Department of Genetics

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The construction of an infectious clone of grapevine virus A (GV A)
(Stellenbosch : University of Stellenbosch, 2005-04) Du Preez, Jacques; Burger, J. T.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
An infectious clone of a viral RNA genome is one that can be used, either as an in vitro transcript or as cDNA, to produce an infection in a susceptible plant. Infectious clones serve as a tool to study viral RNA genomes at a molecular level to gain deeper insight into genome organization, viral gene function, presence of regulatory sequences and gene expression. In the Western Cape (and elsewhere) a new crippling grapevine disease, known as Shiraz disease, is emerging of which the aetiology and pathogenic agents involved are not yet fully understood. Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, is thought to be the associated with this disease. The aim of this study was to construct a full-length infectious cDNA clone of GVA, which will aid in the molecular study of the viral genome. This clone could ultimately be used to investigate GVA’s involvement in Shiraz disease, which could lead to the unravelling of the aetiology and control of the disease. A full-length clone of GVA, named GVA-IC2/T7-2972-3, was constructed in several steps using restriction digestion/ligation and primer overlap extension PCR. Grapevine virus A cDNA fragments were obtained from GVAinfected Nicotiana benthamiana and Vitis vinifera plants using three different techniques, of which the Rapid direct-one-tube RT-PCR was most successful. A 5’ T7 promoter and a 3’ poly-A tail were incorporated and the full-length clone was cloned into pBluescript II SK (+). Full-length sequencing of the clone, revealed two significant frameshift mutations. The first mutation was a single base pair insertion (one G) in a slippery site of 6 G’s at position 1380 – 1385 in open reading frame one (ORF 1) of the viral genome. This mutation was corrected by PCR-based site-directed mutagenesis, which resulted in pSK-GVA-mutagen-3 and pSK-GVA-mutagen-4. The second mutation was a single base pair deletion (one G) at position 6959 in ORF4, which coded for the coat protein (CP). Several techniques were attempted to correct this mutation, but none were successful. Even though the second mutation could not be corrected, in vitro transcriptions were performed on three clones followed by subsequent infections of N. benthamiana plants. The three clones included pSK-GVA-mutagen-3, pSKGVA- mutagen-4 (both hosting the mutation at position 6959) and GVA-IC2/T7-2972-3 (hosting both mutations). At 21 days post-inoculation no significant visual symptoms were observed in plants infected with in vitro RNA or in plants infected with wild type GVA. Rapid direct-one-tube RT-PCR results revealed the presence of viral RNA in infected leaves and apical leaves of infected plants, and provided preliminary evidence that the mutated clones were still capable of systemic infection and viral movement. These results are still inconclusive, and several post-infection studies will have to be performed to confirm these findings. Koch's postulates will also have to be proved in order to confirm the infectious nature of the clones. The effect of the two mutations in the constructed clones will be investigated further and post-infection analysis performed to deduce whether the viral progeny are devoid of the mutations. Three full-length GVA cDNA clones (hosting mutations) seemingly capable of systemic infection in N. benthamiana plants were constructed in this study and have laid the foundation for molecular and mutational analysis of the GVA genome. This could lead to the study of pathogen-host interactions in order to unravel the aetiology of Shiraz disease in the future.
The construction of plant expression vectors for the introduction of leafroll disease resistance in grapevine
(Stellenbosch : Stellenbosch University, 2000-12) Van Straten, Celene Debra; Burger, J. T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Grapevine leafroll is one of the most damaging viral diseases that affect many viticultural regions of the world. Numerous reports over the last few years have associated closterovirus-like particles with leafroll disease. To date, eight serologically distinct closteroviruses have been isolated from leafroll infected vines, of which grapevine leafroll associated closterovirus-3 (GLRaV-3) is the best characterized. Virus resistance in transgenic plants based on the expression of a virusderived gene is known as pathogen-derived resistance. The viral coat protein (CP) gene, which expresses a structural protein responsible for coating the virus particles, was used in the first demonstration of virus-derived resistance. Coat protein-mediated resistance is currently the most feasible and most widely used method to obtain virus resistance in crop plants. The CP gene of a South African isolate of GLRaV-3 infected grapevine was isolated, cloned and sequenced. Double stranded RNA (dsRNA) was extracted from GLRaV-3 infected material and a high molecular weight band, of -18 kb was identified from infected vines. The dsRNA was used as a template in a reverse transcription PCR together with GLRaV-3 CP gene specific primers for the amplification of the GLRaV-3 CP gene (975 bp). The GLRaV-3 CP gene was cloned into the pGem®-T Easy vector. Clones hosting the CP gene in the sense (pLR3CP+) and antisense (pLR3CP-) orientations respectively were obtained. The sequence obtained from these two clones showed 99.26 % similarity to the only other GLRaV-3 CP nucleotide sequence available. The GLRaV-3 CP gene was excised from pLR3CP+ and pLR3CP- and subcloned into a plant expression vector, pCAMBIA 3301 in the sense (pCamBLR3CP+) and antisense (pCamBLR3CP-) orientations respectively, therefore enabling sense and antisense gene expression in transgenic plants. The GLRaV-3 CP gene was also subcloned from pCamBLR3CP+ into another plant expression vector, pCAMBIA 2301 in the sense orientation and designated as pCVSLR3CP+. These three constructs were given to Dr. M. Vivier (Institute for Wine Biotechnology, Stellenbosch) for grapevine transformation experiments. Two of these constructs, pCamBLR3CP+ and pCamBLR3CP- as well as pCAMBIA 3301 were used to transform Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Plants were selected for their ability to withstand the herbicide, Basta. This resistance is due to the presence of a plant selectable marker gene on each of these constructs, known as the bar gene. PCR with GLRaV-3 CP gene specific primers showed no amplification of the GLRaV-3 CP gene in the plants transformed with pCamBLR3CP+ and pCamBLR3CP-. Southern blot analysis with the GLRaV-3 CP gene as hybridization probe showed no signal for these plants, thus confirming the PCR results. PCR with bar gene specific primers showed no amplification of the bar gene in the plants infected with pCAMBIA 3301. The plants transformed with pCamBLR3CP+ and pCamBLR3CP- were also screened for the presence of the bar gene. Three of the eight plants tested showed amplification of the -560 bp bar gene. This result suggests that these plants were transformed with pCAMBIA 3301 (vector without the ligated GLRaV-3 CP gene) and not pCamBLR3CP+ or pCamBLR3CP- as had been expected. This project provides preliminary work for the subsequent transformation of grapevine with the GLRaV-3 CP gene, in an attempt to impart virus resistance.
The detection of Citrus tristeza virus genetic variants using pathogen specific electronic probes
(Stellenbosch : Stellenbosch University, 2017-03) Jooste, Tracey; Maree, H. J.; Burger, J. T.; Visser, M.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Citrus tristeza virus (CTV), a complex pathogen of citrus spp., is endemic to South Africa and has been responsible for great losses locally and internationally. CTV causes severe stem pitting in grapefruit, which forms an important sector of South Africa's citrus production and export market. The limited understanding of CTV’s ability to cause severe disease in one host while no symptoms in another restricts the implementation of effective management strategies. The conservation of plant biosecurity relies on the rapid identification of pathogenic organisms including viruses. While there are many molecular assays available for the detection of plant viruses, these are often limited in their ability to test for multiple viruses simultaneously. However, with next-generation sequencing (NGS) based metagenomic analysis it is possible to detect multiple viruses within a sample, including low-titre and novel viruses, at the same time. Conventional NGS data analysis has computational limitations during contig assembly and similarity searches in sequence databases, which prolongs the time required for a diagnostic result. In this study, an alternative targeted method was explored for the simultaneous detection of 11 recognised citrus viruses in NGS data using electronic probes (e-probes). E-probes were designed, optimised and screened against raw, unassembled NGS data in order to minimise the bioinformatic processing time required. The e-probes were able to accurately detect their cognate viruses in simulated datasets, without any false negatives or positives. The efficiency of the e-probe based approach was validated with NGS datasets generated from different RNA preparations: dsRNA from ‘Mexican’ lime infected with different CTV genotypes, dsRNA from field samples, as well as small RNA and total RNA from grapefruit infected with the CTV T3 genotype. A set of probes were made publically available that is able to accurately detect CTV in NGS data irrespective of which genotype the plants are infected with. The results were confirmed by performing de novo assemblies of the high quality read datasets and subsequent BLAST analyses. This sequence based detection method eliminates the need for NGS data assembly, ultimately reducing the virus-detection turnaround time.
The detection of mycoviral sequences in grapevine using next-generation sequencing
(Stellenbosch : Stellenbosch University, 2013-03) Espach, Yolandi; Burger, J. T.; Maree, H. J.; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Metagenomic studies that make use of next-generation sequencing (NGS) generate large amounts of sequence data, representing the genomes of multiple organisms of which no prior knowledge is necessarily available. In this study, a metagenomic NGS approach was used to detect multiple novel mycoviral sequences in grapevine phloem tissue. Individual sequencing libraries of doublestranded RNA (dsRNA) from two grapevine leafroll diseased (GLD) and three shiraz diseased (SD) vines were sequenced using an Illumina HiScanSQ instrument. Over 3.2 million reads were generated from each of the samples and these reads were trimmed and filtered for quality before being de novo assembled into longer contigs. The assembled contigs were subjected to BLAST (Basic Local Alignment Search Tool) analyses against the NCBI (National Centre for Biotechnology Information) database and classified according to database sequences with which they had the highest identity. Twenty-six putative mycovirus species were identified, belonging to the families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae and Totiviridae. Two of the identified mycoviruses, namely grapevine-associated chrysovirus (GaCV) and grapevine-associated mycovirus 1 (GaMV-1) have previously been identified in grapevine while the rest appeared to be novel mycoviruses not present in the NCBI database. Primers were designed from the de novo assembled mycoviral sequences and used to screen the grapevine dsRNA used for sequencing as well as endophytic fungi isolated from the five sample vines. Only two mycoviruses, related to sclerotinia sclerotiorum partitivirus S and chalara elegans endornavirus 1 (CeEV-1), could be detected in grapevine dsRNA and in fungus isolates. In order to validate the presence of mycoviruses in grapevine phloem tissue, two additional sequencing runs, using an Illumina HiScanSQ and an Applied Biosystems (ABI) SOLiD 5500xl instrument respectively, were performed. These runs generated more and higher quality sequence data than the first sequencing run. Twenty-two of the putative mycoviral sequences initially detected were detected in the subsequent sequence datasets, as well as an additional 29 species not identified in the first HiScanSQ sequence datasets. The samples harboured diverse mycovirus populations, with as many as 19 putative species identified in a single vine. This indicates that the complete virome of diseased grapevines will include a high number of mycoviruses. Additionally, the complete genome of a novel endornavirus, for which we propose the name grapevine endophyte endornavirus (GEEV), was assembled from one of the second HiScanSQ sequence datasets. This is the first complete genome of a mycovirus detected in grapevine. Grapevine endophyte endornavirus has the highest sequence similarity to CeEV-1 and is the same virus that was previously detected in fungus isolates using the mycovirus primers. The virus was detected in two fungus isolates, namely Stemphylium sp. and Aureobasidium pullulans, which is of interest since mycoviruses are not known to be naturally associated with two distinctly different fungus genera. Mycoviral sequence data generated in this study can be used to further investigate the diversity and the effect of mycoviruses in grapevine.
The development and characterisation of grapevine virus-based expression vectors
(Stellenbosch : University of Stellenbosch, 2010-03) Du Preez, Jacques; Burger, J. T.; Goszczynski, D. E.; Stephan, D.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be protected. To achieve this several in vivo tools are needed for the study of this crop and the pathogens that infect it. Recently the grapevine genome has been sequenced and the next important step will be gene annotation and function using these in vivo tools. In this study the use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression and VIGS vector for heterologous protein expression and functional genomics in Nicotiana benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a molecular sequence comparison study. Results confirmed the separation of GVA variants into three groups, with group III (mild variants) being the most distantly related. It showed the high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III respectively. A collaboration study investigating the molecular divergence of GVA variants linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive control by the grapevine industry, was found to contain a 119 nt insert within the native ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA variants suggested that the components in the GVA genome that cause pathogenicity in V. vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana. The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength cDNA clones under control of CaMV 35S promoters. After several strategies were attempted, including a population cloning strategy for GTR1-2, none of the clones generated were able to replicate in N. benthamiana plants. A single amino acid substitution at position 13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce replication of the virus to below a detectable level. Two infectious clones of Israeli variants of GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were infectious, able to replicate, move systemically and induce typical GVA symptoms after agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5- ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector, 35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA- GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N. benthamiana both vectors showed similar GUS expression levels and photobleaching symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118. No GUS expression was observed for the gene exchange vector 35S-GVA-GR5- ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5- ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves after 4 months. This study showed that GVA can be used as gene insertion and gene exchange vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of GVA is not needed for long distance movement in grapevine. To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA clone was removed and subsequently substituted by the corresponding ORFs of four South African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA constructs were able to move systemically through the plant. At this stage no correlation could be found between severity of symptoms, the presence of the P163-M5 insert and the specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral genome or the host plant probably play a crucial role. This study contributed to the pool of available in vivo tools for study and improvement of the valuable grapevine crop. It also opened several exciting research avenues to pursue in the near future.
The development of an enzyme linked immunosorbent assay for the detection of the South African strain(s) of grapevine fanleaf nepovirus
(Stellenbosch : Stellenbosch University, 2008-12) Liebenberg, Annerie; Burger, J. T.; Freeborough, M.-J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
South Africa is one of the top ten wine producing countries in the world. The South African wine industry contributes approximately R16.3 billion to South Africa’s annual gross domestic product with 42.8% of wine being exported. To compete with the top wine producing countries and to ensure a viable export market, South Africa needs to ensure that healthy, virus free propagation material is produced and sold. One of the viruses that need to be tested for is Grapevine fanleaf virus (GFLV). Grapevine fanleaf virus causes degeneration and malformation of berries, leaves and canes and is responsible for significant economic losses by reducing crop yields by as much as 80%, reducing the longevity of the vines and affecting fruit quality. It is widespread in the Breede River Valley of the Western Cape where the nematode vector, Xiphinema index, is prevalent. The Breede River Valley contributes approximately 30% of the total production of the local wine industry, and severe losses in this region could threaten the viticulture. The Plant Improvement Act states that all propagation material sold must be tested for GFLV by a reputable scientific technique. The technique commonly used in South Africa is the Double Antibody Sandwich - Enzyme-linked Immunosorbent Assay (DAS-ELISA) and the kits are imported from Europe at a significant cost to the South African viticulture industry. The objective of this study was to produce a reliable and sensitive diagnostic assay specific for the South African strains of GFLV. This project aimed to develop and optimize a DAS-ELISA, by using recombinant DNA technology to produce antibodies against bacterially expressed viral coat protein. Total RNA was extracted from GFLV infected grapevine material and the viral coat protein (CP) amplified. The CP was cloned into the pGex-6P-2 expression vector, fusing a Glutathione STransferase (GST) partner to the viral coat protein enhancing solubility and protein purification. Insufficient amounts of the soluble protein were expressed and purified, preventing the production of antibodies and thus the development of the DAS-ELISA. An alternative diagnostic rapid-direct-one-tube-RT-PCR assay was developed. This rapid-directone- tube-RT-PCR assay was compared to commercially available DAS-ELISA and ImmunoStrip tests (Agdia) to assess the reliability, sensitivity and specificity of the rapid-direct-one-tube-RTPCR assay. Twelve GFLV isolates from South Africa were sequenced to investigate the variability between the isolates as well as the variability between the South African isolates and GFLV sequences available in Genbank. Sequence identities between clones from different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the coat protein gene sequences showed that the South African isolates form two distinct clades or sub-populations. No significant correlation was found between geographical origin and symptoms, nor between geographical origin and sequence variability or between grapevine cultivar and symptom expression. Of the 23 samples tested with all three tests, 21 tested positive with rapid-direct-one-tube-RT-PCR, 19 with the ImmunoStrips and 17 with an imported DAS-ELISA kit (Agdia). Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for GFLV detection. Although the establishment of a DAS-ELISA directed to the South African strain(s) of GFLV was not successful, an alternative PCR based diagnostic system was developed, and proved to be sensitive and reliable. RT-PCR based diagnostic assays are generally accepted to be more sensitive than DAS-ELISA, but the latter is still used as the diagnostic assay of choice for routine testing due to ease of use. This rapid-direct-one-tube-RT-PCR assay is a rapid, sensitive and reliable diagnostic test, reducing the prevalence of false negatives, contributing to a virus free viticulture industry. The rapid-direct-one-tube-RT-PCR assay is as easy to use as DAS-ELISA, faster and can be performed by semi skilled workers, thus providing all the advantages associated with DAS-ELISA.
Genetic diversity and identification of putative recombination events in Grapevine rupestris stem pitting-associated virus
(Stellenbosch : Stellenbosch University, 2017-12) Mostert, Ilani; Maree, H. J.; Burger, J. T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Phylogenetic analysis for variant classification plays a key role in the characterisation of the aetiological role of viruses. The genomic regions selected to identify viral variants and the occurrence of recombination has the potential to influence tree topologies. To investigate the impact of these factors on variant classification, and to evaluate the success of certification schemes in eliminating virus infection, a diversity study was performed on Grapevine rupestris stem pitting-associated virus (GRSPaV), a ubiquitous virus commonly detected in cultivated vines and reportedly associated with Rupestris Stem Pitting disorder. Three surveys were conducted to characterise and compare the genetic diversity of GRSPaV on a global and local level, using a phylogenetic approach. The first constituted a collection of accessions from various countries to represent global virus diversity. A second survey was carried out on local mother blocks that previously conformed to certification requirements for South Africa. Finally, GRSPaV diversity in South African vineyards established prior to the implementation of current sanitary protocols was investigated. Two genomic areas, the coat protein and replicase domains, were selected for this study as these were used to characterise the sequence diversity of GRSPaV in previous studies. Mixed infections were found to occur within single vines, the genetic diversity of GRSPaV was confirmed with the clustering of sequences into five of the six distinct, currently recognised lineages, and a seventh, previously unclassified lineage was detected. Furthermore, the ability of the two domains to detect and classify variants was compared. Additional evidence for recombination in GRSPaV was provided and a correlation between recombinant sequences and inconsistencies between topologies generated by the two genome regions, was observed. Results indicate that disease control methods were moderately successful, but less effective at eliminating non-symptomatic variants. The study illustrates the effect of recombination on phylogenetic trees, and emphasises the importance of accounting for such factors in the characterisation of virus diversity. Increased knowledge of the recombination events within the GRSPaV genome could promote the development of a standardised method for variant classification and the clarification of the aetiological role of the virus.
Investigating the introduction of a broadspectrum antiviral mechanism into grapevine
(Stellenbosch : Stellenbosch University, 2000-03) Wilsen, Kathleen L. (Kathleen Lucy); Burger, J. T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are potent toxins produced by a wide range of evolutionarily diverse plants. These toxins cause cell death by physically dismantling ribosomal RNA and shutting down protein synthesis. They also have a strong antiviral activity. Some believe that the antiviral property of RIPs is a function of ribosomal inactivation, others believe that the two properties are unrelated. RIPs are non-specific in their antiviral activity. Transgenic RIPexpressing plants are resistant to a wide spectrum of viruses. Many different viruses threaten grapevine. It is not practical to design individual remedies for each of these viruses. In this study, we screen the grapevine genome for the presence of a RIP gene using degenerate PCR primers. If a RIP gene does exist in grapevine, it is not being expressed in a useful way. We also clone several well-documented RIP genes from various plants into pGEM-T Easy: dianthin from Dianthus caryophyllus; p-Iuffin from Luffa octandra and mirabilis antiviral protein (MAP) from Mirabilis jalapa. These isolated genes are then subcloned into a selection of expression vectors: dianthin into pKK223-3, a bacterial expression vector; p-Iuffin into pCambia3301, a plant expression vector; and MAP into pFLAG, a yeast expression vector. The constructs prepared in this project may be used for the synthesis of RIP molecules. The exogenous application of RIPs has been shown to protect plants from viruses. Transformation of grapevine with the RIP-containing plant expression vector may result in a variety of vine that is resistant to a wide range viruses. This thesis describes preliminary work in an attempt to impart broad-spectrum antiviral resistance to grapevine.
A metagenomic approach using next-generation sequencing for viral profiling of a vineyard and genetic characterization of grapevine virus E
(Stellenbosch : University of Stellenbosch, 2010-12) Coetzee, Beatrix; Burger, J. T.; Freeborough, M-J.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: Next-generation sequencing technologies are increasingly used in metagenomic studies, largely due to the high sequence data throughput capacity and unbiased approach in determining the genetic composition of an unknown environmental sample. This study investigated the applicability of the Illumina next-generation sequencing platform for metagenomic sequencing of grapevine viruses to provide the first complete viral profile, or virome, of a diseased vineyard. Leaf material was harvested from 44 randomly selected vines in a leafroll-diseased vineyard in South Africa. Sample material was pooled and double-stranded RNA extracted. The dsRNA was sequenced as a paired-end sequencing run using the Illumina sequencing-by-synthesis technique, and more than 19 million sequence reads, equivalent to approximately 837 megabases of metagenomic sequence data, were obtained. Of these data, approximately 400 megabases could be assembled into 449 scaffolds, using the de novo assembler Velvet. These scaffolds were subjected to BLAST searches against the NCBI databases and top hit scores were used for virus identification. Based on the BLAST results, suitable sequences were selected from the NCBI database and used as reference sequence in MAQ mapping assemblies. The bioinformatic analyses allowed for the determination of the virus species present, the most prominent variants, and the relative abundance of each. Four known grapevine viral pathogens were identified. Grapevine leafroll-associated virus 3, representing 59% of the analyzed short read sequence data, was identified as the most prominent virus species. Three variants of this virus were detected: GP18 was the most abundant, followed by a minor Cl766/NY1 variant and a potential novel grapevine leafroll-associated ampelovirus. A single Grapevine rupestris stem pitting ]associated virus variant, similar to SG1, and a Grapevine virus A variant, a member of molecular group III, were identified. This study is also the first to report the presence of Grapevine virus E (GVE) in South African vineyards. Grapevine virus E was further genetically characterized and the genome sequence of GVE isolate SA94 determined. The GVE SA94 genome sequence, 7568 nucleotides in length, is the first complete genome sequence for the virus species. The genome organization of GVE SA94 is typical of vitiviruses, but in contrast to other RNA viruses, the AlkB domain is located within the helicase domain in open reading frame 1 (ORF 1). Grapevine virus E SA94 shares nearly 100% nucleotide identity with the Japanese TvP15 isolate and GVE 3404, a de novo scaffold generated from the metagenomic sequence data. Bioinformatic analysis of metagenomic sequence data further revealed the presence of three fungus-infecting viral families, Chrysoviridae, Totiviridae and the unclassified dsRNA virus, Fusarium graminearum dsRNA mycovirus 4. A virus from the family Chrysoviridae, similar to Penicillium chrysogenum virus, was the second most abundant virus detected. We demonstrated the successful application of a short read sequencing technology, such as the Illumina platform, for viral profiling of an infected vineyard. To our knowledge this is the first application of the Illumina technology for this purpose.
The molecular and biological characterisation of ORF5 of three South African variants of Grapevine Vitivirus A
(Stellenbosch : University of Stellenbosch, 2009-03) Blignaut, Marguerite; Burger, J. T.; Stephan, D.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
Grapevine Vitivirus A (GVA), genus Vitivirus, family Flexiviridae is a well characterised single-stranded RNA virus that has been implicated in the grapevine diseases, Kober stem grooving and Shiraz disease. The virus infects both its host, Vitis vinifera and the experimental model plant, Nicotiana spp.. Biological studies performed on the virus in its herbaceous host, Nicotiana benthami- ana, revealed that many divergent variants of the virus exists in South Africa and can induce di erent symptoms in the model plant. Further molecular analysis divided the variants into three molecular groups based on molecular heterogeneity and nucleotide identity. The establishment of an infectious full-length cDNA clone of GVA contributed towards the elucidation of gene functions for 4 of the 5 open reading frames (ORF's), and indicated ORF5 as the pathogenicity determinant within the genome. Further studies also showed that ORF5 encodes for a nucleic acid binding protein that exhibits suppression activity of a plants' natural virus silencing mechanism. Many proteins that have previously been identi ed as the pathogenicity determinant within a viral genome have been found to encode for suppression activity. Although suppression activity has been elucidated within the ORF5 of the Italian cDNA clone of GVA, IS 151, no such study has yet been performed on the divergent South African variants of GVA. Three variants, GTR1-1, GTR1- 2 and GTG11-1, which represent each of the molecular groups (Group III, II and I), were selected for this study. The aim of this study was to visually elucidate suppression activity of RNA transgene silencing by the ORF5's of GTR1-1, GTR1-2 and GTG11-1 in a transient expression assays in transgenic N. benthamiana (line 16c). Pathogenicity studies for these variants were also performed. The ORF5 of the infectious full-length clone, GVA118, which can also serve as an expression vector, was deleted and provided with restriction enzyme sites into which the respective ORF5s and the marker genes, GFP and GUS could be cloned directionally. Infectivity, symptom development and systemic movement were compared between the di erent full length clones after co-in ltration in N. benthamiana. Preliminary results obtained in this study failed to visually indicate any suppression activity encoded by the ORF5 of GTR1-1, GTR1-2 and GTG11-1. The deletion of ORF5 within GVA118 was successful and rendered the infectious full length clone asymptomatic. Directional cloning of the ORF5 of GTR1-1 into the unique restriction enzymes provided previously, resulted in much milder symptoms than those observe for GTR1-2 and GTG11-1. No GFP and GUS accumulation could be detected. This study has established an infectious full-length cDNA clone, pBINSN-e35SGVA118 ORF5-1-1-pA, that can possibly induce much milder symptoms in the herbaceous host, N. benthamiana. This construct can be further characterised as a possible expression vector of foreign proteins in herbaceous hosts and grapevine.
Molecular characterisation of South African isolates of grapevine fanleaf virus and a new, associated satellite RNA
(Stellenbosch : Stellenbosch University, 2013-12) Lamprecht, Renate Luise; Burger, J. T.; Stephan, D.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Grapevine fanleaf virus (GFLV) is one of the oldest, most widespread and devastating viruses infecting grapevine, and occurs globally where Vitis vinifera is grown. In South Africa (SA) GFLV is predominant in the Breede River Valley, one of the highest wine producing regions in SA. To date, only three GFLV isolates have been completely sequenced internationally, and limited sequence information is available for SA GFLV isolates. In this study, the first full-length GFLV genome sequence from a South African isolate, GFLV-SAPCS3, was determined. Full-length sequences were used for phylogenetic analysis and revealed that the SA isolates are separate from other sequenced GFLV isolates. Full-length sequences were also used to investigate putative intra- and interspecies recombination events involving GFLV-SAPCS3 RNA1 and RNA2 between GFLV and Arabis mosaic virus (ArMV) isolates. Using two different recombination analysis software packages, the most notable of the putative recombination events involving GFLV-SAPCS3 indicated that the GFLV-SAPCS3 RNA2 5’ UTR might have evolved from an interspecies recombination event between GFLVF13- type and ArMV Ta-type isolates. The presence of satellite RNAs (satRNA) associated with South African GFLV isolates was also investigated. In a collaborative study (see Chapter 4 for details), more than a 100 GFLV- infected grapevine plants were screened for satRNAs. SatRNAs were present in only two plants, containing isolates GFLV-SACH44 and GFLV-SACH47. The full-length nucleotide sequences of the GFLV-SACH44 genomic RNAs 1 and 2, and the associated satRNA were determined. No significant sequence variation could be detected between the GFLV isolates that had the presence of a satRNA and those that had not. The GFLV-SACH44 RNA2 5’ UTR also had the same conserved sequence that was found in GFLVSAPCS3, which suggests that GFLV-SACH44, like GFLV-SAPCS3, may have arisen from a common ancestor, which may have originated from an interspecies recombination event. The GFLV-SACH44 satRNA was found to be more closely related to the ArMV large satRNA than to the satRNA associated with GFLV-F13. A full-length cDNA clone of GFLV-SACH44 satRNA was constructed and its replication and systemic spread in herbaceous hosts, when mechanically co-inoculated with two GFLV isolates as helper viruses, was demonstrated. Replication of the GFLV-SACH44 satRNA cDNA clone was however abolished when co-inoculated with an ArMV helper virus, even though it is phylogenetically more closely related to ArMV satRNAs. The full-length satRNA clones were modified to be used as vectors for expression and/or silencing of foreign genes, by inserting the green fluorescence protein (GFP) full-length or partial sequences downstream of the open reading frame of the satRNA. These constructs were cloned into a binary vector to allow for agro-infiltration into plants. Full-length cDNA clones of GFLV-SAPCS3 RNA1 and RNA2 were constructed to be used in conjunction with modified GFLV-SACH44 satRNA full-length clones. The full length GFLV-SAPCS3 RNA1 and RNA2 clones were however not infectious in Nicotiana benthamiana after agro-infiltration and therefore the evaluation of the modified satRNA expression and silencing constructs had to be aborted. Attempts to understand this failure revealed that, among other point mutations, four frameshifts had occurred in the RNA1 full-length clone, rendering the transcripts untranslatable, and hence noninfectious. Strategies to correct the mutations are discussed. Once these mutations have been corrected this study can continue in evaluating the use of the satRNA component for expression and silencing analysis.
Molecular characterization of grapevine virus E in South Africa
(Stellenbosch : Stellenbosch University, 2012-12) De Koker, Wenhelene Crystal; Burger, J. T.; Maree, H. J.; Stephan, D.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Grapevine virus E (GVE) is a newly identified virus that has been detected in an established vineyard in South Africa. This virus is a member of the genus Vitivirus, family Flexiviridae. Members of this genus are known to infecte grapevine and are associated with various disease complexes, such as the Rugose wood complex (RWC) and Shiraz disease (SD). However, the role and impact of GVE in South African vineyards are still unknown. It is important to study these viruses to determine how they infect and the possible impact they may have on vine health. The accurate and early detection of grapevine viruses is the first important step in disease management. In this study, reverse transcription-polymerase chain reaction (RT-PCR), double antibody sandwich enzyme linked immunesorbent assay (DAS-ELISA) and quantitative (q)RT-PCR were used for the detection of GVE in the vineyard (Vitis vinifera cv Merlot) where GVE was first identified in South Africa. Reverse transcription-PCR was used for detection and determining the incidence of GVE. The incidence was as low as 3% in the vineyard surveyed. All the GVE positive plants were co-infected with GLRaV-3 and no disease association could therefore be made. Evaluation of the Bioreba Grapevine virus A (GVA) DAS-ELISA kit showed that it did not detect GVE. No cross-reactivity occurred with epitopes of GVE, confirming this kit to be a valid and specific assay for GVA infection. The relative virus titer of GVE was calculated over the growing season of 2010/2011, using qRT-PCR. No fluctuation in virus titer was observed during that growing season. Transmission experiments were performed in an attempt to transfer GVE from grapevine to an alternative host. Three different transmission buffers as well as nine different herbaceous plant species, that have shown to be susceptible to several plant viruses in previous studies, were evaluated. In these experiments, GVE could not be transmitted to any of the herbaceous species. To further characterize GVE, chimeric clones were constructed with GVA. The ORF2 and ORF5 of GVE were cloned into previously constructed GVA ORF2 and ORF5 deletion mutants. Construction of the chimeric clones, 35S-GVA-GR5-ΔORF2-GVE-ORF2 and 35S-GVA-118-ΔORF5-GVE-ORF5 were successful and they were evaluated for their infectivity in N. benthamiana. The 35S-GVA-GR5-ΔORF2-GVE-ORF2 chimera was able to infect and replicate in these plants and disease symptoms such as yellowing of veins and leaf curling were observed. Virus, derived from this vector, was detected by TPIA, RT-PCR and DAS-ELISA. The 35S-GVA-118-ΔORF5-GVE-ORF5 chimeric vector was not able to infect N. benthamiana as no disease symptoms were observed in any of the infiltrated plants and virus was not detected with serological analysis and RT-PCR. This study was aimed at further characterizing the recently identified virus GVE. Here, insight is given into the prevalence of this virus in the vineyard where it was first identified and attempts to biologically characterize GVE were made.
The molecular characterization of South African isolates of Grapevine Rupestris Stem Pitting-associated virus (GRSPaV)
(Stellenbosch : University of Stellenbosch, 2010-12) Noach, Liesl Christine; Burger, J. T.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: The first aim of this study was to reliably and rapidly detect Grapevine rupestris stem pittingassociated virus (GRSPaV) in grapevine. This was achieved by screening 94 grapevines using crude plant extracts in both quantitative and conventional reverse transcription polymerase chain reaction (RT-PCR). The second aim was to establish a technique capable of differentiating GRSPaV sequence variants. The application of this technique is for the largescale screening of diseased vines to associate sequence variants of GRSPaV with disease symptoms. Nested quantitative polymerase chain reaction and high resolution melting assays (qPCR-HRM) were developed for three regions of the GRSPaV genome (coat protein, RNAdependant RNA-polymerase and triple gene block movement protein). The qPCR-HRM technique using the high saturation dye, EvaGreen™, and the Rotor-Gene™ 6000 analyzer was validated with a panel of sixteen sequence-characterized viral isolates. Diluted RT-PCR products and cloned cDNA gave the most consistent amplification plots and dissociation profiles. RT-PCR products generated from total RNA extracts were used as template for qPCR-HRM assays and for direct sequencing of sixteen samples in the three aforementioned regions. The average amplification efficiency for qPCR was 1.52±0.04. Auto-calling of userdefine genotypes was performed at a confidence interval of 70%. Phylogenetic analysis of the three regions of the GRSPaV genome was performed with published GenBank sequences to confirm the HRM data. The dominant sequence variants found in the South African sample set radiated with Group II, reference full-length variant GRSPaV-SG1. GRSPaV-infected samples can in future be subjected to qPCR-HRM assays developed during this study. This can be performed to establish similarity to known genotypes and therefore phylogenetic groups. Mixed infection of sequence variants and quasi-species were a common occurrence. The assay will be useful in establishing correlation of specific genotypes to different phenotypical expression of viral disease. This could provide insight into the etiology of diseases associated with GRSPaV.
Real time PCR as a versatile tool for virus detection and transgenic plant analysis
(Stellenbosch : University of Stellenbosch, 2009-12) Malan, Stefanie; Burger, J. T.; Freeborough, M-J.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination.
Screening the genome of grapevine leafroll associated virus-2 for proteins with RNA silencing suppressor activity and the construction of a tandem silencing vector to induce simultaneous silencing of two genes
(Stellenbosch : Stellenbosch University, 2006-04) Engelbrecht, M.; Burger, J. T.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics Institute for Plant Biotechnology (IPB).
ENGLISH ABSTRACT: Grapevine is one of the oldest food plants and was first exploited from in the wild and later cultivated by man. Grapevine viruses are among the most important pathogens of grapevine and one of these viruses termed Grapevine .!:eafroll-§ssociated Y'.irus (GLRaV) is regarded as one of the most harmful grapevine viruses. The virus is responsible for the disease called leafroll disease which affects the South African wine industry causing losses of millions of Rands annually. Existing control measures focus on prevention by utilising virus-free propagation material and integrated control of the insect vectors. Virus resistant grapevine by means of genetic modification seems to be a realistic approach in solving grapevine diseases, especially leafroll disease. A natural occurring plant mechanism called post transcriptional gene silencing (PTGS) can be exploited to help in the process of obtaining transgenic virus resistant grapevine. PTGS is a sequence-specific defence system of the plant that targets alien RNA (transgenes, endogenous genes and cytoplasmically replicating viruses) for degradation (Dunoyer et al., 2002; Vanitharani et al., 2003; Waterhouse et al., 2001a). However viruses have evolved a counter defence mechanism against PTGS by encoding suppressor proteins able to suppress RNA silencing (Thomas et al., 2003). Very few suppressor proteins have been identified in grapevine viruses. In this study the GLRaV-2 genome was screened for a suppressor protein able to reverse or to prevent the onset of PTGS. A constitutively expressed green fluorescent protein (GFP) gene was silenced in transgenic Nicotiana benthamiana plants (line 16c) by agro-infiltration, using a second GFP-construct. The GFP-silenced plants were inoculated with a strain of GLRaV-2 to screen for a suppressor able to reverse PTGS. Individual GLRaV-2 genes were isolated and cloned into an intermediate PCR cloning vector, followed by subsequent cloning into a plant expression vector. These constructs were transformed into Agrobacterium tumefaciens strains GV3101 and C58C1 and were agro-infiltrated into silenced transgenic or co-infiltrated into transgenic N. benthamiana plants (16c) in two different expression assays. It was found in both the silencing reversal assay and the transient assay that the p24 protein of GLRaV-2 possessed suppressor activity. An attempt was made to corroborate the fluorescence assays by screening infiltrated plants for the presence of GFP siRNAs, which would be a telltale sign that silencing has occurred. Unfortunately (and probably due to technical problems) these experiments failed to yield signals in the Northern blot analysis. The second part of this study was to construct a tandem silencing vector to serve as "proof of concept to show that two genes can be silenced simultaneously in a plant. The primary construct, pHanViralGFP-SAS was constructed by performing a rapid direct reverse transcription reaction (RDOT-RT-PCR) with primers containing 5' -extension restriction sites to facilitate subsequent cloning and to amplify a gene fragment from the GLRaV-2 genome. A portion of the GFP was obtained by a polymerase chain reaction (PCR) from the plasmid vector pBIN mGFPS-ER, also using primers containing restriction sites. The fragments obtained in the individual reactions were ligated into an intermediate PCR cloning vector, followed by the subsequent cloning into corresponding sites in the pHannibal vector, in sense and anti-sense orientations. The silencing cassette was removed from the pHannibal vector and ligated into pART27. The final construct, pSilencer-SAS, was transformed into A. tumefaciens strains GV3101 and C58C1 and transgenic (16c) and non-transgenic N. benthamiana plantlets, of which some were infected with GLRaV-2, were agro-infiltrated with these Agrobacterium strains. Results obtained showed that the tandem silencing vector was successful in silencing two genes simultaneously, justifying the construction of a tandem vector. The effectivity of the vector can now be tested by inserting genes from two different viruses.
Sequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa
(Stellenbosch : Stellenbosch University, 2012-12) Bester, Rachelle; Burger, J. T.; Maree, H. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type member of the genus Ampelovirus in the family Closteroviridae and is considered to be the main contributing agent of grapevine leafroll disease (GLD) worldwide. A metagenomic sequencing study of a grapevine leafroll-diseased vineyard led to the discovery of a new variant of GLRaV-3 in South Africa. This new variant was most related to a New Zealand isolate, NZ-1. In this study, we sequenced two isolates, GH11 and GH30, of the new variant group of GLRaV-3. These isolates have less than 70% nucleotide (nt) identity to other known GLRaV-3 variants, indicating that they should be considered variants of a different strain of GLRaV-3. We propose that the GLRaV-3-like virus identified in this study be grouped together with NZ-1 and some Napa Valley isolates as Group VI of GLRaV-3. This study also provided further evidence that next-generation sequencing is an invaluable approach to identify novel viruses and variants, in that the draft sequence generated with bioinformatic tools in this study was 98% identical to the GH11 sequence generated using Sanger sequencing. The study further confirmed that the industry standard ELISA is still an effective GLRaV-3 diagnostic method and that it is able to detect all known variant groups of GLRaV-3. However, this assay is not able to differentiate between GLRaV-3 variant groups. In the current study therefore, a real-time RT-PCR was designed that is able to detect GLRaV-3 variant groups I, II, III and VI, using a single primer pair targeting the Hsp70h gene of GLRaV-3. If high-resolution melting (HRM) curve analysis is added to the real-time RT-PCR, it is possible to differentiate between variant groups based on three melting point intervals. The RT-PCR HRM assay provides a more sensitive and rapid tool to detect and differentiate between different GLRaV-3 variant groups. Finally, a multiplex RT-PCR was designed to differentiate between the variant groups present in South Africa. This multiplex RT-PCR offers a validation method for the RT-PCR HRM and provides an end-point PCR alternative for variant identification. In order to investigate the spread and impact of different GLRaV-3 variants in vineyards, sensitive diagnostic techniques are a necessity. The abovementioned tools will contribute to the understanding of the pathogenesis of GLD and aid epidemiological studies to investigate how these different GLRaV-3 variant groups are spreading, the association of specific GLRaV-3 variants to disease symptoms and the mealybug vector transmission efficiency for each GLRaV-3 variant.
Small RNA profiling of grapevine leafroll-associated virus 3 infected grapevine plants
(Stellenbosch : Stellenbosch University, 2016-12) Bester, Rachelle; Maree, H. J.; Burger, J. T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: One of the most important viral diseases of grapevine worldwide is grapevine leafroll disease (GLD). A number of viruses from the family Closteroviridae have been associated with this disease, though Grapevine leafroll-associated virus 3 is considered the leading causative agent due to its consistent association with GLD. To better understand the disease and develop effective control strategies, it is necessary to characterise the molecular interactions between the virus and the plant. Small RNA (sRNA) molecules have been shown to play an important role in gene regulation of normal development and defence responses to biotic and abiotic stresses in plants. Therefore, the aim of this study was to characterise the sRNA species in healthy and infected grapevine to contribute to the growing database of sRNAs present in Vitis vinifera. Microarray analysis and next-generation sequencing was used to identify sRNA species in Chardonnay, Chenin blanc, Cabernet Sauvignon and own-rooted Cabernet Sauvignon plants. Differential expression of sRNAs was evaluated to identify sRNAs associated with GLRaV-3 infection. The modulation of the differentially expressed microRNAs (miRNAs) was validated with stemloop RT-qPCR assays. Transcriptome NGS was also performed to validate the differential expression of the predicted miRNA targets, and to identify metabolic pathways modulated in response to GLRaV-3 independently from sRNA regulation. The transcriptome NGS transcripts that were differentially expressed in all cultivar groups, and transcripts that anti-correlated with miRNA expression, were validated with RT-qPCR assays. These highthroughput approaches identified several differentially expressed sRNAs and (target) genes in infected plants. The anti-correlation of miRNA expression and putative target expression were shown for two miRNAs. Cultivar specificity was identified in the sRNA and gene expression analyses, and both approaches identified Chenin blanc-specific responses. This comparison of symptomatic and asymptomatic GLRaV-3-infected plants provides the first insight into the disease symptom inhibition observed in certain cultivars. The differentially expressed genes identified in all cultivar groups, using the NGS transcriptome data, provides a collection of genes displaying a potentially universal molecular response against GLRaV-3. These genes showed strong associations with cell wall biosynthesis and signalling during pathogen recognition. This study has contributed significantly to the knowledge of sRNAs produced in grapevine and significantly extended the existing sRNA reference database for grapevine. The knowledge generated in this study can be utilised as potential targets for grapevine functional studies, and be translated into potential management strategies to control the disease. A better understanding of both the host defence and viral counter-defence strategies can lead to the prevention of virus replication or the impaired ability of the virus to induce pathogenesis in plants.

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