Extraction of RNA from plant tissue containing high levels of polyphenols and polysaccharides is tedious and difficult in grapes. Although several protocols have been published for plant RNA isolation, most have failed to yield high quality RNA in sufficient quantity from mature and diseased grape tissue. We describe a protocol for isolating intact and high quality RNA from various grape tissues as evident by high A 260 /A 280 absorbance ratio (1.8 to 1.9) and electrophoretic profile on denaturing formaldehyde agarose gel. On an average, 205 µg RNA per g of fresh tissue were obtained using this modified protocol. RNA quality was further *Corresponding author assessed through RT-PCR, differential display RT-PCR and subtractive hybridization, and found to be suitable for molecular studies.

Pierce's disease (PD) is a destructive bacterial disease of grapes caused by Xylella fastidiosa which is xylem-confined. The tolerance level to this disease varies among Vitis species. Our research was aimed at identifying unique xylem sap proteins present in PD-tolerant Vitis species. The results showed wide variation in the xylem sap protein composition, where a set of polypeptides with pI between 4.5 and 4.7 and M(r) of 31 kDa were present in abundant amount in muscadine (Vitis rotundifolia, PD-tolerant), in reduced levels in Florida hybrid bunch (Vitis spp., PD-tolerant) and absent in bunch grapes (Vitis vinifera, PD-susceptible). Liquid chromatography/mass spectrometry/mass spectrometry analysis of these proteins revealed their similarity to beta-1, 3-glucanase, peroxidase, and a subunit of oxygen-evolving enhancer protein 1, which are known to play role in defense and oxygen generation. In addition, the amount of free amino acids and soluble sugars was found to be significantly lower in xylem sap of muscadine genotypes compared to V. vinifera genotypes, indicating that the higher nutritional value of bunch grape sap may be more suitable for Xylella growth. These data suggest that the presence of these unique proteins in xylem sap is vital for PD tolerance in muscadine and Florida hybrid bunch grapes.

Muscadine grapes (Vitis rotundifolia Michx) are considered as excellent genetic resources for grape breeding programs as they are known for their hardiness and resistance to pests and diseases. However, contrary to popular belief, our study indicated that not all muscadine cultivars are resistant to anthracnose disease. In order to identify a source of genetic tolerance towards anthracnose among muscadine cultivars, a series of in-situ and ex-situ experiments were conducted through strict and sensitive screening processes. Two consecutive years of field evaluation of 54 grape cultivars showed various levels of anthracnose incidence among the cultivars between a scale of 0 (tolerant) to 5 (highly-susceptible). Resistance bioassay by inoculation of different spore densities of Elsinoë ampelina on 40 cultivars presented similar results and was consistent with those obtained from the field test. A real-time PCR analysis was conducted to investigate differences of gene expression between susceptible and tolerant cultivars and to confirm results by phenotypic identification. Expression of genes encoding chalcone synthase, stilbene synthase, polygalacturonase-inhibiting protein, chitinase and lipid transfer-protein was only detected in tolerant cultivars. Resistant muscadine cultivars identified in this study could be excellent candidates for grape disease resistance breeding programs.

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Florida hybrid bunch grape (Vitis spp.) cultivars with commercial value are susceptible to anthracnose disease caused by the fungi, Elsinoe¨ampelina. The Florida grape industry is keen on improving anthracnose tolerance characteristics of these grape cultivars as the cultivation of European grapes is limited in Southeastern United States due to Pierce's disease incidence. In this connection, a molecular approach was taken to identify and characterize the gene/s differentially expressed upon infestation with E. ampelina in selected Florida hybrid bunch grape cultivars differing in their disease tolerance level. Variation in gene expressions was monitored by employing Differential Display RT-PCR. The results showed significant up-regulation of transcripts ranging between 200 and 800 bp in anthracnose-tolerant (cvs. Blue Lake and Lake Emerald) grape cultivars compared to anthracnose-susceptible (cvs. Suwannee and Blanc du Bois) cultivars. The transcripts that were uniquely induced in tolerant cultivars were identified as chitinase, stilbene synthase, protein/sugar kinases and transcriptional factor. Expression of chitinase and stilbene synthase genes was validated using real time PCR analysis. The results showed that these genes were more rapidly expressed in tolerant cultivars within a day after inoculating with E. ampelina and peaking by day 3, compared to susceptible cultivars. Induction of these antifungal genes appears to enable tolerant cultivars to withstand fungal infestation compared to susceptible cultivars. Rapid expression of chitinase and stilbene synthase genes along with others genes may be partly responsible for anthracnose-tolerance in Florida hybrid bunch grapes.Further analysis may shed light on their specific role in anthracnose tolerance.

Anthracnose is a major disease in Florida hybrid bunch grapes, caused by a fungus viz. Elsinoe ampelina. Florida hybrid bunch grapes are grown in southeastern USA for their superior wine characteristics. However, the effect of anthracnose on grape productivity and wine quality is a major concern to grape growers. Our research is aimed at determining biochemical basis of anthracnose tolerance in Florida hybrid bunch grape. Leaf samples were collected from the plants infected with E. ampelina at different periods and analyzed for differential protein expression using high throughput two-dimensional gel electrophoresis. Among the 32 differentially expressed leaf proteins, two were uniquely expressed in tolerant genotypes in response to E. ampelina infection. These proteins were identified as mitochondrial adenosine triphosphate synthase and glutamine synthetase, which are known to play a major role in carbohydrate metabolism and defense. Several proteins including ribulose 1-5 bisphosphate-carboxylase involved in photosynthesis were found to be suppressed in susceptible genotypes compared to tolerant genotypes following E. ampelina infection. The results indicate that the anthracnose-tolerant genotypes have the ability to up-regulate and induce new proteins upon infection to defend the invasion of the pathogen as well as maintain the normal regulatory processes.

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Among grape species muscadines are the important grape crops of the southeastern United States,where cultivation of Vitis vinifera is limited due to Pierce's disease.Muscadine grapes are less desirable because of their low sugar content and leathery skin. Sugar accumulation in berry depends on the activity of sugar metabolizing enzymes during ripening. The present study was carried out to investigate differences in invertase activity among Muscadinia rotundifolia (Muscadine), Vitis spp. (Florida hybrid bunch) and V. vinifera (Bunch) grape genotypes and to characterize muscadine invertase/s. Bunch grape showed highest invertase activity followed by Florida hybrid bunch and muscadine. Muscadine invertase showed a pH optimum between 2.5 and 3.5 and substrate optimum at 150 mM. These data suggest that low sugar content of muscadine berry may be related to its low invertase activity, and enhancement of invertase activity may be one way to increase the sugar content of muscadine grape. PRACTICAL APPLICATIONSSugar accumulation is an important event in the berry-ripening physiology of grapevine, and is an important characteristic essential for superior enological characteristics and product value. Sugars are synthesized in leaves as a result of photosynthesis and imported into the berry in the form of sucrose. Any limitations or restrictions in sucrose breakdown can adversely affect the sugar content and composition of grape berry as well as its quality. Our studies has revealed that one of the key enzyme acid invertase required for sucrose breakdown is low in muscadine grape compared to bunch and Florida hybrid bunch grapes indicating a positive correlation between invertase activity and berry sugar content. The data also suggest that increasing acid invertase activity may be a way to increase sugar content of muscadine grape to improve its enological characteristics.