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

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

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

Isolation by PCR-based methods of a plant antifungal polygalacturonase-inhibiting protein gene

A polygalacturonase-inhibiting protein (pgip) gene from Malus domestica cv Granny Smith apple plants was cloned by degenerate oligo-primed polymerase chain reaction (PCR) and Inverse PCR. An alignment of the pear and bean pgip sequences was used to design degenerate PCR primers in highly conserved regions. Degenerate PCR allowed the amplification of a 351bp internal fragment of the pgip gene, termed ipgip. The DNA sequence of ipgip was used to design Inverse PCR primers. A Southern blot of apple genomic DNA probed with the ipgip fragment was used to identify restriction enzyme sites for Inverse PCR. Inverse PCR enabled cloning of the remainder of the gene, from which a composite pgip gene sequence was constructed. A new set of PCR primers were designed to the 5' and 3' ends of the gene, which allowed amplification of the full-length gene from apple genomic DNA. This method has broad application to isolation of homologues of any gene for which some sequence information is known

Molecular characterisation of two homoeologous elicitor-responsive lipin genes in cotton

The identification and molecular characterisation of two lipin-like gene copies (GhLIPN) in cotton, Gossypium hirsutum, an allotetraploid derived from two progenitor diploid Gossypium species, is described. Sequence analyses of the GhLIPN copies, designated GhLIPN-1 and -2, revealed that they contain 11 exons, separated by ten introns. They each have a 2,643 bp open reading frame that encodes 880 aa proteins, and share a 97.7 and 95.5 % sequence similarity at the translated nucleotide and amino acid level, respectively. The GhLIPN genes have a distinct domain architecture consisting of an archetypical N-terminal lipin domain, followed by a haloacid dehalogenase (HAD) domain towards the C-terminus. A Southern blot did not distinguish between the two gene copies, which suggests that they may be homoeologs rather than paralogs. GhLIPN-2 is more similar to a homoeologous sequence from G. raimondii, representing the ancestral D-genome, compared to GhLIPN-1 that matches G. herbaceum and that represents the A-genome. Our data indicate that GhLIPN-1 and GhLIPN-2 are homoeologs that derive from the A- and the D-diploid genomes, respectively. The promoter sequences of GhLIPN-1 and -2 differ by 56 %, as a result of multiple indels. In silico analysis of the promoter regions revealed that both genes contain numerous putative defence-related and elicitor-responsive cis-elements that support a role for GhLIPN in defence responses. Relative quantification real-time PCR confirmed the up-regulation in response to a cell-wall-derived V. dahliae elicitor, which supported the association of GhLIPN with defence signalling. The results add a new dimension to the proposed roles of lipins in plants by suggesting that lipins may have a role in defence signalling.The South African National Research Foundation and the University of Johannesburg.http://link.springer.com/journal/438hb2013ab201

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

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

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

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

Rhizobacteria-induced systemic resilience in Sorghum bicolor (L.) moench against Fusarium pseudograminearum crown rot under drought stress conditions

The potential of 77 rhizobacterial isolates to elicit induced systemic resilience (ISResilience) against combined biotic (Fusarium pseudograminearum crown rot) and abiotic (drought) stress in Sorghum bicolor was investigated. ISResilience was determined by assessing disease incidence and severity, plant height and biomass (root and shoots) in rhizobacteria-primed and untreated (naïve) plants inoculated with F. pseudograminearum and subjected to drought stress. Three rhizobacterial isolates (Paenibacillus alvei NAS-6G6, Pseudomonas taiwanensis N66 and Bacillus velezensis N54) showed significant protection of S. bicolor seedlings against biotic, abiotic and combined biotic and abiotic stress. Isolate N54, identified in this study as B. velezensis by 16S rRNA sequencing, was considered as the best performing rhizobacterial isolate to elicit ISResilience. Untargeted ultra-high performance liquid chromatography-high definition mass spectrometry (UHPLC-HDMS) based metabolomics was used to investigate the mechanism by which ISResilience was elicited in S. bicolor by strain N54 (B. velezensis). Comparisons were made with isolates that were previously selected for induced systemic tolerance (ISTolerance) against drought stress (strain N66, Ps. taiwanensis) and induced systemic resistance (ISResistance) against F. pseudograminearum crown rot (strain NAS-6G6, Pa. alvei). The stress alleviation that resulted from treatment with the respective rhizobacterial isolates, was visually confirmed by the use of infrared (IR) thermography. For the metabolomics study, intracellular metabolites were methanol-extracted from rhizobacteria-primed and untreated (naïve) S. bicolor shoots. Extracts were analyzed on an UHPLC-HDMS platform, and the data were chemometrically analyzed to determine metabolite bio-markers related to ISResistance, ISTolerance and ISResilience. The results demonstrated significant treatment-related differences, reflecting differential metabolic reprogramming in S. bicolor in response to the biotic, abiotic and combined stresses. Synergistic effects involved in the lowered susceptibility to crown rot of rhizobacteria-primed S. bicolor seedlings, compared to those left naïve (untreated control) under drought stress conditions and the upregulation of the signatory molecules myo-inositol and riboflavin, provided evidence for the role of crosstalk in the ISResilience observed.The National Research Foundation (NRF) of South Africa and the University of Pretoria.http://www.elsevier.com/locate/ybcon2021-08-01hj2021Plant Production and Soil Scienc