Browsing by Author "Visser, Marike"

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    An evaluation of the efficacy of antimicrobial peptides against grapevine pathogens
    (Stellenbosch : University of Stellenbosch, 2011-03) Visser, Marike; Burger, Johan T.; Stephan, D.; University of Stellenbosch. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: This study investigated the use of antimicrobial peptides (AMPs) as possible source of resistance against a range of pathogens in grapevine. Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time consuming and therefore pre-screening of potential AMPs is necessary. These small molecules, of less than 50 amino acids in length, are expressed by almost all organisms as part of their non-specific defence system. In vitro pre-screening of AMP activity is valuable but is limited since the activity on artificial media may differ from the AMP activity in planta. These tests are also restricted to pathogens which can be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against pathogens of interest. In this study transient systems were used to express AMPs in developed plant tissue to test their efficacy against grapevine pathogens such as Agrobacterium vitis, Xylophilus ampelinus and aster yellows phytoplasma. Aster yellows phytoplasma, which was recently discovered in local vineyards, is known to cause extensive damage and therefore pose a great threat to the South African grapevine industry. To study the in planta effect of AMPs against the abovementioned pathogens, transient expression vectors were constructed expressing either of the AMPs D4E1 or Vv-AMP1. D4E1 is a synthetically designed AMP known to be active against bacteria and fungi, while Vv-AMP1, isolated from grapevine berries, has already shown activity against fungi. In a transient approach in grapevine, the expression of foreign genes from viral and non-viral vectors was confirmed by expression of the marker genes β-glucuronidase and Green Fluorescent Protein, while tissue-printing immunoassays confirmed viral replication and systemic spread in Nicotiana benthamiana. The viral vectors were based on the phloem-limited virus grapevine virus A. Only Agrobacterium-mediated 35S transient expression vectors were used for AMP in planta activity screening since the viral-mediated expression in grapevine was insufficient for screening against A. vitis and X. ampelinus as it was restricted to phloem tissues after whole-leaf infiltration. No phytoplasma-infected material could be established and as a result AMP activity screening was only performed against the A. vitis and X. ampelinus. Quantification of the bacteria was performed by qPCR. Vv-AMP1 did not show activity against either of the two bacteria in planta while D4E1 was found to be active against both. The observed in planta activity of D4E1 correlated with the in vitro activity as measured in an AMP plate bioassay. In contrast to in vitro screenings, the in planta AMP activity screening might give a more accurate representation of the potential antimicrobial activity of the peptide in a transgenic plant environment. This study proved that transient expression systems can be used as a pre-screening method of AMP activity in planta against grapevine pathogens, allowing the screening of various AMPs in a relatively short period of time before committing to transgenic grapevine development.
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    Extending the sRNAome of Apple by next-generation sequencing
    (Public Library of Science, 2014-04) Visser, Marike; Van der Walt, Anelda P.; Maree, Hans J.; Rees, D. Jasper G.; Burger, Johan T.
    The global importance of apple as a fruit crop necessitates investigations into molecular aspects of the processes that influence fruit quality and yield, including plant development, fruit ripening and disease resistance. In order to study and understand biological processes it is essential to recognise the range of molecules, which influence these processes. Small non-coding RNAs are regulatory agents involved in diverse plant activities, ranging from development to stress response. The occurrence of these molecules in apple leaves was studied by means of next-generation sequencing. 85 novel microRNA (miRNA) gene loci were predicted and characterized along with known miRNA loci. Both cis- and trans-natural antisense transcript pairs were identified. Although the trans-overlapping regions were enriched in small RNA (sRNA) production, cis-overlaps did not seem to agree. More than 150 phased regions were also identified, and for a small subset of these, potential miRNAs that could initiate phasing, were revealed. Repeat-associated siRNAs, which are generated from repetitive genomic regions such as transposons, were also analysed. For this group almost all available repeat sequences, associated with the apple genome and present in Repbase, were found to produce siRNAs. Results from this study extend our current knowledge on apple sRNAs and their precursors significantly. A rich molecular resource has been created and is available to the research community to serve as a baseline for future studies.
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    High-throughput sequencing reveals small RNAs involved in ASGV infection
    (BioMed Central, 2014) Visser, Marike; Maree, Hans J.; Rees, D. Jasper G.; Burger, Johan T.
    Background : Plant small RNAs (sRNAs) associated with virulent virus infections have been reported by previous studies, while the involvement of sRNAs in latent virus infection remains largely uncharacterised. Apple trees show a high degree of resistance and tolerance to viral infections. We analysed two sRNA deep sequencing datasets, prepared from different RNA size fractions, to identify sRNAs involved in Apple stem grooving virus (ASGV) infection. Results sRNA analysis revealed virus-derived siRNAs (vsiRNAs) originating from two ASGV genetic variants. A vsiRNA profile for one of the ASGV variants was also generated showing an increase in siRNA production towards the 3′ end of the virus genome. Virus-derived sRNAs longer than those previously analysed were also observed in the sequencing data. Additionally, tRNA-derived sRNAs were identified and characterised. These sRNAs covered a broad size-range and originated from both ends of the mature tRNAs as well as from their central regions. Several tRNA-derived sRNAs showed differential regulation due to ASGV infection. No changes in microRNA, natural-antisense transcript siRNA, phased-siRNA and repeat-associated siRNA levels were observed. Conclusions This study is the first report on the apple sRNA-response to virus infection. The results revealed the vsiRNAs profile of an ASGV variant, as well as the alteration of the tRNA-derived sRNA profile in response to latent virus infection. It also highlights the importance of library preparation in the interpretation of high-throughput sequencing data.
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    In silico analysis of the grapefruit sRNAome, transcriptome and gene regulation in response to CTV-CDVd co-infection
    (BioMed Central, 2017) Visser, Marike; Cook, Glynnis; Burger, Johan T.; Maree, Hans J.
    Background: Small RNA (sRNA) associated gene regulation has been shown to play a significant role during plant-pathogen interaction. In commercial citrus orchards co-infection of Citrus tristeza virus (CTV) and viroids occur naturally. Methods: A next-generation sequencing-based approach was used to study the sRNA and transcriptional response in grapefruit to the co-infection of CTV and Citrus dwarfing viroid. Results: The co-infection resulted in a difference in the expression of a number of sRNA species when comparing healthy and infected plants; the majority of these were derived from transcripts processed in a phased manner. Several RNA transcripts were also differentially expressed, including transcripts derived from two genes, predicted to be under the regulation of sRNAs. These genes are involved in plant hormone systems; one in the abscisic acid, and the other in the cytokinin regulatory pathway. Additional analysis of virus- and viroid-derived small-interfering RNAs (siRNAs) showed areas on the pathogen genomes associated with increased siRNA synthesis. Most interestingly, the starting position of the p23 silencing suppressor’s sub-genomic RNA generated a siRNA hotspot on the CTV genome. Conclusions: This study showed the involvement of various genes, as well as endogenous and exogenous RNA-derived sRNA species in the plant-defence response. The results highlighted the role of sRNA-directed plant hormone regulation during biotic stress, as well as a counter-response of plants to virus suppressors of RNA-silencing.
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    Next-generation sequencing for virus detection : covering all the bases
    (BioMed Central, 2016-06) Visser, Marike; Bester, Rachelle; Burger, Johan T.; Maree, Hans J.
    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.
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    Small RNA profiling of virus-infected apple plants
    (Stellenbosch : Stellenbosch University, 2014-12) Visser, Marike; Burger, Johan T.; Maree, H. J.; Rees, Jasper; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Apple stem grooving virus (ASGV) is globally associated with latent infection in commercial apple trees. Little is known about this plant-­‐virus interaction. This study made use of next-­‐generation sequencing to investigate the effect of virus-­‐infection on the expression of the different small RNA (sRNA) species namely, miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐derived sRNAs and vsiRNAs. Broad and narrow size-­‐range datasets were generated using sRNA libraries prepared from total and size-­‐selected RNA, respectively. Through bioinformatic data analyses, 130 genomic loci were predicted to give rise to miRNAs, 85 of which were novel MIR genes. Targets were predicted for the majority of miRNAs, a few of which could be validated with a publicly available degradome dataset. Cis-­‐ and trans-­‐natural antisense transcripts (NATs) were identified, of which only the latter were highly enriched for sRNAs in their overlapping regions. Transcript as well as genomic regions were also identified that can give rise to phasiRNAs. For 25 of these loci an in-­‐phase miRNA target site was identified, half of which could be validated with the degradome dataset. Nearly all apple repeat sequences in Repbase were associated with sRNA synthesis. sRNAs derived from both ends of mature tRNAs were identified. These sRNAs corresponded to tRFs and tRNA-­‐halves. Reads associated with tRNA-­‐halves were prominent in the broad range datasets. sRNAs, originating from the central regions of tRNAs, were also observed. Analysis of the vsiRNAs suggested the presence of two ASGV genetic variants in two of the samples, while the third sample was infected with only one variant. Comparison of the vsiRNA profiles generated from the two datasets highlighted the influence of library preparation on the interpretation of results. Differential expression analysis of the identified apple sRNA species showed no variation between healthy and infected plants, except for the tRNA-­‐derived sRNAs, which did show altered expression levels. Taken together, the various sRNA species characterised in this study significantly extended the existing knowledge of apple sRNAs and provide a broad platform for future functional studies in apple. This study also presents the first and most comprehensive report on sRNAs involved in ASGV infection in apple.