Transcriptomic analysis of the interaction geminivirus-tomato

Geminiviridae family is one of the main families of plant pathogenic viruses with large relevance as they cause great losses worldwide in commercial crops and crops destined to food production. Geminiviruses present a little single-stranded DNA genome and a capsid composed of two twin icosahedral parts. Tomato Yellow Leaf Curl Virus (TYLCV) belongs to the Begomovirus genus and is transmitted by the whitefly Bemisia tabaci. With only 6 viral proteins, this geminivirus must create a proper environment for viral replication, transcription and propagation. Behind the apparent simplicity of geminiviruses lies a complex network of molecular interactions with their host and even their natural vector, which induces a wide variety of transcriptional, post-transcriptional and chromatinic changes in both the plant and the geminivirus. In order to study these changes and decipher the effects of the transmission vector on the infection, we carried out a global approximation of the TYLCV-tomato interaction to generate integrated single-base resolution maps by NGS (next-generation sequencing) of the transcriptome, smallRNAome and methylome of the pathogen and the host. Tomato plants (Moneymaker) were infected with TYLCV under controlled conditions of light and temperature using Agrobacterium tumefaciens or its natural vector. Apical tissue from these plants was collected at different time points (2, 7, 14 and 21 days after inoculation), and three biological replicas were generated for each treatment and time. Total RNA and DNA was extracted and analysed by RNA-Seq, smallRNA-Seq and Bisulfite-Seq. The transcriptome of the tomato-TYLCV interaction will be presented and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Improvement of root architecture under abiotic stress through control of auxin homeostasis in Arabidopsis and Brassica crops

Auxin plays an important role in many aspects of plant development including stress responses. Here we briefly summarize how auxin is involved in salt stress, drought (i.e. mostly osmotic stress), waterlogging and nutrient deficiency in Brassica plants. In addition, some mechanisms to control auxin levels and signaling in relation to root formation (under stress) will be reviewed. Molecular studies are mainly described for the model plant Arabidopsis thaliana, but we also like to demonstrate how this knowledge can be transferred to agriculturally important Brassica species, such as Brassica rapa, Brassica napus and Brassica campestris. Moreover, beneficial fungi could play a role in the adaptation response of Brassica roots to abiotic stresses. Therefore, the possible influence of Piriformospora indica will also be covered since the growth promoting response of plants colonized by P. indica is also linked to plant hormones, among them auxin

Bisociative knowledge discovery for microarray data analysis

Peer reviewe

Network modeling unravels mechanisms of crosstalk between ethylene and salicylate signaling in potato

To develop novel crop breeding strategies, it is crucial to understand the mechanisms underlying the interaction between plants and their pathogens. Network modeling represents a powerful tool that can unravel properties of complex biological systems. In this study, we aimed to use network modeling to better understand immune signaling in potato (Solanum tuberosum). For this, we first built on a reliable Arabidopsis (Arabidopsis thaliana) immune signaling model, extending it with the information from diverse publicly available resources. Next, we translated the resulting prior knowledge network (20,012 nodes and 70,091 connections) to potato and superimposed it with an ensemble network inferred from time-resolved transcriptomics data for potato. We used different network modeling approaches to generate specific hypotheses of potato immune signaling mechanisms. An interesting finding was the identification of a string of molecular events illuminating the ethylene pathway modulation of the salicylic acid pathway through Nonexpressor of PR Genesi gene expression. Functional validations confirmed this modulation, thus supporting the potential of our integrative network modeling approach for unraveling molecular mechanisms in complex systems. In addition, this approach can ultimately result in improved breeding strategies for potato and other sensitive crops

RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases.

Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.LJRH was supported by a studentship co-funded by the James Hutton Institute (formerly Scottish Crop Research Institute) and the UK Biotechnological and Biological Sciences Research Council (BBSRC). Work in the JPC lab is funded by The Leverhulme Trust (RPG-2012-667), BBSRC (BB/D014376/1, BB/J011762/1) and the Cambridge University Newton Trust. SFB was funded by Leverhulme grant F/09-741/G to Professor Beverley Glover. KG was funded by an EMBO Short Term Fellowship. Work in the PP lab is funded by grant number NRF-2013R1A2A2A01016282 from the Korean National Research Foundation.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/srep2308

RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases

Cellular RNA-dependent RNA polymerases (RDR) catalyze synthesis of double stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.LJRH was supported by a studentship co-funded by the James Hutton Institute (formerly Scottish Crop Research Institute) and the UK Biotechnological and Biological Sciences Research Council (BBSRC). Work in the JPC lab is funded by The Leverhulme Trust (RPG-2012-667), BBSRC (BB/D014376/1, BB/J011762/1) and the Cambridge University Newton Trust. SFB was funded by Leverhulme grant F/09-741/G to Professor Beverley Glover. KG was funded by an EMBO Short Term Fellowship. Work in the PP lab is funded by grant number NRF-2013R1A2A2A01016282 from the Korean National Research Foundation.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/srep2308

Toward learning the principles of plant gene regulation

Advanced machine learning (ML) algorithms produce highly accurate models of gene expression, uncovering novel regulatory features in nucleotide sequences involving multiple cis-regulatory regions across whole genes and structural properties. These broaden our understanding of gene regulation and point to new principles to test and adopt in the field of plant science

Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms

<p>Abstract</p> <p>Background</p> <p>The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing TaqMan^®and SYBR^®Green real-time PCR chemistries. In our study four alternative chemistries: Lux™, Plexor™, Cycling Probe Technology and LNA^®were extensively evaluated and compared using TaqMan^®chemistry as a reference system.</p> <p>Results</p> <p>Amplicons were designed on the maize invertase gene and the 5'-junction of inserted transgene and plant genomic DNA in MON 810 event. Real-time assays were subsequently compared for their efficiency in PCR amplification, limits of detection and quantification, repeatability and accuracy to test the performance of the assays. Additionally, the specificity of established assays was checked on various transgenic and non-transgenic plant species. The overall applicability of the designed assays was evaluated, adding practicability and costs issues to the performance characteristics.</p> <p>Conclusion</p> <p>Although none of the chemistries significantly outperformed the others, there are certain characteristics that suggest that LNA^®technology is an alternative to TaqMan^®when designing assays for quantitative analysis. Because LNA^®probes are much shorter they might be especially appropriate when high specificity is required and where the design of a common TaqMan^®probe is difficult or even impossible due to sequence characteristics. Plexor™ on the other hand might be a method of choice for qualitative analysis when sensitivity, low cost and simplicity of use prevail.</p

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

BACKGROUND: Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. RESULTS: Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. CONCLUSION: The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification