Project description:Grapevine line pattern virus (GLPV) was described 30 years ago from Hungary, and in the lack of its sequence until now no additional information about its presence was reported. However High-Throughput Sequencing (HTS) applied on dsRNAs extracts recovered from a grapevine plant (accession Baco22A) infected with GLPV Grapevine line pattern virus (GLPV) allowed us to sequence it with different High-Throughput Sequencing (HTS) methods andthe assembleing of the full genome sequence of this virus. The availability of the sequence allowed us to validate the presence of the virus bot with RT-PCR and with Northern blot hybridization. These methods were also used to test its graft and seed transmission. In accordance as it was originally suggested its genome was found to comprise three RNA segments.Its RNA1 (3.160 bp), RNA2 (2.493 bp) and RNA3 (2.529 bp), encode four proteins, denoted 1a (Methyltransferase, helicase), 2a (RNA-dependent RNA Polymerase), 3a (Movement protein, MP) and 3b (Coat protein, CP). GLPV showed the highest amino acid identity (92%–99%) with all domains of Hop yellow virus (HYV), which is a tentative member of the genus Anulavirus of the family Bromoviridae. The phylogenetic trees constructed based on the amino acid sequences of 2a and 3b also confirmed the belongingness of GLPV to the genus Anulavirus, allocating it in one cluster together with the anulaviruses, and close to HYV. The very high sequence identity found between GLPV and HYV leaves no doubt that both are two isolates of the same viral species.
Project description:Grapevine red blotch is a recently identified viral disease that was first recognized in the Napa Valley of California. Infected plants showed foliar symptoms similar to leafroll, another grapevine viral disease, on vines testing negative for known grapevine leafroll-associated virus. Later, the Grapevine red blotch virus (GRBV) was independently discovered in the US states of California and New York and was demonstrated to be the causal agent of red blotch disease. Due to its wide occurrence in the US, vector transmission and impacts on grape industry, this virus has the potential to cause serious economic losses. Despite numerous attempts, it was not possible to isolate or visualize viral particles from GRBV infected plants. Consequently, this has hampered the development of a serological assay that would facilitate GRBV detection in grapevine. We therefore decided to explore mass spectrometry approaches in order to quantify GRBV in infected plants and to identify potential biomarkers for viral infection. We present for the first time the physical detection on the protein level of the two GRBV genes V1 (coat protein) and V2 in grapevine tissue lysates. The GRBV coat protein load in leaf petioles was determined to be in the range of 100 to 900 million copies per milligram wet weight by using three heavy isotope labeled reference peptides as internal standards. The V1 copy number per unit wet tissue weight in leaves appeared to be about six times lower, and about 200-times lower in terms of protein concentration in the extractable protein mass than in petioles. We found a consistent upregulation of several enzymes involved in flavonoid biosynthesis in leaf and petiole extracts of GRBV-infected plants by label-free shotgun proteomics, indicating the activation of a defense mechanism against GRBV, a plant response already described for grapevine leafroll associated virus infection on the transcriptome level. Last but not least, we identified some other microorganisms belonging to the grapevine leaf microbiota, two bacterial species (Novosphingobium sp. Rr 2-17 and Methylobacterium) and one virus, Grapevine rupestris stem pitting associated virus.
Project description:Meristem culture and somatic embryogenesis is an effective tool for virus elimination of vegetatively propagated crops including grapevine. While they both are proved to be useful to eliminate the main grapevine viruses their efficiency differs according to the virus and the variety. In our work we investigated their efficiency using small RNA high-throughput sequencing as virus diagnostic method. Field grown mother plants of four clones representing three cultivars, infected with different viruses and viroids were selected for sanitation via somatic embryogenesis and meristem culture. Our results show that the sanitation with SE was efficient against all of the presenting viruses, including grapevine Pinot gris virus, grapevine rupestris vein feathering virus and grapevine Syrah virus 1, having no data using somatic embryogenesis for their elimination. In case of other viruses and viroids such as GFkV, GRSPaV, GYSVd-1, HSVd this study confirms the findings of earlier researches, that SE is a possible way for elimination. While the efficiency of the elimination of different viruses was high, in case of viroids this ratio was lower. Our work demonstrated that efficiency of SE is comparable to the technically difficult meristem culture technique, and show promising way for the high demand of the production of virus-free grapevine in the future.
Project description:Grapevine red blotch is a recently identified viral disease that was first recognized in the Napa Valley of California. Infected plants showed foliar symptoms similar to leafroll, another grapevine viral disease, on vines testing negative for known grapevine leafroll-associated virus. Later, the Grapevine red blotch virus (GRBV) was independently discovered in the US states of California and New York and was demonstrated to be the causal agent of red blotch disease. Due to its wide occurrence in the US, vector transmission and impacts on grape industry, this virus has the potential to cause serious economic losses. Despite numerous attempts, it was not possible to isolate or visualize viral particles from GRBV infected plants. Consequently, this has hampered the development of a serological assay that would facilitate GRBV detection in grapevine. We therefore decided to explore mass spectrometry approaches in order to quantify GRBV in infected plants and to identify potential biomarkers for viral infection. We present for the first time the physical detection on the protein level of the two GRBV genes V1 (coat protein) and V2 in grapevine tissue lysates. The GRBV coat protein load in leaf petioles was determined to be in the range of 100 to 900 million copies per milligram wet weight by using three heavy isotope labeled reference peptides as internal standards. The V1 copy number per unit wet tissue weight in leaves appeared to be about six times lower, and about 200-times lower in terms of protein concentration in the extractable protein mass than in petioles. We found a consistent upregulation of several enzymes involved in flavonoid biosynthesis in leaf and petiole extracts of GRBV-infected plants by label-free shotgun proteomics, indicating the activation of a defense mechanism against GRBV, a plant response already described for grapevine leafroll associated virus infection on the transcriptome level. Last but not least, we identified some other microorganisms belonging to the grapevine leaf microbiota, two bacterial species (Novosphingobium sp. Rr 2-17 and Methylobacterium) and one virus, Grapevine rupestris stem pitting associated virus.
Project description:MicroRNAs (miRNAs) are a class of non-coding RNA molecules which have significant gene regulation roles in organisms. The advent of new high throughput sequencing technologies has enabled the revelation of novel miRNAs. Although there are two recent reports on high throughput sequencing analysis of small RNA libraries from different organs of two grapevine wine varieties, there were significant divergence in the number and kinds of miRNAs sequenced in these studies. More sequencing of small RNA libraries is still important for the discovery of novel miRNAs in grapevine. Here, we initially constructed a small RNA library of flower and fruit tissues of a table grapevine cultivar ‘Summer Black’ and performed sequencing and analysis of sRNAs using the Illumina Solexa platform, expecting to discover more miRNAs related to the development of grapevine flowers and berries and the formation of dessert quality in grapevine berries. Totally, 130 conserved grapevine miRNA (Vv-miRNA) belonging to 28 Vv-miRNA families were validated, and 92 novel potential grapevine-specific ones representing 80 unique ones were first discovered. Forty-two (48.84%) of the novel miRNAs possessed differential semi-quantitative PCR expression profiles in various grapevine tissues that could further confirm their existence in the grapevine, among which twenty were expressed only in grapevine berries, indicating some fruit-specificity. 130 target genes for 46 novel miRNAs could be predicted. The locations of these potential target genes on grapevine chromosomes and their complementary levels with the corresponding miRNAs were also analyzed.
Project description:Purpose and strategy: Grapevine fanleaf virus (GFLV) causes variable symptoms in most vineyards worldwide. To better understand GFLV-grapevine interactions in relation to symptom development, field and greenhouse trials were conducted with a grapevine genotype that exhibits distinct symptoms in response to a severe and a mild strain of GFLV. Results: After validation of the infection status of the experimental vines by high throughput sequencing, the transcriptomic and metabolomic profiles in plants infected with the two viral strains were tested and compared by RNA-Seq and LC-MS, respectively, in the differentiating grapevine genotype. In vines infected with the severe GFLV strain, 1,023 genes, among which some are implicated in the regulation of the hypersensitive-type response, were specifically de-regulated, and a higher accumulation of resveratrol and phytohormones was observed. Interestingly, some experimental vines restricted the virus to the rootstock and remained symptom-less. Our results suggest that GFLV induces a strain- and cultivar-specific defense reaction similar to a hypersensitive reaction. This type of defense leads to a severe stunting phenotype in some grapevines whereas others are resistant. This work is the first evidence of a hypersensitive-like reaction in grapevine during virus infection. Conclusion: Our results suggest that GFLV induces a strain- and cultivar-specific defense reaction similar to a hypersensitive reaction. This type of defense leads to a severe stunting phenotype in some grapevines whereas others are resistant. This work is the first evidence of a hypersensitive-like reaction in grapevine during virus infection.
Project description:Nicotiana benthamiana was infected with several strains of grapevine fanleaf virus (GFLV). Apical tissue was collected 4, 7, and 12 days after inoculation, with identical samples for shotgun proteomics and transcriptomics analysis. Five leaf discs per leaf were collected a pooled by three plants into a single tube at each time point. Five biological replicates represent each treatment at each time point for a total of 75 samples. Two samples were lost between sample processing and data acquisition. The analysis methods between proteomics and transcriptomics were then cross-analyzed for host genes responsible for phenotypic differences upon infection.
Project description:microRNAs(miRNAs) play critical regulatory roles mainly through cleaving targeted mRNAs or repressing gene translation during plant developments. Grapevine is amongst the most economically important fruit crops with whole genome available, and the study on grapevine miRNAs (Vv-miRNAs) have also been emphasized. However, the regulation mode of Vv-miRNAs on their target mRNAs during grapevine development has not been studied well, especially on a transcriptome-wide level. Here, six small RNA (sRNA) and mRNA libraries from various grapevine tissues were constructed for Illumina and Degradome sequencing. Subsequently, the spatiotemporal variation in the Vv-miRNAs’ regulation on their target genes was systematically analyzed. Totally, 242 known and 132 novel Vv-miRNAs were identified, and 193 target mRNAs including 103 for known and 90 for novel miRNAs were validated in one or more of tissues examined. The interesting finding was that over 50% of novel miRNAs were expressed exclusively in flowers or berries where they had tissue-specific cleavage roles on their target genes, especially, the breadth of their cleavage sites in flower tissues. Moreover, six novel miRNAs in berries were found to response to exogenous gibberellin (GA) and/or ethylene by real time RT-PCR (qRT-PCR) analysis, confirming their regulatory functions during berry development. Other finding was that about 93.6% of the known miRNAs possessed the high conservation in various tissues where their expression levels exhibited some dynamic variations during grapevine development. Significantly, it was found the phenomena that some Vv-miRNA families exist one key member that act as the main regulator of their target genes during grapevine development.
Project description:Several pathogens infect grapevine, including viruses and viroids. Considering that there are no effective plant protection treatments against these pathogens and vineyards are cultivated through decades usage of high quality and pathogen-free propagation material (rootstocks and scions) is essential. Although presence of regulated pests is routinely checked using ELISA or rarely RT-PCR, these diagnostics methods can detect only particular pathogens moreover can fail to detect variant strains. High-throughput sequencing of small RNAs can be an effective, alternative method to avoid these disadvantages. Since for production of grafts, pathogen free cultivars and rootstocks must be used, 17 grapevine rootstock plantations and 2 rootstock variety collections were selected for characterisation of their virom by high throughput sequencing of virus derived small RNAs.
Project description:MicroRNAs (miRNAs) are a class of non-coding RNA molecules which have significant gene regulation roles in organisms. The advent of new high throughput sequencing technologies has enabled the revelation of novel miRNAs. Although there are two recent reports on high throughput sequencing analysis of small RNA libraries from different organs of two grapevine wine varieties, there were significant divergence in the number and kinds of miRNAs sequenced in these studies. More sequencing of small RNA libraries is still important for the discovery of novel miRNAs in grapevine. Here, we initially constructed a small RNA library of flower and fruit tissues of a table grapevine cultivar M-bM-^@M-^XSummer BlackM-bM-^@M-^Y and performed sequencing and analysis of sRNAs using the Illumina Solexa platform, expecting to discover more miRNAs related to the development of grapevine flowers and berries and the formation of dessert quality in grapevine berries. Totally, 130 conserved grapevine miRNA (Vv-miRNA) belonging to 28 Vv-miRNA families were validated, and 92 novel potential grapevine-specific ones representing 80 unique ones were first discovered. Forty-two (48.84%) of the novel miRNAs possessed differential semi-quantitative PCR expression profiles in various grapevine tissues that could further confirm their existence in the grapevine, among which twenty were expressed only in grapevine berries, indicating some fruit-specificity. 130 target genes for 46 novel miRNAs could be predicted. The locations of these potential target genes on grapevine chromosomes and their complementary levels with the corresponding miRNAs were also analyzed. Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.