules, salicylic acid (SA), ethephon (Eth) and methyl jasmonate (MeJA) all triggered an induction of VqPR-1. However, the induction by addition of MeJA was weaker than that induced by SA and Eth. In addition, the response to inoculation with E. ampelina or treatment with signaling molecules, was sometimes a suppression of VqPR-1 gene expression. The highest expression of VqPR- 1 was observed in flowers, stems and leaves, while low-level or no obvious transcripts were detected in pericarps and tendrils, respectively. Key words: Vitis quinquangularis, PR-1 gene, Elsinoe ampelina, expression analysis.