Dirk Stephan

Grapevine virus A-mediated gene silencing in Nicotiana benthamiana and Vitis vinifera.

Virus-induced gene silencing (VIGS) is an attractive approach for studying gene function. Although the number of virus vectors available for use in VIGS experiments has increased in recent years, most of these vectors are applied in annual or herbaceous plants. The aim of this work was to develop a VIGS vector based on the Grapevine virus A (GVA), which is a member of the genus Vitivirus, family Flexiviridae. The GVA vector was used to silence the endogenous phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. In addition, an Agrobacterium-mediated method for inoculating micropropagated Vitis vinifera cv. Prime plantlets via their roots was developed. Using this method, it was possible to silence the endogenous PDS gene in V. vinifera plantlets. The GVA-derived VIGS vector may constitute an important tool for improving functional genomics in V. vinifera.

The grapevine-infecting vitiviruses, with particular reference to grapevine virus A

A number of vitiviruses infect grapevine, arguably the most important fruit crop, and the host from which they derive their genus name. In contrast to most grapevine viruses, the etiological role of these viruses is unclear, albeit that they are associated with several well-known – and a number of emerging – diseases of grapevine. Here, we review the genus Vitivirus, with special reference to its most omnipresent member, grapevine virus A. We discuss the latest taxonomic status of the genus, as well as the genome and genomic organisation, replication mechanism, and genetic variability of GVA, and we also present the latest research progress with vitivirus-based vectors; the identification of a new vitivirus, GVE, the discovery of VIGG, a unique GVA-induced host protein, the molecular characterisation of hitherto unknown or puzzling genetic elements in the GVA genome, and the latest developments in vitivirus diagnostics.

MicroRNAs in fruit trees: discovery, diversity and future research directions

Since the first description of microRNAs (miRNAs) 20 years ago, the number of miRNAs identified in different eukaryotic organisms has exploded, largely due to the recent advances in DNA sequencing technologies. Functional studies, mostly from model species, have revealed that miRNAs are major post-transcriptional regulators of gene expression in eukaryotes. In plants, they are implicated in fundamental biological processes, from plant development and morphogenesis, to regulation of plant pathogen and abiotic stress responses. Although a substantial number of miRNAs have been identified in fruit trees to date, their functions remain largely uncharacterised. The present review aims to summarise the progress made in miRNA research in fruit trees, focusing specifically on the economically important species Prunus persica, Malus domestica, Citrus spp, and Vitis vinifera. We also discuss future miRNA research prospects in these plants and highlight potential applications of miRNAs in the on-going improvement of fruit trees.

Complete Nucleotide Sequence of a South African Isolate of Grapevine Fanleaf Virus and Its Associated Satellite RNA

The complete sequences of RNA1, RNA2 and satellite RNA have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SACH44). The two RNAs of GFLV-SACH44 are 7,341 nucleotides (nt) and 3,816 nt in length, respectively, and its satellite RNA (satRNA) is 1,104 nt in length, all excluding the poly(A) tail. Multiple sequence alignment of these sequences showed that GFLV-SACH44 RNA1 and RNA2 were the closest to the South African isolate, GFLV-SAPCS3 (98.2% and 98.6% nt identity, respectively), followed by the French isolate, GFLV-F13 (87.3% and 90.1% nt identity, respectively). Interestingly, the GFLV-SACH44 satRNA is more similar to three Arabis mosaic virus satRNAs (85%–87.4% nt identity) than to the satRNA of GFLV-F13 (81.8% nt identity) and was most distantly related to the satRNA of GFLV-R2 (71.0% nt identity). Full-length infectious clones of GFLV-SACH44 satRNA were constructed. The infectivity of the clones was tested with three nepovirus isolates, GFLV-NW, Arabis mosaic virus (ArMV)-NW and GFLV-SAPCS3. The clones were mechanically inoculated in Chenopodium quinoa and were infectious when co-inoculated with the two GFLV helper viruses, but not when co-inoculated with ArMV-NW.

The first complete nucleotide sequence of a grapevine virus E variant

Grapevine virus E (GVE) was first isolated from a Japanese table grape cultivar (Vitis labrusca cv. Aki Queen and Pione) and classified as a new member of the genus Vitivirus in 2008 [1]. Although one of the plants used as a source of virus had stem pitting disease, no relationship between GVE and any disease symptoms could be established. Grapevine virus E is a positive-sense single-stranded RNA virus with a genome organization resembling that of grapevine virus A (GVA). Currently, the only sequence data available are the incomplete sequences of GVE isolates TvAQ7 (AB432910) and TvP15 (AB432911). Both isolates are transmissible by the mealybug Pseudococcus comstocki [1]. Recently, a metagenomic sequencing study reported the presence of GVE in South African vineyards for the first time [2]. The virus was detected in a severely diseased vineyard (cv. Merlot) in the Stellenbosch region of South Africa. Sequence data obtained using an Illumina Genome Analyzer II were subjected to de novo assembly, and two scaffolds were found to align preferentially to GVE when doing BLAST searches against the NCBI’s non-redundant nucleotide databases. The GVE variant present in the metagenomic sample was homologous to the incomplete GVE TvP15 sequence [1]. A scaffold assembled from metagenomic sequencing data [2], GVE Node 3404, was edited to 4,216 nt to contain only high-quality sequence data and submitted to GenBank (accession number GU903011). This scaffold aligned to TvAQ7 at nt positions 373–4,589. Based on the available sequence information (GVE Node 3404, TvP15 and TvAQ7 sequence data), the following set of diagnostic primers was designed: GVE-1-For 50-AA TGGAGTCAAAAGCGATCC-30 and GVE-Rev 50-GTAG GGTCAATCAACCAACA-30. To identify a GVE-infected plant, total RNA was isolated from individual plants using a CTAB method adapted for grapevine [3]. The extracted RNA was used as a template in RT-PCR using the diagnostic primers. A single grapevine plant, SA94 (Vitis vinifera cv. Shiraz), from a different vineyard and cultivar than the metagenomic study, was identified. The vine displayed typical Shiraz disease symptoms (e.g., canes with a lack of lignification, delayed leaf fall and reduced vigor) and tested positive for GVE. Sequencing of the PCR product confirmed the GVE infection status. The vine also tested positive for grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine leafroll-associated virus 3 (GLRaV-3) and GVA infections. RNA extracted from this vine was used to determine the complete nucleotide sequence of the South African GVE isolate SA94. A set of nine primer pairs was designed to amplify overlapping regions spanning the GVE genome. Primer pairs were used to synthesize cDNA from total RNA using AMV reverse transcriptase. The cDNA was then used as a template in a PCR with a high-fidelity DNA polymerase. Annealing temperatures and extension times were adapted according to the primer pairs and the expected sizes of the amplified fragments. PCR amplicons of the expected sizes were excised from agarose gels, purified and sequenced directly using the primers used for amplification. Sequence data from the 9 overlapping amplicons generated a contig of 7,042 nt using BioEdit [4]. The 30-terminal nucleotide sequence of the virus genome was determined by cDNA synthesis from total RNA with the oligo(dT) primer 5-TACGATGGCTGCAG(T)17-3 0 [5]. B. Coetzee H. J. Maree D. Stephan M.-J. Freeborough J. T. Burger (&) Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa e-mail: jtb@sun.ac.za

The use of high-throughput small RNA sequencing reveals differentially expressed microRNAs in response to aster yellows phytoplasma-infection in Vitis vinifera cv. ‘Chardonnay’

Phytoplasmas are cell wall-less plant pathogenic bacteria responsible for major crop losses throughout the world. In grapevine they cause grapevine yellows, a detrimental disease associated with a variety of symptoms. The high economic impact of this disease has sparked considerable interest among researchers to understand molecular mechanisms related to pathogenesis. Increasing evidence exist that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation during plant development and responses to biotic and abiotic stresses. Thus, we aimed to dissect complex high-throughput small RNA sequencing data for the genome-wide identification of known and novel differentially expressed miRNAs, using read libraries constructed from healthy and phytoplasma-infected Chardonnay leaf material. Furthermore, we utilised computational resources to predict putative miRNA targets to explore the involvement of possible pathogen response pathways. We identified multiple known miRNA sequence variants (isomiRs), likely generated through post-transcriptional modifications. Sequences of 13 known, canonical miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a unique genomic location, were predicted, of which 23 were differentially expressed. A homology search revealed that some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of randomly selected known and novel miRNAs was determined with real-time RT-qPCR analysis, thereby validating the trend of expression seen in the normalised small RNA sequencing read count data. Among the putative miRNA targets, we identified genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. Our results may assist in understanding the role that miRNA pathways play during plant pathogenesis, and may be crucial in understanding disease symptom development in aster yellows phytoplasma-infected grapevines.

Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging

BackgroundTransient expression of proteins in plants has become a favoured method over the production of stably transformed plants because, in addition to enabling high protein yields, it is both fast and easy to apply. An enhancement of transient protein expression can be achieved by plant virus-encoded RNA silencing suppressor proteins. Since viral suppressor proteins differ in their efficiency to enhance transient protein expression in plants, we developed a whole-leaf green fluorescent protein (GFP)-based imaging assay to quantitatively assess suppressor protein activity.ResultsIn a transient GFP-expression assay using wild-type and GFP-transgenic N. benthamiana, addition of the plant viral suppressors Beet mild yellowing virus (BMYV-IPP) P0 or Plum pox virus (PPV) HC-Pro was shown to increase fluorescent protein expression 3-4-fold, 7 days post inoculation (dpi) when compared to control plants. In contrast, in agroinfiltrated patches without suppressor activity, near complete silencing of the GFP transgene was observed in the transgenic N. benthamiana at 21 dpi. Both co-infiltrated suppressors significantly enhanced GFP expression over time, with HC-Pro co-infiltrations leading to higher short term GFP fluorescence (at 7 dpi) and P0 giving higher long term GFP fluorescence (at 21 dpi). Additionally, in contrast to HC-Pro co-infiltrations, an area of complete GFP silencing was observed at the edge of P0 co-infiltrated areas.ConclusionsFluorescence imaging of whole intact leaves proved to be an easy and effective method for spatially and quantitatively observing viral suppressor efficiency in plants. This suppressor assay demonstrates that plant viral suppressors greatly enhanced transient GFP expression, with P0 showing a more prolonged suppressor activity over time than HC-Pro. Both suppressors could prove to be ideal candidates for enhancing target protein expression in plants.