Grapevine vein clearing virus Is Prevalent and Genetically Variable in Grape Aphid (Aphis illinoisensis Shimer) Populations.

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.

North American Grape 'Norton' is Resistant to Grapevine Vein Clearing Virus.

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.

A Natural Reservoir and Transmission Vector of Grapevine Vein Clearing Virus.

Grapevine vein clearing virus (GVCV) is associated with a vein-clearing and vine-decline disease. In this study, we surveyed wild Ampelopsis cordata from the Vitaceae family and found that 31% (35 of 113) of native A. cordata plants are infected with GVCV. The full-length genome sequence of one GVCV isolate from A. cordata shared 99.8% identical nucleotides with an isolate from a nearby cultivated 'Chardonel' grapevine, suggesting the occurrence of an insect vector. To identify a vector, we collected Aphis illinoisensis (common name: grape aphids) from wild A. cordata plants and detected GVCV in the aphid populations. We found that A. illinoisensis is capable of transmitting GVCV from infected A. cordata to Chardonel grapevines in the greenhouse. Upon transmission, GVCV caused severe symptoms on the infected Chardonel 45 days post transmission. We conclude that wild GVCV isolates from A. cordata are capable of inducing a severe disease on cultivated grapevines once they spread from native A. cordata to vineyards via grape aphids. The discovery of a natural reservoir and an insect vector of GVCV provides timely knowledge for disease management in vineyards and critical clues on viral evolution and epidemiology.

Viral small RNAs reveal the genomic variations of three grapevine vein clearing virus quasispecies populations.

Viral small RNAs (vsRNAs) include viral small interfering RNAs (vsiRNAs) that are initiators and products of RNA silencing, and small RNAs that are derived from viral RNAs with function still unknown. Sequencing of vsRNAs allows assembling of viral genomes and revelation of viral population variations at genomic levels. Grapevine vein clearing virus (GVCV) is a new member of the family Caulimoviridae whose DNA genome is replicated by reverse transcription of pre-genomic RNA molecules. In this short report, three genomic sequences of GVCV were assembled from vsRNAs that were isolated and sequenced from three individual grapevines in commercial vineyards and compared to the GVCV-CHA reference genome. Profiles of single nucleotide polymorphism among three viral populations indicated a closer relatedness between two populations in different grape cultivars at the same location than those in the same grape cultivar at different locations, suggesting the spread of GVCV populations among vineyards of close proximity. Classic types of vsiRNAs (21-nt, 22-nt, and 24-nt) were found in the three GVCV vsiRNA populations, but these did not produce alignment hotspots on the GVCV-CHA reference genome. The number of 36-nt reads is the highest among vsRNAs, the role of these vsRNAs remains unclear. The analysis of vsRNAs provides a first holistic picture of genomic variations among GVCV viral quasispecies populations that help monitor epidemics and evolution of GVCV populations, an emerging virus that is becoming a threat to grape production in the Midwest region of the USA.

Transcriptional expression of Stilbene synthase genes are regulated developmentally and differentially in response to powdery mildew in Norton and Cabernet Sauvignon grapevine.

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.

Berry skin development in Norton grape: distinct patterns of transcriptional regulation and flavonoid biosynthesis.

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.

A functional EDS1 ortholog is differentially regulated in powdery mildew resistant and susceptible grapevines and complements an Arabidopsis eds1 mutant.

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'.

Resistance to Erysiphe necator in the grapevine 'Kishmish vatkana' is controlled by a single locus through restriction of hyphal growth.

Vitis vinifera 'Kishmish vatkana', a cultivated grapevine from Central Asia, does not produce visible symptoms in response to natural or artificial inoculation with the fungus Erysiphe necator Schwein., the casual agent of powdery mildew. 'Kishmish vatkana' allowed pathogen entry into epidermal cells at a rate comparable to that in the susceptible control Vitis vinifera 'Nimrang', but was able to limit subsequent hyphal proliferation. Density of conidiophores was significantly lower in 'Kishmish vatkana' (33.6+/-8.7 conidiophores mm(-2)) than in 'Nimrang' (310.5+/-24.0 conidiophores mm(-2)) by 120 h after inoculation. A progeny of 310 plants from a 'Nimrang 'Kishmish vatkana' cross were scored for the presence or absence of visible conidiophores throughout two successive seasons. Phenotypic segregation revealed the presence of a single dominant allele termed Resistance to Erysiphe necator 1 (REN1), which was heterozygous in 'Kishmish vatkana'. A bulked segregant analysis was carried out using 195 microsatellite markers uniformly distributed across the entire genome. For each marker, association with the resistance trait was inferred by measuring in the bulks the ratio of peak intensities of the two alleles inherited from 'Kishmish vatkana'. The phenotypic locus was assigned to linkage group 13, a genomic region in which no disease resistance had been reported previously. The REN1 position was restricted to a 7.4 cM interval by analyzing the 310 offspring for the segregation of markers that surrounded the target region. The closest markers, VMC9H4-2, VMCNG4E10-1 and UDV-020, were located 0.9 cM away from the REN1 locus.