Yali Zhang

Whole genome wide expression profiles of Vitis amurensis grape responding to downy mildew by using Solexa sequencing technology

BackgroundDowny mildew (DM), caused by pathogen Plasmopara viticola (PV) is the single most damaging disease of grapes (Vitis L.) worldwide. However, the mechanisms of the disease development in grapes are poorly understood. A method for estimating gene expression levels using Solexa sequencing of Type I restriction-endonuclease-generated cDNA fragments was used for deep sequencing the transcriptomes resulting from PV infected leaves of Vitis amurensis Rupr. cv. Zuoshan-1. Our goal is to identify genes that are involved in resistance to grape DM disease.ResultsApproximately 8.5 million (M) 21-nt cDNA tags were sequenced in the cDNA library derived from PV pathogen-infected leaves, and about 7.5 M were sequenced from the cDNA library constructed from the control leaves. When annotated, a total of 15,249 putative genes were identified from the Solexa sequencing tags for the infection (INF) library and 14,549 for the control (CON) library. Comparative analysis between these two cDNA libraries showed about 0.9% of the unique tags increased by at least five-fold, and about 0.6% of the unique tags decreased more than five-fold in infected leaves, while 98.5% of the unique tags showed less than five-fold difference between the two samples. The expression levels of 12 differentially expressed genes were confirmed by Real-time RT-PCR and the trends observed agreed well with the Solexa expression profiles, although the degree of change was lower in amplitude. After pathway enrichment analysis, a set of significantly enriched pathways were identified for the differentially expressed genes (DEGs), which associated with ribosome structure, photosynthesis, amino acid and sugar metabolism.ConclusionsThis study presented a series of candidate genes and pathways that may contribute to DM resistance in grapes, and illustrated that the Solexa-based tag-sequencing approach was a powerful tool for gene expression comparison between control and treated samples.

Phenolic Contents and Compositions in Skins of Red Wine Grape Cultivars among Various Genetic Backgrounds and Originations

In order to analyze and compare the phenolic characteristics of red wine grapes with diverse genetic backgrounds, skin phenolics among 21 different cultivars belonging to Vitis vinifera L., East Asian and North American Vitis species and hybrids, as well as 2 varieties of muscadine grapes were estimated by HPLC-MS/MS. There were 45 anthocyanins, 28 flavonols, 8 flavan-3-ols, 9 cinnamic acids, 5 benzoic acids, 5 ellagic acids and 2 stilbenes detected in all the samples. Total contents of each phenolic type varied significantly among the different grape cultivars investigated. There was also a large variability in the phenolic compositions of different grape groups. The differences in anthocyanin composition were obvious between V. vinifera and non-V. vinifera grapes and also between the grapes originating from Eurasia and North America. Quercetin-3-glucuronide and quercetin-3-glucoside were marker flavonol compounds for Euvitis grape skins. Flavan-3-ol monomers were dominant in the skins of muscadine and non-V. amurensis East Asian grapes, whereas polymers were more common in V. vinifera and North American grapes. The muscadine grapes were very rich in flavonols, flavan-3-ols and ellagic acids. Via principal component analysis, these grape cultivars were clustered into three groups according to their characteristic phenolic content and composition.

Influence of growing season on phenolic compounds and antioxidant properties of grape berries from vines grown in subtropical climate.

The influence of growing season (winter vs summer) on the synthesis and accumulation of phenolic compounds and antioxidant properties was studied in five grape cultivars for three consecutive years. Four phenolic compound parameters (total phenols, flavonoids, flavan-3-ols, and anthocyanins) and three antioxidant property parameters [2,2-diphenyl-1-picrylhydrazyl radical scavenging, 2,2-azinobis(3-ethylbenzothiazolinesulfonic acid) radical scavenging, and ferric reducing antioxidant power] were investigated. Results showed that both phenolic compounds and antioxidant properties in the seed and skin of winter berries were significantly (p < 0.05) higher than those of summer berries for all of the cultivars investigated. The anthocyanin profiles of berry skins appeared to be extremely consistent in different years for the same crop, whereas they varied greatly between the two crops within the same year (winter vs summer). Winter berries contained richer glucosides of delphinidin, cyanidin, peonidin, and malvidin than summer berries. These seasonal variations of phenolic compounds and antioxidant properties on grape berries were largely contributed by climatic factors such as temperature, solar radiation, rainfall, and hydrothermic coefficient between different growing seasons.

The Mode of Host Resistance to Plasmopara viticola Infection of Grapevines

ABSTRACT The resistance and susceptibility of grapevines to downy mildew (DM) disease caused by Plasmopara viticola were compared among different cultivars/accessions belonging to Vitis vinifera, V. rotundifolia, and 10 oriental Vitis species. After inoculation with P. viticola pathogen, no symptom was found in V. rotundifolia grapevines at all, while oriental species V. davidii and V. piasezkii, like V. vinifera, were susceptible to DM disease. The other eight oriental Vitis species showed various resistance levels to DM disease. Intraspecific resistant variations were also observed in V. amurensis. Microscopy studies were conducted on various time courses after pathogen infection on grape leaves. P. viticola hyphae were not observed in V. rotundifolia cultivars, while symptoms with varying degrees of severity were observed among the Euvitis species. In general, the DM resistant oriental species showed a slower development of hypha and less formation of haustoria than DM susceptible V. vinifera grapevines. Cells with distinctive fluorescence were observed in V. rotundifolia and the oriental species V. pseudoreticulata, and callose deposits were observed in V. rotundifolia, V. pseudoreticulata, and V. amurensis grapevines. Based on the results of morphological observations and microscopy studies, we concluded that there were five levels of grapevine resistance to P. viticola pathogen: (i) immune, (ii) extremely resistant, (iii) resistant, (iv) partly resistant, and (v) susceptible.

Phenolic concentrations and antioxidant properties of wines made from north american grapes grown in china.

The characteristics of wine phenolics found in several North American and (for comparison) European grape cultivars grown in China were analyzed. This was done to find non-Vitis vinifera wines with prominent features in order to diversify the kinds of wines. The phenolic richness and antioxidant activity decreased in the order: red > rose > white wines. In the red wines, the American grape ‘Cynthiana’ had the highest total concentrations of phenols, anthocyanins, flavonols and phenolic acids, as well as antioxidant capacity, followed by the French hybrid ‘Chambourcin’, the lowest were detected in two European grape varieties, ‘Merlot’ and ‘Cabernet Sauvignon’, while the total flavon-3-ols levels were reversed among these red grape cultivars. The highest concentration of stilbenes out of all the wines analyzed was found in the ‘Merlot’ variety. There were significant differences among wine phenolic compositions between North American and European grape cultivars. The antioxidant activities were significantly related to the concentrations of total phenols (r2 = 0.996), anthocyanins (r2 = 0.984), flavonols (r2 = 0.850) and gallic acid (r2 = 0.797). The prominent features of wine aroma and nutrition could make the American grape wines attractive to consumers. It is therefore necessary to perform further research on cultural practices and wine making involving these grapes.

Studying the Mechanism of Plasmopara viticola RxLR Effectors on Suppressing Plant Immunity

The RxLR effector family, produced by oomycete pathogens, may manipulate host physiological and biochemical events inside host cells. A group of putative RxLR effectors from Plasmopara viticola have been recently identified by RNA-Seq analysis in our lab. However, their roles in pathogenesis are poorly understood. In this study, we attempted to characterize 23 PvRxLR effector candidates identified from a P. viticola isolate “ZJ-1-1.” During host infection stages, expression patterns of the effector genes were varied and could be categorized into four different groups. By using transient expression assays in Nicotiana benthamiana, we found that 17 of these effector candidates fully suppressed programmed cell death elicited by a range of cell death-inducing proteins, including BAX, INF1, PsCRN63, PsojNIP, PvRxLR16 and R3a/Avr3a. We also discovered that all these PvRxLRs could target the plant cell nucleus, except for PvRxLR55 that localized to the membrane. Furthermore, we identified a single effector, PvRxLR28, that showed the highest expression level at 6 hpi. Functional analysis revealed that PvRxLR28 could significantly enhance susceptibilities of grapevine and tobacco to pathogens. These results suggest that most P. viticola effectors tested in this study may act as broad suppressors of cell death to manipulate immunity in plant.

Effect of suppression of ethylene biosynthesis on flavor products in tomato fruits

To elucidate the role of ethylene in the production of flavor compounds by tomato fruits, wild-type tomato (Lycopersicon esculentum L., cv. Lichun) and its transgenic antisense LeACS2 line with suppressed ethylene biosynthesis were used. The metabolism of individual sugars was ethylene-independent. However, citric acid and malic acid were under ethylene regulation. The content of these acids was higher in transgenic tomato fruits and returned to normal level after transgenic fruits were treated with ethylene. Because most of amino acids, which are important precursors of volatiles, were shown to be correlated with ethylene, we surmise that amino acid-related aroma volatiles were also affected by ethylene. Headspace analysis of volatiles showed a significant accumulation of aldehydes in wild-type tomato fruits during fruit ripening and showed a dramatic decrease in most aroma volatiles in transgenic tomato fruits as compared with wild-type fruits. The production of hexanal, hexanol, trans-2-heptenal, cis-3-hexanol, and carotenoid-related volatiles, except β-damascenone and β-ionone, was inhibited by suppression of ethylene biosynthesis. No remarkable differences were observed in the concentrations of cis-3-hexenal and trans-2-hexenal between transgenic and wild-type tomato fruits, indicating these two volatiles to be independent of ethylene. Thus, there are various regulation patterns of flavor profiles in tomato fruits by ethylene.

Overexpression of pathogen-induced grapevine TIR-NB-LRR gene VaRGA1 enhances disease resistance and drought and salt tolerance in Nicotiana benthamiana

The NBS-LRR proteins encoded by the majority of R genes represent important intracellular receptors that directly or indirectly recognize pathogen effector proteins, which subsequently activate plant defense responses. In this study, a novel Plasmopara viticola-induced TIR-NBS-LRR gene, named VaRGA1, was cloned from leaf tissues of a highly downy mildew-resistant Vitis amurensis “Shuanghong” grapevine. The fluorescence signal of the VaRGA1-GFP fusion protein was clearly partitioned to the cytoplasm and nucleus. The expression of the VaRGA1 gene was strongly induced during early stages of infection by P. viticola, and was also significantly upregulated after drought and salt treatments. Accordingly, grapevine leaves transiently expressing the VaRGA1 gene manifested increased resistance to P. viticola, and the overexpression of the VaRGA1 gene in Nicotiana benthamiana conferred enhanced resistance to Phytophthora parasitica through the activation of salicylic acid (SA) signaling and phenylpropanoid pathways and could also increase tolerance to drought and salt stresses at the germination and vegetable growth stages. These findings indicate that the grapevine VaRGA1 gene may function as the immune and non-immune receptors against biotic and abiotic stresses and that there may be signaling overlap between biotic and abiotic responses.

Isolation of a WRKY30 gene from Muscadinia rotundifolia (Michx) and validation of its function under biotic and abiotic stresses

WRKY transcription factors (TFs) play important roles in many plant processes, including responses to biotic and abiotic stresses. In the present study, Muscadinia rotundifolia MrWRKY30 dramatically accumulated in grapevine leaves in response to inoculation of Plasmopara viticola, a pathogen causing grapevine downy mildew disease. Similar responses were also found on grapevines treated with exogenous SA/JA/ET. Ectopic expression of MrWRKY30 in Arabidopsis thaliana “COL0” enhanced its resistance to downy mildew pathogen Peronospora parasitica. Pathogenesis-related (PR) genes, including AtPR1, AtPR4, AtPR5, and AtPDF1.2, were significantly upregulated in transgenic A. thaliana after P. parasitica inoculation. In the mean time, two critical genes in SA and JA signaling pathways, AtEDS5 and AtJAR1, were abundantly expressed as well, indicating that MrWRKY30 may enhance disease resistance of A. thaliana through SA and JA defense system. The transgenic A. thaliana plants also enhanced tolerance to cold stress accompanied with upregulation of AtCBF1, AtCBF3, AtICE1, and AtCOR47. MrWRKY30 might protect A. thaliana from cold damage by activating the AtCBF-mediated signaling pathway to induce the downstream AtCOR47 gene. Interestingly, the transgenic seedlings had a negative effect on salt tolerance. Reverse transcription PCR (RT-PCR) analysis revealed that antioxidant enzyme genes AtAPX (ascorbate peroxidase), AtCAT (catalase), and AtGST (glutathione-S-transferase) were suppressed in transgenic plants, which may lead to reactive oxygen species (ROS)-mediated sensitivity to salt stress.

Overexpression of a thaumatin-like protein gene from Vitis amurensis improves downy mildew resistance in Vitis vinifera grapevine

Downy mildew is a highly destructive disease in grapevine production. A gene encoding pathogenesis-related (PR) thaumatin-like protein was isolated from the downy mildew-resistant grapevine “Zuoshan-1,” a clonal selection from wild Vitis amurensis Rupr. The predicted thaumatin-like protein (VaTLP) has 225 amino acids and it is acidic, with a calculated isoelectric point of 4.8. The full length of the VaTLP gene was transformed into somatic embryogenic calli of V. vinifera ‘Thompson Seedless’ via Agrobacterium tumefaciens. Real-time RT-PCR confirmed that the VaTLP gene was expressed at a high level in the transgenic grapevines. Improved resistance of the transgenic lines against downy mildew was evaluated using leaf disks and whole plants inoculated with Plasmopara viticola, the pathogen causing grapevine downy mildew disease. Bioassay of the pathogen showed that both hyphae growth and asexual reproduction were inhibited significantly among the transgenic plants. Histological analysis also confirmed this disease resistance by demonstrating the inhibition and malformation of hyphae development in leaf tissue of the transgenic plants. These results indicated that the accumulation of VaTLP could enhance resistance to P. viticola in transgenic ‘Thompson Seedless’ grapevines.