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Grapevine vein clearing virus (GVCV) causes severe stunting and death of cultivated grapevines and is prevalent in native Vitis spp. and Ampelopsis cordata in the Midwest region of the United States. GVCV can be transmitted from wild A. cordata to Vitis spp. by grape aphid (Aphis illinoisensis) under greenhouse conditions, but its prevalence, genetic composition, and genome number in native grape aphids are unknown. In this study, we collected grape aphids from native Vitaceae across the state of Missouri in 2018 and 2019, and conducted diagnostic, genetic, and quantitative analyses. GVCV was detected in 91 of the 105 randomly sampled communities on 71 Vitaceae plants (87%). It was present in 211 of 525 single grape aphids (40%). Diverse GVCV variants from aphids were present on both GVCV-negative and GVCV-positive plants. Identical GVCV variants were found in grape aphids sampled from wild and cultivated Vitaceae, indicating that viruliferous aphids likely migrate and disperse GVCV variants among wild and cultivated Vitaceae. In addition, we found that the number of GVCV genomes varies largely in the stylet and body of individual aphids. Our study provides a snapshot of GVCV epidemics and genetic structure in its mobile vector and sessile hosts. This presents a good model for studying the epidemiology, ecology, and evolution of a plant virus.

Grapevines are frequently infected by multiple viruses. Our previous study showed that Norton grapevine (Vitis aestivalis) is resistant to grapevine vein clearing virus (GVCV), a DNA virus in the family Caulimoviridae. To study the reaction of Norton to RNA viruses, we transferred seven RNA viruses to Norton from Kishmish Vatkana (KV) (V. vinifera) via graft transmission. We profiled viral small RNAs (vsRNAs) of the seven viruses and compared viral titers in Norton and KV. Total vsRNAs of grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, grapevine virus A (GVA), and grapevine Pinot gris virus (GPGV) were significantly less abundant in Norton than in KV, but total vsRNAs of grapevine fleck virus (GFkV) were more abundant in Norton than in KV. Total vsRNAs of grapevine rupestris stem pitting-associated virus (GRSPaV) were not different between Norton and KV. Grafting direction of Norton to KV or KV to Norton did not affect the quantity of vsRNAs. The genome coverage of GLRaV-1, GLRaV-2, GLRaV-3, and GVA vsRNAs was lower in Norton than KV. The 21- and 22-nucleotide classes of vsRNAs were predominant for all seven viruses. Virus quantification by quantitative PCR indicated that GLRaV-1 was undetectable in Norton; GLRaV-2, GLRaV-3, and GVA were less abundant in Norton; and GFkV was more abundant in Norton than in KV. These results demonstrated that Norton grapevine suppresses GLRaV-1, GLRaV-2, GLRaV-3, and GVA but supports GFkV in comparison with KV. This study revealed new facets of complex molecular interactions between grapevines and multiple viruses.

Grapevines (Vitis spp.) host viruses belonging to 17 families. Virus-associated diseases are a constant challenge to grape production. Genetic resources for breeding virus-resistant grape cultivars are scarce. ‘Norton’ is a hybrid grape of North American Vitis aestivalis and is resistant to powdery mildew and downy mildew. In this study, we assessed resistance of ‘Norton’ to grapevine vein clearing virus (GVCV), which is prevalent in native, wild Vitaceae and in vineyards in the Midwest region of the U.S. We did not detect GVCV in ‘Norton’ as either the scion or the rootstock up to 3 years after it was grafted with a GVCV-infected ‘Chardonel’ grapevine. Upon sequencing of small RNAs, we were able to assemble the GVCV genome from virus small RNAs in GVCV-infected ‘Chardonel’ scion or rootstock, but not from grafted ‘Norton’ scion and rootstock. This study unveils a new trait of ‘Norton’ that can be used in breeding GVCV-resistant grape cultivars, and to investigate genetic mechanisms of ‘Norton’ resistance to GVCV.

Among the grapevine viruses recently identified, grapevine vein clearing virus (GVCV), grapevine Pinot gris virus (GPGV) and grapevine red blotch virus (GRBV) have emerged in the last decade as new threats to the grape and wine industry. Initially reported in Italy (GPGV) and the USA (GVCV and GRBV), GPGV and GRBV have a wider distribution at present, likely as a result of an extensive exchange of infected, propagative material, while GVCV seems to remain restricted to the mid-western regions in the USA. Much progress has been made on the ecology of these three emerging viruses since the last reviews were published in 2017 (Cieniewicz et al. 2017a; Qiu and Schoelz 2017; Saldarelli et al. 2017). Here we compile and critically analyze the latest information on these three viruses with a special emphasis on the (i) association between the genetic make-up of GPGV isolates and chlorotic mottling and leaf deformation symptoms, (ii) epidemiological and ecological attributes of GVCV and GRBV, and (iii) impacts of GRBV, particularly on molecular underpinnings of fruit ripening physiological pathways. Common trends among these three emerging grapevine viruses but also some unique characteristics are highlighted. Finally, we conclude on how critical it is to embrace 'believing is seeing' in the case of emerging grapevine viruses to remain relevant and impactful in providing research-based management solutions, while leading a sustained dialogue with grape grower's communities, extension educators, policy makers and regulatory authorities.

Maize (Zea mays) and itch grass (Rottboellia cochinchinensis) plants exhibiting a mild mosaic or mottle were collected from a farmer's field near Mokwa, Nigeria, in 1993. Icosahedral virions (approximately 28 to 30 nm) were purified from symptomatic tissue by differential centrifugation in 0.1 M phosphate buffer, pH 7.0. The virions are composed of one single-stranded positive-sense RNA of approximately 4,000 nucleotides and, as estimated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a capsid protein of approximately 28 kDa. The virus was readily detected in infected plants by enzyme-linked immunosorbent assay and immunoblot assays with a polyclonal rabbit antibody derived from purified virions. A partial cDNA library was generated with random primers. Sequence analyses of a cDNA clone representing a portion of the putative replicase gene aligned most closely with related sequences of viruses within the Tombusviridae. In particular, a region of 78 predicted amino acids surrounding the “GDD” replicase motif shares 73% identity with panicum mosaic virus and 61% identity with maize chlorotic mottle virus. The virus is readily transmitted by mechanical inoculation to sweet and dent corn, millet, and wheat. Currently it is not considered of economic importance in Nigeria. The data suggest that “maize mild mottle virus” is a newly identified virus infecting maize.

Grapevine leaf roll disease (GLRD) is one of the most prevalent viral diseases in vineyards worldwide. At least 10 Grapevine leafroll-associated viruses (GLRaV-1 to -7, and -9, -10, and -11) are associated with GLRD. GLRaV-2 has a number of distinct isolates that are associated not only with GLRD, but also with graft incompatibility, young vine decline, and rootstock stem lesions. In the United States, GLRaV-2 isolates have been reported in the states of California (4), New York (1), Oregon, and Washington (3). A survey of grapevine viruses was conducted on seven grape cultivars, Norton, Chambourcin, Chardonel, Vignoles, Vidal Blanc, Traminette, and Cayuga White, in a vineyard located at Mountain Grove, MO during the falls of 2009 and 2010 and late summer of 2011. Vines from each cultivar were assigned into three blocks. From each block, six individual vines were randomly selected. Phloem scrapings were collected for detecting viruses by ELISA and reverse-transcription (RT)-PCR. To test for GLRaV-2, a set of primers, GLRaV-2-Forward (5′-GGTGATAACCGACGCCTCTA, nt 6745 to 6764) and GLRaV-2-Reverse (5′-CCTAGCTGACGCAGATTGCT, nt 7268 to 7287), were designed from the coat protein coding region as reported previously (2). GRLaV-2 was detected only in the samples of ‘Vidal Blanc’. The detection of GLRaV-2 was further verified by ELISA using GRLaV-2-specific antisera (Bioreba AG, Reinach, Switzerland). The GLRaV-2-infected ‘Vidal Blanc’ vines did not exhibit visible symptoms. The RT-PCR amplified 543-bp cDNA fragments were isolated from agarose gel and cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). Plasmid DNA was purified from three individual clones and sequenced from both directions. Comparison of the 543-bp sequences showed that the sequences shared 99% nt identity with the corresponding regions of 21 GLRaV-2 isolates, including PMC-083 isolate from Croatia (GenBank Accession No. HM185277), LN isolate from China (GenBank No. FJ786017), SE isolate from Brazil (GenBank No. EU204909), and four isolates from Washington (GenBank Nos. EU760836, 760838, 760843, and 760848). ‘Vidal Blanc’ is a white interspecific hybrid grape. Absence of visible symptoms on this cultivar emphasizes the importance of planting certified grapevines that have been indexed for viruses. Discovery of GLRaV-2 in asymptomatic white grape cultivars will warn growers on the potential damages and epidemics of GLRD in Midwest vineyards and promote planting clean grapevines. To our knowledge, this is the first report of GLRaV-2 in grapevines in Missouri. References: (1) M. Fuchs et al. Plant Dis. 93:395, 2009. (2) G. Gambino and I. Gribaudo. Phytopathology 96:1223, 2006. (3) R. R. Martin et al. Plant Dis. 89:763, 2005. (4) A. Rowhani et al. Phytopathology (Abstr.) 92:(suppl.):S71, 2002.

Guo, Q., Honesty, S., Xu, M. L., Zhang, Y., Schoelz, J., and Qui, W. 2014. Genetic diversity and tissue and host specificity of Grapevine vein clearing virus. Phytopathology 104:539-547. Grapevine vein clearing virus (GVCV) is a new badnavirus in the family Caulimoviridae that is closely associated with an emerging veinclearing and vine decline disease in the Midwest region of the United States. It has a circular, double-stranded DNA genome of 7,753 bp that is predicted to encode three open reading frames (ORFs) on the plus-strand DNA. The largest ORF encodes a polyprotein that contains domains for a reverse transcriptase (RT), an RNase H, and a DNA-binding zinc-finger protein (ZF). In this study, two genomic regions, a 570-bp region of the RT domain and a 540-bp region of the ZF domain were used for an analysis of the genetic diversity of GVCV populations. In total, 39 recombinant plasmids were sequenced. These plasmids consisted of three individual clones from each of 13 isolates sampled from five grape varieties in three states. The sequence variants of GVCV could not be phylogenetically grouped into clades according to geographical location and grape variety. Codons of RT or ZF regions are subject to purifying selection pressure. Quantitative polymerase chain reaction assays indicated that GVCV accumulates abundantly in the petioles and least in the root tip tissue. Upon grafting of GVCV-infected buds onto four major grape cultivars, GVCV was not detected in the grafted ‘Chambourcin’ vine but was present in the grafted ‘Vidal Blanc’, ‘Cayuga White’, and ‘Traminette’ vines, suggesting that Chambourcin is resistant to GVCV. Furthermore, seven nucleotides were changed in the sequenced RT and ZF regions of GVCV from a grafted Traminette vine and one in the sequenced regions of GVCV from grafted Cayuga White but no changes were found in the sequenced regions of GVCV in the grafted Vidal Blanc. The results provide a genetic snapshot of GVCV populations, which will yield knowledge important for monitoring GVCV epidemics and for preventing the loss of grape production that is associated with GVCV.

Non-expressor of pathogenesis-related genes 1 (NPR1) plays a significant role in the defense responses of plants to pathogens by regulating the expression of defense-related genes. In the present study, we isolated two NPR1 genes from Vitis aestivalis cv. Norton and Vitis vinifera cv. Cabernet Sauvignon, which were referred to as VaNPR1.1 and VvNPR1. 1-CS, respectively. They encode a protein of 584 amino acids with a predicted molecular weight of 64.8 kDa and a theoretical isoelectric point (pI) of 5.74. The predicted amino acid sequences of VaNPR1.1 and VvNPR1.1-CS differ by only one amino acid. Over-expression of VaNPR1.1 gene in Arabidopsis npr1-1 mutant plants restores the transcriptional expression of AtPR-1 gene, though not to the full scale. This result demonstrated that a grapevine VaNPR1.1 possesses a similar function to the Arabidopsis NPR1 in the regulation of defense-related genes. Over-expression of VaNPR1.1 in transgenic Arabidopsis plant increased tolerance to salinity, but had no effect on the drought tolerance. We conclude that VaNPR1.1 is a functional ortholog of AtNPR1 and also involved in grapevine's response to the salt stress.

Grapevine vein clearing virus (GVCV), a new member of the genus Badnavirus in the family Caulimoviridae, is associated with a vein clearing and vine decline disease that severely affects grape production and berry quality in commercial vineyards in the Midwest region of the United States. In this paper, the genetic and phenotypic characteristics of GVCV-VRU1 and GVCV-VRU2, two isolates from wild Vitis rupestris grapevines in their native habitat, are described. The GVCV-VRU1 genome is 7,755 bp long while the GVCV-VRU2 genome consists of 7,725 bp, both of which are different from the genome of the GVCV-CHA isolate (7,753 bp), which was originally discovered in the grape cultivar ‘Chardonel’. The nucleotide sequence identity among GVCV-VRU1, GVCV-VRU2, and GVCV-CHA ranges from 91.6 to 93.4%, and open reading frame (ORF) II is the most divergent ORF with only 83.3 to 88.5% identity. Sequence analysis of the ORF II indicated that GVCV isolates genetically similar to GVCV-VRU1 and GVCV-VRU2 also are present in commercial vineyards. Symptoms of GVCV-VRU1- or GVCV-VRU2-infected wild V. rupestris grapevine appeared initially as translucent vein clearing on young leaves and progressed to vein necrosis on mature leaves. Inoculation of GVCV-VRU1 or GVCV-VRU2 by grafting onto grape cultivar Chardonel resulted in mild mottle and leaf distortion. The natural range of wild V. rupestris grapevines overlaps with commercial vineyards in the Midwestern United States. Therefore, the discovery of GVCV isolates in wild V. rupestris grapevines has important implications for epidemics and management of the GVCV-associated disease.

Stilbenic compounds are natural phytoalexins that have antimicrobial activities in plant defense against pathogens. Stilbene synthase (STS) is the key enzyme that catalyzes the biosynthesis of stilbenic compounds. Grapevine genome contains a family of preliminarily annotated 35 STS genes, the regulation of each STS gene needs to be studied to define their roles. In this study, we selected eight STS genes, STS8, STS27/31, STS16/22, STS13/17/23, and applied quantitative polymerase chain reaction (qPCR) to characterize their transcriptional expression profiles in leaf tissues upon infection by the powdery mildew fungus (PM), Erysiphe necator (Schw.) Burr. Their transcripts were also compared in young and old leaves as well as in the berry skin at five developmental stages in Vitis vinifera 'Cabernet Sauvignon' and Vitis aestivalis 'Norton'. The results showed that transcripts of selected STS genes increased significantly in Cabernet Sauvignon leaves at 24 and 48 h post inoculation with PM spores and remained unchanged in Norton leaves in response to the PM infection. Transcripts of STS8, STS27/31 and STS13/17/23 were more abundant in the old leaves of Norton than in Cabernet Sauvignon. STS genes showed lower expression levels in young leaves than in old leaves. Transcript levels of the eight STS genes increased drastically in the berry skin of Cabernet Sauvignon and Norton post véraison. In addition, the content of trans-resveratrol in the berry skin rapidly increased post véraison and reached the highest level at harvest. These assays demonstrated that individual STS genes are regulated differentially in response to PM infection and during development in the two grape varieties. The present study yields basic knowledge for further investigation of the regulation and function of each STS gene in grapevine and provides experimental evidences for the functional annotation of the STS gene family in the grapevine genome.

A severe vein-clearing and vine decline syndrome has emerged on grapevines (Vitis vinifera) and hybrid grape cultivars in the Midwest region of the United States. The typical symptoms are translucent vein-clearing on young leaves, short internodes and decline of vine vigor. Known viral pathogens of grapevines were not closely associated with the syndrome. To obtain a comprehensive profile of viruses in a diseased grapevine, small RNAs were enriched and two cDNA libraries were constructed from a symptomatic grapevine and a symptomless grapevine, respectively. Deep sequencing of the two cDNA libraries showed that the most abundant viral small RNAs align with the genomes of viruses in the genus Badnavirus, the family Caulimoviridae. Amplification of the viral DNA by polymerase chain reaction allowed the assembly of the whole genome sequence of a grapevine DNA virus, which shared the highest homology with the Badnavirus sequences. This is the first report of a DNA virus in grapevines. The new DNA virus is closely associated with the vein-clearing symptom, and thus has been given a provisional name Grapevine vein clearing virus (GVCV). GVCV was detected in six grapevine cultivars showing vein-clearing and vine decline syndrome in Missouri, Illinois, and Indiana, suggesting its wide distribution in the Midwest region of the United States. Discovery of DNA viruses in grapevines merits further studies on their epidemics and economic impact on grape production worldwide.

Grapevine red blotch is a relatively new disease of grapevines that was first described on ‘Cabernet Sauvignon’ at the Oakville, CA, Research Field Station in 2008. A geminivirus, grapevine red blotch virus (GRBV, genus Grablovirus, family Geminiviridae), has consistently been isolated from symptomatic grapevines (Al Rwahnih et al. 2013), and Koch’s postulates have recently been fulfilled, proving that GRBV is the causal agent of red blotch disease (Yepes et al. 2018). Since its initial characterization, GRBV has been found throughout the United States (Krenz et al. 2014), as well as in germplasm repositories (Al Rwahnih et al. 2015; Thompson et al. 2018). However, the incidence of GRBV in hybrid grapes grown in the U.S. Midwest is not known. In 2016, potted rooted cuttings of a Vitis sp. hybrid ‘Crimson Cabernet’ (Cabernet Sauvignon crossed with ‘Norton’) near Union, MO, displayed typical symptoms of GRBV. Three potted vines were screened by polymerase chain reaction (PCR) for GRBV by Agdia Testing Services (Elkhart, IN), and all three tested positive for GRBV. In July 2017, leaf tissue was collected from eight asymptomatic Crimson Cabernet grapevines from a vineyard located near Hermann, MO. Total nucleic acids were isolated from each of the samples, and a DNA segment of 231 bp corresponding to the predicted coat protein region of GRBV was amplified by PCR from two of the eight samples. The PCR primers were CP-Forward (5′-AGCGGAAGCATGATTGAGACATTGACG-3′, nucleotide positions 1,073 to 1,099) and CP-Reverse (5′-AACGTATGTCCACTTGCAGAAGCCGC-3′, nucleotide positions 1,329 to 1,304). In a follow-up visit to the vineyard in August, we detected GRBV in two out of 10 samples with the set of primers for the CP gene and a second set of primers for the replicase gene, as described in Krenz et al. (2014). In a subsequent visit in late October, we observed red blotch symptoms in many of the vines in this vineyard. An additional 10 samples were collected during the late October visit for PCR analysis: eight samples from symptomatic vines and two from asymptomatic vines of the Crimson Cabernet hybrid cultivar. Seven of the eight symptomatic vines were positive, and one of the asymptomatic vines was also positive for GRBV. The entire GRBV genome sequence was determined for one sample (MO-CC7) and compared with other GRBV sequences by a Basic Local Alignment Search Tool (BLAST). The genome of MO-CC7 (GenBank accession no. MH732736) has a size of 3,205 nucleotides and is similar to GRBV isolates ONRB4 (GenBank accession no. KY316022.1) and 93-26 (GenBank accession no. KY426922), with sequence identities of 99%. A comprehensive survey has been planned to examine the incidence of GRBV in the commonly grown hybrid grapes in Missouri.

Grapevines exhibit a wide spectrum of resistance to the powdery mildew fungus (PM), Erysiphe necator (Schw.) Burr., but little is known about the transcriptional basis of the defense to PM. Our microscopic observations showed that PM produced less hyphal growth and induced more brown-colored epidermal cells on leaves of PM-resistant Vitis aestivalis ‘Norton’ than on leaves of PM-susceptible Vitis vinifera ‘Cabernet sauvignon’. We found that endogenous salicylic acid levels were higher in V. aestivalis than in V. vinifera in the absence of the fungus and that salicylic acid levels increased in V. vinifera at 120 h postinoculation with PM. To test the hypothesis that gene expression differences would be apparent when V. aestivalis and V. vinifera were mounting a response to PM, we conducted a comprehensive Vitis GeneChip analysis. We examined the transcriptome at 0, 4, 8, 12, 24, and 48 h postinoculation with PM. We found only three PM-responsive transcripts in V. aestivalis and 625 in V. vinifera. There was a significant increase in the abundance of transcripts encoding ENHANCED DISEASE SUSCEPTIBILITY1, mitogen-activated protein kinase kinase, WRKY, PATHOGENESIS-RELATED1, PATHOGENESIS-RELATED10, and stilbene synthase in PM-infected V. vinifera, suggesting an induction of the basal defense response. The overall changes in the PM-responsive V. vinifera transcriptome also indicated a possible reprogramming of metabolism toward the increased synthesis of the secondary metabolites. These results suggested that resistance to PM in V. aestivalis was not associated with overall reprogramming of the transcriptome. However, PM induced defense-oriented transcriptional changes in V. vinifera.

Comparative microarray (Vitis GeneChip) analysis in young leaves revealed considerable variation in gene expression between Vitis vinifera L. and Vitis aestivalis Michx. Approximately 12% of the genes were differentially expressed in the two grapevine species (P < 0.001). Over 200 probe sets were identified which consistently detected transcripts in one grapevine species, but not in the other. We were unable to identify any broad functional category in which transcript abundance was overall different in any one species. Of the genes expressed only in V. aestivalis leaves, we identified a class IV chitinase which was previously shown by others to have a flower- and fruit-specific expression in V. vinifera. Among the transcripts which were differentially expressed (P < 0.001) in both species, we identified genes encoding key enzymes in flavonoid, monolignol, and proanthocyanindin biosynthesis. Statistical exploration of the data suggested that sequence divergence between the predominantly V. vinifera-derived GeneChip probes and the V. aestivalis cRNA did not confound the hybridization data and that the reliability of the microarray results was similar in the two grapevine species.

The most economically important disease of cultivated grapevines worldwide is powdery mildew (PM) caused by the ascomycete fungus Erysiphe necator. The majority of grapevine cultivars used for wine, table grape, and dried fruit production are derived from the Eurasian grape species Vitis vinifera because of its superior aroma and flavor characteristics. However, this species has little genetic resistance against E. necator meaning that grape production is highly dependent on the frequent use of fungicides. The integration of effective genetic resistance into cultivated grapevines would lead to significant financial and environmental benefits and represents a major challenge for viticultural industries and researchers worldwide. This review will outline the strategies being used to increase our understanding of the molecular basis of V. vinifera susceptibility to this fungal pathogen. It will summarize our current knowledge of different resistance loci/genes that have evolved in wild grapevine species to restrict PM infection and assess the potential application of these defense genes in the generation of PM-resistant grapevine germplasm. Finally, it addresses future research priorities which will be important in the rapid identification, evaluation, and deployment of new PM resistance genes which are capable of conferring effective and durable resistance in the vineyard.

The molecular interactions between grapevine and the obligate biotrophic fungus Erysiphe necator are not understood in depth. One reason for this is the recalcitrance of grapevine to genetic modifications. Using defense-related Arabidopsis mutants that are susceptible to pathogens, we were able to analyze key components in grapevine defense responses. We have examined the functions of defense genes associated with the salicylic acid (SA) pathway, including ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), EDS1-LIKE 2 (EDL2), EDL5 and PHYTOALEXIN DEFICIENT 4 (PAD4) of two grapevine species, Vitis vinifera cv. Cabernet Sauvignon, which is susceptible to E. necator, and V. aestivalis cv. Norton, which is resistant. Both VaEDS1 and VvEDS1 were previously found to functionally complement the Arabidopsis eds1-1 mutant. Here we show that the promoters of both VaEDS1 and VvEDS1 were induced by SA, indicating that the heightened defense of Norton is related to its high SA level. Other than Va/VvEDS1, only VaEDL2 complemented Arabidopsis eds1-1, whereas Va/VvPAD4 did not complement Arabidopsis pad4-1. Bimolecular fluorescence complementation results indicated that Vitis EDS1 and EDL2 proteins interact with Vitis PAD4 and AtPAD4, suggesting that Vitis EDS1/EDL2 forms a complex with PAD4 to confer resistance, as is known from Arabidopsis. However, Vitis EDL5 and PAD4 did not interact with Arabidopsis EDS1 or PAD4, correlating with their inability to function in Arabidopsis. Together, our study suggests a more complicated EDS1/PAD4 module in grapevine and provides insight into molecular mechanisms that determine disease resistance levels in Vitis species native to the North American continent.

Background The complex and dynamic changes during grape berry development have been studied in Vitis vinifera, but little is known about these processes in other Vitis species. The grape variety 'Norton', with a major portion of its genome derived from Vitis aestivalis, maintains high levels of malic acid and phenolic acids in the ripening berries in comparison with V. vinifera varieties such as Cabernet Sauvignon. Furthermore, Norton berries develop a remarkably high level of resistance to most fungal pathogens while Cabernet Sauvignon berries remain susceptible to those pathogens. The distinct characteristics of Norton and Cabernet Sauvignon merit a comprehensive analysis of transcriptional regulation and metabolite pathways. Results A microarray study was conducted on transcriptome changes of Norton berry skin during the period of 37 to 127 days after bloom, which represents berry developmental phases from herbaceous growth to full ripeness. Samples of six berry developmental stages were collected. Analysis of the microarray data revealed that a total of 3,352 probe sets exhibited significant differences at transcript levels, with two-fold changes between at least two developmental stages. Expression profiles of defense-related genes showed a dynamic modulation of nucleotide-binding site-leucine-rich repeat (NBS-LRR) resistance genes and pathogenesis-related (PR) genes during berry development. Transcript levels of PR-1 in Norton berry skin clearly increased during the ripening phase. As in other grapevines, genes of the phenylpropanoid pathway were up-regulated in Norton as the berry developed. The most noticeable was the steady increase of transcript levels of stilbene synthase genes. Transcriptional patterns of six MYB transcription factors and eleven structural genes of the flavonoid pathway and profiles of anthocyanins and proanthocyanidins (PAs) during berry skin development were analyzed comparatively in Norton and Cabernet Sauvignon. Transcriptional patterns of MYB5A and MYB5B were similar during berry development between the two varieties, but those of MYBPA1 and MYBPA2 were strikingly different, demonstrating that the general flavonoid pathways are regulated under different MYB factors. The data showed that there were higher transcript levels of the genes encoding flavonoid-3'-O-hydroxylase (F3'H), flavonoid-3',5'-hydroxylase (F3'5'H), leucoanthocyanidin dioxygenase (LDOX), UDP-glucose:flavonoid 3'-O-glucosyltransferase (UFGT), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR) 1 and LAR2 in berry skin of Norton than in those of Cabernet Sauvignon. It was also found that the total amount of anthocyanins was markedly higher in Norton than in Cabernet Sauvignon berry skin at harvest, and five anthocyanin derivatives and three PA compounds exhibited distinctive accumulation patterns in Norton berry skin. Conclusions This study provides an overview of the transcriptome changes and the flavonoid profiles in the berry skin of Norton, an important North American wine grape, during berry development. The steady increase of transcripts of PR-1 and stilbene synthase genes likely contributes to the developmentally regulated resistance during ripening of Norton berries. More studies are required to address the precise role of each stilbene synthase gene in berry development and disease resistance. Transcriptional regulation of MYBA1, MYBA2, MYB5A and MYBPA1 as well as expression levels of their putative targets F3'H, F3'5'H, LDOX, UFGT, ANR, LAR1, and LAR2 are highly correlated with the characteristic anthocyanin and PA profiles in Norton berry skin. These results reveal a unique pattern of the regulation of transcription and biosynthesis pathways underlying the viticultural and enological characteristics of Norton grape, and yield new insights into the understanding of the flavonoid pathway in non-vinifera grape varieties.

Vitis vinifera (grapevine) is the most economically important deciduous fruit crop, but cultivated grapevine varieties lack adequate innate immunity to a range of devastating diseases. To identify genetic resources for grapevine innate immunity and understand pathogen defense pathways in a woody perennial plant, we focus in this study on orthologs of the central Arabidopsis thaliana defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). The family of EDS1-like genes is expanded in grapevine, and members of this family were previously found to be constitutively upregulated in the resistant variety 'Norton' of the North American grapevine species Vitis aestivalis, while they were induced by Erysiphe necator, the causal agent of grapevine powdery mildew (PM), in the susceptible V. vinifera variety 'Cabernet Sauvignon'. Here, we determine the responsiveness of individual EDS1-like genes in grapevine to PM and salicylic acid, and find that EDS1-like paralogs are differentially regulated in 'Cabernet Sauvignon', while two are constitutively upregulated in 'Norton'. Sequencing of VvEDS1 and VaEDS1 cDNA and genomic clones revealed high conservation in the protein-encoding sequence and some divergence of the promoter sequence in the two grapevine varieties. Complementation of the Arabidopsis eds1-1 mutant showed that the EDS1-like gene with highest predicted amino acid sequence similarity to AtEDS1 from either grapevine varieties is a functional ortholog of AtEDS1. Together, our analyses show that differential susceptibility to PM is correlated with differences in EDS1 expression, not differences in EDS1 function, between resistant 'Norton' and susceptible 'Cabernet Sauvignon'.

A disease complex was observed on grapevine var. Chardonnay (Vitis vinifera) in a commercial vineyard in Missouri that destroyed the affected vineyard. Conspicuous vein-clearing symptoms on the leaves of originally diseased Chardonnay vines and bud-grafted Chardonnay, V. vinifera ‘Cabernet Franc’, V. vinifera ‘Baco Blanc’, and hybrid ‘LN-33’ vines are characteristics of the disease complex, which is referred to as the grapevine vein-clearing complex (GVCC). By applying reverse-transcription polymerase chain reaction (RT-PCR) using virus-specific primers, we detected combinations of Grapevine fanleaf virus (GFLV), Tomato ringspot virus (ToRSV) and Grapevine rupestris stem pitting-associated virus (GRSPaV), in symptomatic Chardonnay vines. Sequencing of RT-PCR amplified DNA fragments confirmed the identity of each virus, indicating the occurrence of ToRSV yellow vein strain, and two distinct strains of GRSPaV in the GVCC-affected Chardonnay vines. This is the first report of the co-infection of two nepoviruses and GRSPaV in var. Chardonnay. This study demonstrated that mixed infections of grapevine viruses belonging to different taxonomic groups pose a great threat to vineyards under certain climatic and soil conditions.

Pathogen- and host-derived resistance have been shown to suppress infection by many plant viruses. Tomato spotted wilt tospovirus (TSWV) is among these systems; however, it has easily overcome nearly all host resistance genes and has recently been shown to overcome resistance mediated by the TSWV N gene. To better understand the resistance-breaking mechanisms, we have chosen TSWV N gene-derived resistance (TNDR) as a model to study how plant viruses defeat resistance genes. A defined viral population of isolates TSWV-D and TSWV-10, both suppressed by TNDR, was subjected to TNDR selection by serial passage in an N-gene transgenic plant. The genotype analysis demonstrated that the mixed viral population was driven to form a specific reassortant, L10M10SD, in the presence of TNDR selection, but remained as a heterogeneous mixture in the absence of the selection. A genotype assay of 120 local lesion isolates from the first, fourth, and seventh transfers confirmed the shift of genomic composition. Further analysis demonstrated that the individual L10, M10, and SD RNA segments were each selected independently in response to TNDR selection rather than to a mutation or recombination event. Following the seventh transfer on the N-gene transgenic plants, TSWV S RNA remained essentially identical to the S RNA from TSWV-D, indicating that no intermolecular recombination occurred between the two S RNAs from TSWV-10 and TSWV-D nor with the transferred N gene. These results support the hypothesis that TSWV utilizes genome reassortment to adapt to new host genotypes rapidly and that elements from two or more segments of the genome are involved in suppression of the resistance reaction.

A severe virus-like disease emerged on Chardonnay vines in a Missouri vineyard that resulted in significant reduction of vine vigor and yield. The pathogens can be transmitted via cutting and grafting. Chardonnay, Cabernet franc and Baco franc vines that were grafted with the buds of the original diseased Chardonnay vines exhibited distinct vein-clearing symptom. The result of reverse transcription polymerase chain reaction indicated the association of Grapevine fanleaf virus with this disease.Accepted for publication 26 September 2007. Published 19 November 2007.