86 research outputs found

    Molecular Approaches to Address Intended and Unintended Effects and Substantial Equivalence of Genetically Modified Crops

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    The release of GM organisms into the environment and marketing of GM crops have resulted in public debate in many parts of the world. This debate is likely to continue, probably in the broader context of plant biotechnology and consequences for human societies. The general issues under debate include cost–benefit analysis and safety issues, but might exhibit regional differences and crop-specific nuances. This chapter addresses an in-depth understanding of events involved in transgene insertion, but also the unintended effects of transformation following the production of genetically enhanced plants. In order to dissect this topic, a foundational overview is given on biolistic- and Agrobacterium-based techniques. Background information of possible transformation-induced unintended alterations to transgenic plant genomes is reviewed and aspects that collectively constitute possible unintended transformation - and post-transformation events are described. This is followed by an overview of molecular techniques to study gene insertion and – expression with special focus on differential gene expression analysis techniques to investigate unintended effects of genetic transformation. Historical and current safety assessment guidelines and requirements are also briefly discussed

    Lipopolysaccharide perception leads to dynamic alterations in the microtranscriptome of Arabidopsis thaliana cells and leaf tissues

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    MicroRNAs (miRNAs) are non-coding RNA molecules which have recently emerged as important gene regulators in plants and their gene expression analysis is becoming increasingly important. miRNAs regulate gene expression at the post-transcriptional level by translational repression or target degradation of specific mRNAs and gene silencing. In order to profile the microtranscriptome of Arabidopsis thaliana leaf and callus tissues in response to bacterial lipopolysaccharide (LPS), small RNA libraries were constructed at 0 and 3 h post induction with LPS and sequenced by Illumina sequencing technology.National Research Foundation (NRF) of South AfricaUniversity of Johannesbur

    Unravelling the metabolic reconfiguration of the post-challenge primed state in Sorghum bicolor responding to Colletotrichum sublineolum infection

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    Priming is a natural phenomenon that pre-conditions plants for enhanced defence against a wide range of pathogens. It represents a complementary strategy, or sustainable alternative that can provide protection against disease. However, a comprehensive functional and mechanistic understanding of the various layers of priming events is still limited. A non-targeted metabolomics approach was used to investigate metabolic changes in plant growth-promoting rhizobacteria (PGPR)-primed Sorghum bicolor seedlings infected with the anthracnose-causing fungal pathogen, Colletotrichum sublineolum, with a focus on the post-challenge primed state phase. At the 4-leaf growth stage, the plants were treated with a strain of Paenibacillus alvei at 108 cfu mL1. Following a 24 h PGPR application, the plants were inoculated with a C. sublineolum spore suspension (106 spores mL1), and the infection monitored over time: 1, 3, 5, 7 and 9 days post-inoculation. Non-infected plants served as negative controls. Intracellular metabolites from both inoculated and non-inoculated plants were extracted with 80% methanol-water. The extracts were chromatographically and spectrometrically analysed on an ultra-high performance liquid chromatography (UHPLC) system coupled to high-definition mass spectrometry. The acquired multidimensional data were processed to create data matrices for chemometric modelling. The computed models indicated time-related metabolic perturbations that reflect primed responses to the fungal infection. Evaluation of orthogonal projection to latent structure-discriminant analysis (OPLS-DA) loading shared and unique structures (SUS)-plots uncovered the di erential stronger defence responses against the fungal infection observed in primed plants. These involved enhanced levels of amino acids (tyrosine, tryptophan), phytohormones (jasmonic acid and salicylic acid conjugates, and zeatin), and defence-related components of the lipidome. Furthermore, other defence responses in both naïve and primed plants were characterised by a complex mobilisation of phenolic compounds and de novo biosynthesis of the flavones, apigenin and luteolin and the 3-deoxyanthocyanidin phytoalexins, apigeninidin and luteolinidin, as well as some related conjugates.Supplementary Material: Figure S1: Evaluation of disease symptoms in Colletotrichum sublineolum infected sorghum plants; Figure S2: Representative BPI MS chromatograms of ESI(+) data (3 d.p.i.); Figure S3: Unsupervised chemometric modelling of ESI(-) data; Figure S4: OPLS-DA modelling and variable/feature selection. Table S1: Annotated (MSI-level 2) metabolites reported in Table 1, with fragmentation information.The South African National Research Foundation (NRF)http://www.mdpi.com/journal/metabolitesam2020Plant Production and Soil Scienc

    Differential metabolic reprogramming in paenibacillus alvei-primed sorghum bicolor seedlings in response to fusarium pseudograminearum infection

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    Metabolic changes in sorghum seedlings in response to Paenibacillus alvei (NAS-6G6)-induced systemic resistance against Fusarium pseudograminearum crown rot were investigated by means of untargeted ultra-high performance liquid chromatography-high definition mass spectrometry (UHPLC-HDMS). Treatment of seedlings with the plant growth-promoting rhizobacterium P. alvei at a concentration of 1 × 108 colony forming units mL- 1 prior to inoculation with F. pseudograminearum lowered crown rot disease severity significantly at the highest inoculum dose of 1 × 106 spores mL-1. Intracellular metabolites were subsequently methanol-extracted from treated and untreated sorghum roots, stems and leaves at 1, 4 and 7 days post inoculation (d.p.i.) with F. pseudograminearum. The extracts were analysed on an UHPLC-HDMS platform, and the data chemometrically processed to determine metabolic profiles and signatures related to priming and induced resistance. Significant treatment-related differences in primary and secondary metabolism post inoculation with F. pseudograminearum were observed between P. alvei-primed versus naïve S. bicolor seedlings. The differential metabolic reprogramming in primed plants comprised of a quicker and/or enhanced upregulation of amino acid-, phytohormone-, phenylpropanoid-, flavonoid- and lipid metabolites in response to inoculation with F. pseudograminearum.Supplementary Materials: Figure S1. (A) Microscopic identification of F. pseudograminearum at 400 × magnification. (B) Conidial morphology of F. pseudograminearum taken from Aoki et al. [65]. Figure S2. UHPLC-HDMS BPI chromatograms of ESI-positive data indicating the metabolomic profiles of untreated (black), naïve infected (blue) and primed infected (green) stems obtained at 1 d.p.i. with F. pseudograminearum. Figure S3. UHPLC-HDMS BPI chromatograms of ESI-positive data indicating the metabolomic profiles of untreated (black), naïve infected (blue) and primed infected (green) leaves obtained at 1 d.p.i. with F. pseudograminearum. Figure S4. PCA score/scatter plot of stem samples computed from ESI-positive data. Figure S5. PCA score/scatter plot of leaf samples computed from ESI-positive data. Figure S6. PCA score/scatter plot of root samples computed from ESI-negative data. Figure S7. PCA score/scatter plot of stems samples computed from ESI-negative data. Figure S8. PCA score/scatter plot of leaves samples computed from ESI-negative data. Figure S9. OPLS-DA modelling and variable/feature selection ESI-positive data (stem samples). Figure S10. OPLS-DA modelling and variable/feature selection ESI-positive data (leaf samples). Table S1. Summary of the description and validation of all the generated OPLS-DA models separating naïve versus primed S. bicolor plants. Figure S11. Summary of pathway analysis with MetPA. Figure S12. Venn diagram comparing the number of metabolites shown in Table 2 that were significantly upregulated at 1 d.p.i. (blue), 4 d.p.i. (yellow) and 7 d.p.i. (green) with F. pseudograminearum in primed versus naïve S. bicolor seedlings.https://www.mdpi.com/journal/metaboliteshj2020Plant Production and Soil Scienc

    Metabolomic Analysis of Defense-Related Reprogramming in Sorghum bicolor in Response to Colletotrichum sublineolum Infection Reveals a Functional Metabolic Web of Phenylpropanoid and Flavonoid Pathways

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    The metabolome of a biological system provides a functional readout of the cellular state, thus serving as direct signatures of biochemical events that define the dynamic equilibrium of metabolism and the correlated phenotype. Hence, to elucidate biochemical processes involved in sorghum responses to fungal infection, a liquid chromatography-mass spectrometry-based untargeted metabolomic study was designed. Metabolic alterations of three sorghum cultivars responding to Colletotrichum sublineolum, were investigated. At the 4-leaf growth stage, the plants were inoculated with fungal spore suspensions and the infection monitored over time: 0, 3, 5, 7, and 9 days post inoculation. Non-infected plants were used as negative controls. The metabolite composition of aqueous-methanol extracts were analyzed on an ultra-high performance liquid chromatography system coupled to high-definition mass spectrometry. The acquired multidimensional data were processed to create data matrices for multivariate statistical analysis and chemometric modeling. The computed chemometric models indicated time- and cultivar-related metabolic changes that reflect sorghum responses to the fungal infection. Metabolic pathway and correlation-based network analyses revealed that this multi-component defense response is characterized by a functional metabolic web, containing defense-related molecular cues to counterattack the pathogen invasion. Components of this network are metabolites from a range of interconnected metabolic pathways with the phenylpropanoid and flavonoid pathways being the central hub of the web. One of the key features of this altered metabolism was the accumulation of an array of phenolic compounds, particularly de novo biosynthesis of the antifungal 3-deoxyanthocynidin phytoalexins, apigeninidin, luteolinidin, and related conjugates. The metabolic results were complemented by qRT-PCR gene expression analyses that showed upregulation of defense-related marker genes. Unraveling key characteristics of the biochemical mechanism underlying sorghum—C. sublineolum interactions, provided valuable insights with potential applications in breeding crop plants with enhanced disease resistance. Furthermore, the study contributes to ongoing efforts toward a comprehensive understanding of the regulation and reprogramming of plant metabolism under biotic stress

    Metabolomic evaluation of PGPR defence priming in wheat (Triticum aestivum L.) cultivars infected with Puccinia striiformis f. sp. tritici (stripe rust)

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    Plant-microbe interactions are a phenomenal display of symbiotic/parasitic relationships between living organisms. Plant growth-promoting rhizobacteria (PGPR) are some of the most widely investigated plant-beneficial microbes due to their capabilities in stimulating plant growth and development and conferring protection to plants against biotic and abiotic stresses. As such, PGPR-mediated plant priming/induced systemic resistance (ISR) has become a hot topic among researchers, particularly with prospects of applications in sustainable agriculture. The current study applies untargeted ultra-high performance liquid chromatography-high-definition mass spectrometry (UHPLC-HDMS) to investigate PGPR-based metabolic reconfigurations in the metabolome of primed wheat plants against Puccinia striiformis f. sp. tricti (Pst). A seed bio-priming approach was adopted, where seeds were coated with two PGPR strains namely Bacillus subtilis and Paenibacillus alvei (T22) and grown under controlled conditions in a glasshouse. The plants were infected with Pst one-week post-germination, followed by weekly harvesting of leaf material. Subsequent metabolite extraction was carried out for analysis on a UHPLC-HDMS system for data acquisition. The data was chemometrically processed to reveal the underlying trends and data structures as well as potential signatory biomarkers for priming against Pst. Results showed notable metabolic reprogramming in primary and secondary metabolism, where the amino acid and organic acid content of primed-control, primed-challenged and non-primed-challenged plants were differentially reprogrammed. Similar trends were observed from the secondary metabolism, in which primed plants (particularly primed-challenged) showed an up-regulation of phenolic compounds (flavonoids, hydroxycinnamic acids-HCAs- and HCA amides) compared to the non-primed plants. The metabolomics-based semi-quantitative and qualitative assessment of the plant metabolomes revealed a time-dependent metabolic reprogramming in primed-challenged and primed-unchallenged plants, indicating the metabolic adaptations of the plants to stripe rust infection over time

    The Effect of Geometrical Isomerism of 3,5-Dicaffeoylquinic Acid on Its Binding Affinity to HIV-Integrase Enzyme: A Molecular Docking Study

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    A potent plant-derived HIV-1 inhibitor, 3,5-dicaffeoylquinic acid (diCQA), has been shown to undergo isomerisation upon UV exposure where the naturally occurring 3trans,5trans-diCQA isomer gives rise to the 3cis,5trans-diCQA, 3trans,5cis-diCQA, and 3cis,5cis-diCQA isomers. In this study, inhibition of HIV-1 INT by UV-induced isomers was investigated using molecular docking methods. Here, density functional theory (DFT) models were used for geometry optimization of the 3,5-diCQA isomers. The YASARA and Autodock VINA software packages were then used to determine the binding interactions between the HIV-1 INT catalytic domain and the 3,5-diCQA isomers and the Discovery Studio suite was used to visualise the interactions between the isomers and the protein. The geometrical isomers of 3,5-diCQA were all found to bind to the catalytic core domain of the INT enzyme. Moreover, the cis geometrical isomers were found to interact with the metal cofactor of HIV-1INT, a phenomenon which has been linked to antiviral potency. Furthermore, the 3trans,5cis-diCQA isomer was also found to interact with both LYS156 and LYS159 which are important residues for viral DNA integration. The differences in binding modes of these naturally coexisting isomers may allow wider synergistic activity which may be beneficial in comparison to the activities of each individual isomer

    A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data

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    MicrobeMASST, a taxonomically-informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbial-derived metabolites and relative producers, without a priori knowledge, will vastly enhance the understanding of microorganisms’ role in ecology and human health
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