Search

Ornithogalum thyrsoides, a widely cultivated bulbous ornamental plant endemic to South Africa, has significant commercial value as a pot plant and for the production of cut flowers. However, infection by viruses threatens the success of commercial cultivation, as symptoms negatively affect the appearance of the plant and flowers. To date, four Ornithogalum-infecting viruses have been reported. Complete genome sequence data are available for three of these viruses, but the genome of the potyvirus ornithogalum virus 3 (OV3) has not been fully sequenced. In this study, the complete sequence of OV3 was determined by high-throughput sequencing (HTS) and validated by Sanger sequencing. Based on recognition of protease cleavage patterns and multiple sequence alignments with closely related viruses, the polyprotein of OV3 was predicted to be proteolytically cleaved to produce 10 mature peptides containing domains conserved in members of the genus Potyvirus. Phylogenetic analysis and species demarcation criteria confirm the previous classification of OV3 as a member of a separate species in this genus. This is the first report of a complete genome sequence of OV3.

The molecular interactions between grapevine leafroll-associated virus 3 (GLRaV-3) and three grapevine cultivars were characterised by identifying small RNAs and genes with differential expression between healthy and infected grapevine using next-generation sequencing and RT-qPCR assays. Cultivar specificity was identified in the sRNA and gene expression analyses, and both approaches identified Chenin blanc-specific responses providing the first insight into the differential symptom expression observed between cultivars. Differentially expressed genes identified in all three cultivars, provide a potential pool of genes displaying a molecular response to GLRaV-3 infection, contributing to the characterisation of this virus-plant interaction.

The economic importance of the grapevine industry creates the need for the generation of genomic resources to ensure the sustainability of the industry. These resources will extend the knowledge of grapevine physiology, cultivar-specific characteristics and understanding how diseases and environmental conditions affect the plant. Small non-coding RNAs (sRNAs) are regulator molecules that can influence normal development and/or responses to environmental stimuli. In this study, next-generation sequencing (NGS) and bioinformatic analyses were used to identify sRNA species in grapevine phloem tissue. Forty-five novel mature Vitis vinifera miRNAs were identified and the presence of ten was validated using stemloop RT-qPCRs. Both non-coding and protein-coding gene regions were identified as putative phased loci of which the majority of the protein-coding gene loci were identified as disease resistance proteins. Potential phase-initiating miRNAs were also identified. A high number of sRNAs originating from repeat sequences were identified in all cultivar groups. This study also confirmed the non-random manner in which tRNA-derived sRNAs originate. The number of tRNA halves identified in Chenin blanc was lower compared to the other cultivar groups investigated. The identification of these different sRNAs extends the grapevine sRNA knowledge base significantly and can contribute to the unravelling of sRNA regulation in plants, potentially creating tools for grapevine functional studies.

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs (sRNA) that play an essential role in the regulation of target mRNAs expressed during plant development and in response to stress. MicroRNA expression profiling has helped to identify miRNAs that regulate a range of processes, including the plant's defence response to pathogens. In this study, differential miRNA expression in own-rooted Vitis vinifera cv. Cabernet Sauvignon plants infected with grapevine leafroll-associated virus 3 was investigated with microarrays and next-generation sequencing (NGS) of sRNA and mRNA. These high-throughput approaches identified several differentially expressed miRNAs. Four miRNAs, identified by both approaches, were validated by stemloop RT-PCRs. Three of the predicted targets of the differentially expressed miRNAs were also differentially expressed in the transcriptome data of infected plants, and were validated by RT-qPCR. Identification of these miRNAs and their targets can lead to a better understanding of host-pathogen interactions involved in grapevine leafroll disease and the identification of possible targets for virus resistance.

The complete genome sequences of two newly identified genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) are described. Two isolates, GH11 (18671 nt) and GH30 (18576 nt) were sequenced and found to be less than 70 % similar to other South African GLRaV-3 variants. The genome organization of GH11 and GH30 is similar to that of previously described GLRaV-3 isolates. However, no corresponding open reading frame 2 (ORF2) could be identified. Phylogenetic analysis indicates that GH11 and GH30 cluster in a subgroup (group VI) that is separate from the previously described GLRaV-3 isolates and should be regarded as a different strain of GLRaV-3.

The evolutionary history of the exclusively grapevine (Vitis spp.) infecting, grapevine leafroll-associated virus 3 (GLRaV-3) has not been studied extensively, partly due to limited available sequence data. In this study we trace the evolutionary history of GLRaV-3, focussing on isolate GH24, a newly discovered variant. GH24 was discovered through the use of next-generation sequencing (NGS) and the whole genome sequence determined and validated with Sanger sequencing. We assembled an alignment of all 13 available whole genomes of GLRaV-3 isolates and all other publicly available GLRaV-3 sequence data. Using multiple recombination detection methods we identified a clear signal for recombination in one whole genome sequence and further evidence for recombination in two more, including GH24. We inferred phylogenetic trees and networks and estimated the ages of common ancestors of GLRaV-3 clades by means of relaxed clock models calibrated with asynchronous sampling dates. Our results generally confirm previously identified variant groups as well as two new groups (VII and VIII). Higher order groups were defined as supergroups designated A to D. Supergroup A includes variant groups I-V and supergroup B group VI and its related unclassified isolates. Supergroups C and D are less well known, including the newly identified groups VII (including isolate GH24) and VIII respectively. The inferred node ages suggest that the origins of the major groups of GLRaV-3, including isolate GH24, may have occurred prior to worldwide cultivation of grapevines, whilst the current diversity represents closely related isolates that diverged from common ancestors within the last century.

Background The use of next-generation sequencing has become an established method for virus detection. Efficient study design for accurate detection relies on the optimal amount of data representing a significant portion of a virus genome. Findings In this study, genome coverage at different sequencing depths was determined for a number of viruses, viroids, hosts and sequencing library types, using both read-mapping and de novo assembly-based approaches. The results highlighted the strength of ribo-depleted RNA and sRNA in obtaining saturated genome coverage with the least amount of data, while even though the poly(A)-selected RNA yielded virus-derived reads, it was insufficient to cover the complete genome of a non-polyadenylated virus. The ribo-depleted RNA data also outperformed the sRNA data in terms of the percentage of coverage that could be obtained particularly with the de novo assembled contigs. Conclusion Our results suggest the use of ribo-depleted RNA in a de novo assembly-based approach for the detection of single-stranded RNA viruses. Furthermore, we suggest that sequencing one million reads will provide sufficient genome coverage specifically for closterovirus detection. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0539-x) contains supplementary material, which is available to authorized users.