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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 (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.

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.

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.

Powdery mildew is the most economically important disease of cultivated grapevines worldwide. In the agricultural community, there is a great need for better understanding of the complex genetic basis of powdery mildew (PM) resistance by delineating possible gene biomarkers associated with the plants' defense mechanisms. Machine learning techniques can be applied to analysis of gene expression data to aid knowledge discovery of disease fighting genes. In this work, we apply a data-driven computational model, utilizing a graph-based clustering algorithm - Node-Based Resilience Clustering (NBR- Clust), to analyze grapevine gene expression data to identify possible gene biomarkers associated with powdery mildew disease defense mechanisms. We investigated two graph representations (geometric and kNN) on the mean differences of PM inoculated vs. mock inoculated gene expression values of Cabernet and Norton (PM disease resistant) species across 6 time points. By applying the contrarian approach, we hypothesized that smaller sized clusters will contain genes that do not follow general patterns, hence, could display distinct expression patterns of PM- induced transcripts across the time points that may insinuate biological relevance. We compared the smaller clusters obtained in Norton in contrast with the ones from Cabernet in terms of the genes that clustered in common between both (intersection of sets) as well as the differences of the sets. The results obtained demonstrate the usefulness of the geometric graphs for this domain application in contrast to the kNN graphs. Some genes that belong to biologically relevant pathways were identified that displayed differences in patterns across the time points between Norton and Cabernet species.

Grapevine vein clearing virus (GVCV) is a recently discovered DNA virus in grapevine that is closely associated with the grapevine vein clearing syndrome observed in vineyards in Missouri and surrounding states. The genome sequence of GVCV indicates that it belongs to the genus Badnavirus in the family Caulimoviridae. To identify the GVCV promoter, we cloned portions of the GVCV large intergenic region in front of a GFP gene present in an Agrobacterium tumefaciens binary vector. GFP expression was assessed by ELISA 3 days after agroinfiltration of Nicotiana benthamiana leaves. We found that the GVCV DNA segment between nts 7332 and 7672 directed expression of GFP and this expression was stronger than expression using the Cauliflower mosaic virus 35S promoter. It was revealed by 5' and 3' RACE that transcription was initiated predominantly at nt 7571 and terminated at nt 7676.

The molecular interactions between grapevine and the obligate biotrophic fungus Erysiphe necator have not been understood in depth because of the recalcitrance of grapevine to genetic modifications. Defense-related mutants have been available for Arabidopsis thaliana, which is an excellent model for elucidating molecular mechanisms underlying host-pathogen interactions. Arabidopsis mutants that are susceptible to pathogens provide valuable genetic tools for grapevine gen¬eticists to identify key components in perception, signal transduction and regulatory circuits in the mounting of defense responses in grapevine. We have employed these mutants to verify the functions of salicylic acid (SA)-mediated defense genes including Enhanced Disease Susceptibility1 (EDS1) of grapevine, and to characterize the functionality of promoters in response to SA and E. necator. In addition, we are using the Arabidopsis npr1-1 mutant to confirm the regulatory role of grapevine NPR1 in the activation of defense-related genes. With these results, we begin to get a glimpse of molecular events that determine disease resistance levels in Vitis species native to the North American continent. Our results demonstrate the utility of Arabidopsis genetic resources in understanding the genetic mechanism of grapevine disease resistance. Currently we are applying these mutants in the genetic dissection of grapevine defense components and in verifying the roles of defense-related genes that have been discovered by functional genomics analysis of grapevine-pathogen interactions.

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.

The paper presents a new reliability-evaluation method to distribution system, based on interval analysis. In this method, the available capacity of feeder is defined and is considered when evaluation the load-point reliability. By utilizing the minimal-path method, the element is divided treatment principle is established. The basic interval formulas for the load point are presented, considering the uncertainty of the time-varying. The procedure is illustrated by application to a relatively simple but practical system example.

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) is a recently discovered DNA virus that is closely associated with a severe disease that poses a great threat to the sustainable growth and productivity of grapevines in the Midwest region of the USA. The most damaged vineyards have been removed because of GVCV infection. Diagnostic symptoms are translucent vein clearing along the second and tertiary veins on young leaves and mosaic patterns on mature leaves of the affected grapevine. GVCV genome comprises a circular, double-stranded DNA, which is characteristic of the viruses in the genus Badnavirus, family Caulimoviridae. Three large open reading frames (ORFs) are predicted on the plus-strand of the genome. The promoter region and transcription start and termination sites have been mapped. GVCV is replicated through transcription of a terminally redundant transcript as other members of the Caulimoviridae family. Increasing incidences of GVCV on grapevines over the last few years suggest the transmission of the virus by a vector, whose identity is still under investigation. Two new isolates of GVCV were also found in wild grapevines native in Missouri (USA). The reservoirs of genetically complex GVCV populations in wild grapevines create challenges to the management of GVCV-associated disease. The rising prevalence of GVCV and the severity of the emerging disease with which this virus is associated with warrant that it should be tested routinely in the grapevine certification program.

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.

Recombinant plant viruses have the propensity to remove foreign inserts during replication. This process is virusspecific and occurs in a host-dependent manner. In the present study, we investigated the integrity of foreign inserts in recombinant plant viruses using a model system consisting of Tomato bushy stunt virus (TBSV) and its defective interfering RNA (DI). These were tested in Nicotiana benthamiana plants that were either wild type or transgenic for the green fluorescent protein (GFP) gene. GFP-derived inserts were retained in the recombinant TBSV and DI population that were inoculated onto GFPtransgenic N. benthamiana plants in which silencing of the GFP transgene was initiated, but they were removed from the virus and DIs that were maintained on wild-type plants. A foreign insert derived from an endogenous N. benthamiana gene encoding the H subunit of the magnesium chelatase (NbChlH) was deleted, whereas the fragment of an RNA-dependent RNA polymerase gene (NbRdRP1m) was retained in the recombinant TBSV population. These results demonstrate that the recombination of TBSV to remove nonviral fragments is influenced by silencing and the type of inserts.

The p19 protein (P19) of Tomato bushy stunt virus (TBSV) is a pathogenicity determinant with host-dependent effects on virus spread and symptom induction. In addition, results in this study confirm that Potato virus X-mediated delivery of P19 suppresses posttranscriptional gene silencing (PTGS). To study the relevance of this activity for TBSV biology, we evaluated whether TBSV activates virus-induced gene silencing (VIGS) and if this process is suppressed by P19. TBSV vectors with the green fluorescent protein (GFP) gene, either active or inactive for P19 expression, were inoculated onto GFP-transgenic Nicotiana bentha-miana plants. In the absence of P19 expression, VIGS was activated, as evidenced by the disappearance of GFP mRNA and green fluorescence. Coexpression of GFP and P19 from the TBSV vector suppressed VIGS, except in the newly emerging leaves. The suppressor activity required a central P19 region that is also known to be essential for host-dependent virus spread and symptom induction. Defective interfering RNAs (DIs) that contained the 3′ end of the GFP gene induced silencing very effectively. The concomitant DI-instigated reduction in P19 accumulation failed to suppress this process, analogous to the known P19 dosage effects for other biological activities. In conclusion, (i) TBSV and its DIs are very effective inducers of VIGS, (ii) P19 is a strong suppressor of PTGS, (iii) P19 is a moderate suppressor of VIGS, and (iv) the suppressor activity is influenced by genetic and dosage features that are also important for P19-associated pathogenesis.

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.

Accumulation of RNA of Tomato bushy stunt virus (TBSV) was examined within the first few hours after infection of Nicotiana benthamiana protoplasts to determine the influence of the coat protein (CP), the movement-associated proteins P22 and P19 and RNA sequences at very early stages of replication. The results showed that P19 had no effect on early RNA replication, whereas the absence of CP and/or P22 expression delayed RNA accumulation only marginally. Removal of CP-coding sequences had no added negative effects, but when the deletion extended into the downstream p22 gene, it not only eliminated synthesis of subgenomic RNA2 but also delayed accumulation of genomic RNA by 10 h. At times beyond 20 h post-transfection, RNA accumulated to normal high levels for all mutants. This illustrates that TBSV RNA sequences that have negligible impact on overall RNA levels observed late in infection can actually have pronounced effects at very early stages.

Panicum mosaic virus (PMV) is a recently molecularly characterized RNA virus with the unique feature of supporting the replication of two subviral RNAs in a few species of the family Gramineae . The subviral agents include a satellite RNA (satRNA) that is devoid of a coding region and the unrelated satellite panicum mosaic virus (SPMV) that encodes its own capsid protein. Here we report the association of this complex with a new entity in the RNA world, a defective-interfering RNA (DI) of a satellite virus. The specificity of interactions governing this four-component viral system is illustrated by the ability of the SPMV DIs to strongly interfere with the accumulation of the parental SPMV. The SPMV DIs do not interfere with PMV satRNA, but they do slightly enhance the rate of spread and titer of PMV. The SPMV-derived DIs provide an additional avenue by which to investigate fundamental biological questions, including the evolution and interactions of infectious RNAs.

A viral genetic system was used to map the determinants of the ability of Tomato spotted wilt virus (TSWV) to overcome the R gene (Sw-5) in tomato and the resistance conferred by the nucleocapsid gene of TSWV (N gene) in tobacco. A complete set of reassortant genotypes was generated from TSWV isolates A and D. TSWV-A was able to overcome the Sw-5 gene in tomato and the TSWV N gene in tobacco, whereas TSWV-D was repressed by both forms of resistance. The ability to overcome both forms of resistance was associated with the M RNA segment of TSWV-A (MA). Overcoming the Sw-5 gene was linked solely to the presence of MA, to overcome the TSWV N gene was modified by the L RNA and the S RNA of TSWV-A, which is consistent with previous reports that suggest that the nucleocapsid gene is not the primary determinant for overcoming the nucleocapsid-mediated resistance. Sequence analysis of the M RNA segment of TSWV-A, -D, and the type isolate BR-01 revealed multiple differences in the coding and noncoding regions, which prevented identification of the resistance-breaking nucleotide sequences.

Satellite panicum mosaic virus (SPMV) depends on its helper virus, panicum mosaic virus (PMV), to provide trans -acting proteins for replication and movement. The 824-nucleotide (nt) genome of SPMV possesses an open reading frame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensable for SPMV replication. To localize cis -acting RNA elements required for replication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3′ untranslated region (UTR) plus 73 nt upstream of the CP stop codon and the first 16 nt in the 5′ UTR are required for SPMV RNA amplification and/or systemic spread. A region from nt 17 to 67 within the 5′ UTR may have an accessory role in RNA accumulation, and a fragment bracketing nt 68 to 104 appears to be involved in the systemic movement of SPMV RNA in a host-dependent manner. Unexpectedly, defective RNAs (D-RNAs) accumulated de novo in millet plants coinfected with PMV and either of two SPMV mutants: SPMV-91, which is incapable of expressing the 17.5-kDa CP, and SPMV-GUG, which expresses low levels of the 17.5-kDa CP. The D-RNA derived from SPMV-91 was isolated from infected plants and used as a template to generate a cDNA clone. RNA transcripts derived from this 399-nt cDNA replicated and moved in millet plants coinoculated with PMV. The characterization of this D-RNA provided a biological confirmation that the critical RNA domains identified by the reverse genetic strategy are essential for SPMV replication and movement. The results additionally suggest that a potential “trigger” for spontaneous D-RNA accumulation may be associated with the absence or reduced accumulation of the 17.5-kDa SPMV CP. This represents the first report of a D-RNA associated with a satellite virus.

Small RNAs (sRNAs) have emerged as one of the most important regulators of gene expression in eukaryotes. sRNAs are intermediate molecules as well as end products in the antiviral defense pathway called RNA interference in plants and animals. Profiling of sRNAs using next-generation sequencing technologies has identified a number of plant viruses that have never been reported previously, and has provided a deeper view of virus populations in a plant that cannot be achieved by conventional methods like PCR and ELISA. In this chapter, we describe the methodology of deep sequencing of sRNAs. The high-throughput and highly sensitive method will revolutionize the identification of plant viruses and the study of molecular plant-virus interactions.

Grapevine is one of the economically and culturally important perennial fruit crops. More than 60 viruses infect grapevines and adversely affect their growth and development. Latent infection of most viruses in grapevines leads to chronic modulation of gene expression at transcriptional and post-transcriptional levels. Plant small RNAs (sRNAs) consist of microRNA (miRNA) and small interfering RNA (siRNA). miRNAs are expressed from the plant genome while most siRNAs are derived from double-stranded RNA molecules which are intermediates during virus replication. In a previous study, we constructed four cDNA libraries of sRNAs that were enriched from three virus-infected grapevines and one virus-free grapevine. Majority of siRNAs align most closely with the genomes of DNA viruses in the genus Badnavirus, family Caulimoviridae that led to the discovery of a new Grapevine vein clearing virus in grapevines. In this study, we conducted a comprehensive analysis of miRNAs in the four cDNA libraries and identified novel and stress-related miRNAs. The results indicated that miRNA abundance was influenced by virus infection. A total of 54 new miRNAs were identified and characterized, six of which, VITIS-MIR17, 18, 19, 20, 21, and 22, were detected only in virus-infected samples. One target of VITIS-MIR18 is the gene coding a non-apical meristem protein (GSVIVT00035370001), a transcription factor in the regulation of plant development and stress responses. Among the virus infection-induced known miRNAs, miRNA168 and miRNA3623 likely regulate grapevine's defense response, miRNA319 and miRNA395 modulate the expression of genes that are involved in nutrient metabolisms while miRNA396 plays a role in the regulation of cell division and cell cycle. The abiotic stress-induced miR169 and mi398 were negatively regulated by virus infection in grapevines. In addition, variety-specific miRNAs were discovered and compiled. The newly discovered miRNAs expand the miRNA profiles in the Vitis species. The characteristics of variety-specific and virus infection-associated miRNAs help understand the biology underlying the development and defense response of grapevines.

Small RNAs (sRNAs) have emerged as one of the most important regulators of gene expression in eukaryotes. sRNAs are intermediate molecules as well as end products in the antiviral defense pathway called RNA interference in plants and animals. Profiling of sRNAs using next-generation sequencing technologies has identified a number of plant viruses that have never been reported previously, and has provided a deeper view of virus populations in a plant that cannot be achieved by conventional methods like PCR and ELISA. In this chapter, we describe the methodology of deep sequencing of sRNAs. The high-throughput and highly sensitive method will revolutionize the identification of plant viruses and the study of molecular plant-virus interactions.

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.

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus within the new family Betaflexiviridae. GRSPaV is distributed among grapevines worldwide and is implicated in the disease rupestris stem pitting (RSP) of the rugose wood complex and two other disorders. GRSPaV is composed of a wide range of sequence variants, and so far, the complete genomes of five sequence variants have been sequenced. Quick and reliable detection of different GRSPaV variants is a critical step in the elimination and control of GRSPaV. Previously, primers designed from various genomic regions have been used in RT-PCR for the detection of GRSPaV variants. The efficiency of RT-PCR varied widely depending on the spectrum of the primers that were used. In this study, we designed a pair of degenerate primers based on the consensus sequence of the genomic region encoding the highly conserved RNA-dependent RNA polymerase domain from five reference isolates of GRSPaV for which the genome sequence are available. We demonstrate that this set of primers is comparable, if not superior, to the broad-spectrum primers RSP13&14 in detecting multiple GRSPaV variants. Using these degenerate primers, we identified two new and distinct sequence variants. The 3′ terminal genomic region of one of the new variants, GRSPaV-ML, spanning the 3′ part of ORF1, through the entire open reading frames 2-4, and the 5′ region of ORF5 were sequenced. Sequence comparison demonstrates that GRSPaV-ML is distinct from each of the five reference isolates. © 2011 Association of Applied Biologists.

A comparative analysis of differentially expressed proteins in a susceptible grapevine (Vitis vinifera ‘Cabernet Sauvignon’) during the infection of Erysiphe necator, the causal pathogen of grapevine powdery mildew (PM), was conducted using iTRAQ. The quantitative labeling analysis revealed 63 proteins that significantly changed in abundance at 24, 36, 48, and 72 h post inoculation with powdery mildew conidiospores. The functional classification of the PM-responsive proteins showed that they are involved in photosynthesis, metabolism, disease/defense, protein destination, and protein synthesis. A number of the proteins induced in grapevine in response to E. necator are associated with the plant defense response, suggesting that PM-susceptible Cabernet Sauvignon is able to initiate a basal defense but unable to restrict fungal growth or slow down disease progression.

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'.