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

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

Metabolomics in Plant Priming Research: The Way Forward?

A new era of plant biochemistry at the systems level is emerging, providing detailed descriptions of biochemical phenomena at the cellular and organismal level. This new era is marked by the advent of metabolomics—the qualitative and quantitative investigation of the entire metabolome (in a dynamic equilibrium) of a biological system. This field has developed as an indispensable methodological approach to study cellular biochemistry at a global level. For protection and survival in a constantly-changing environment, plants rely on a complex and multi-layered innate immune system. This involves surveillance of ‘self’ and ‘non-self,’ molecule-based systemic signalling and metabolic adaptations involving primary and secondary metabolites as well as epigenetic modulation mechanisms. Establishment of a pre-conditioned or primed state can sensitise or enhance aspects of innate immunity for faster and stronger responses. Comprehensive elucidation of the molecular and biochemical processes associated with the phenotypic defence state is vital for a better understanding of the molecular mechanisms that define the metabolism of plant–pathogen interactions. Such insights are essential for translational research and applications. Thus, this review highlights the prospects of metabolomics and addresses current challenges that hinder the realisation of the full potential of the field. Such limitations include partial coverage of the metabolome and maximising the value of metabolomics data (extraction of information and interpretation). Furthermore, the review points out key features that characterise both the plant innate immune system and enhancement of the latter, thus underlining insights from metabolomic studies in plant priming. Future perspectives in this inspiring area are included, with the aim of stimulating further studies leading to a better understanding of plant immunity at the metabolome level

Plasma Membrane-Associated Proteins Identified in Arabidopsis Wild Type, <i>lbr2-2</i> and <i>bak1-4</i> Mutants Treated with LPSs from <i>Pseudomonas</i> <i>syringae</i> and <i>Xanthomonas campestris</i>

Plants recognise bacterial microbe-associated molecular patterns (MAMPs) from the environment via plasma membrane (PM)-localised pattern recognition receptor(s) (PRRs). Lipopolysaccharides (LPSs) are known as MAMPs from gram-negative bacteria that are most likely recognised by PRRs and trigger defence responses in plants. The Arabidopsis PRR(s) and/or co-receptor(s) complex for LPS and the associated defence signalling remains elusive. As such, proteomic identification of LPS receptors and/or co-receptor complexes will help to elucidate the molecular mechanisms that underly LPS perception and defence signalling in plants. The Arabidopsis LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI)-related-2 (LBR2) have been shown to recognise LPS and trigger defence responses while brassinosteroid insensitive 1 (BRI1)-associated receptor kinase 1 (BAK1) acts as a co-receptor for several PRRs. In this study, Arabidopsis wild type (WT) and T-DNA knock out mutants (lbr2-2 and bak1-4) were treated with LPS chemotypes from Pseudomonas syringae pv. tomato DC3000 (Pst) and Xanthomonas campestris pv. campestris 8004 (Xcc) over a 24 h period. The PM-associated protein fractions were separated by liquid chromatography and analysed by tandem mass spectrometry (LC-MS/MS) followed by data analysis using ByonicTM software. Using Gene Ontology (GO) for molecular function and biological processes, significant LPS-responsive proteins were grouped according to defence and stress response, perception and signalling, membrane transport and trafficking, metabolic processes and others. Venn diagrams demarcated the MAMP-responsive proteins that were common and distinct to the WT and mutant lines following treatment with the two LPS chemotypes, suggesting contributions from differential LPS sub-structural moieties and involvement of LBR2 and BAK1 in the LPS-induced MAMP-triggered immunity (MTI). Moreover, the identification of RLKs and RLPs that participate in other bacterial and fungal MAMP signalling proposes the involvement of more than one receptor and/or co-receptor for LPS perception as well as signalling in Arabidopsis defence responses

Concurrent metabolic profiling and quantification of aromatic amino acids and phytohormones in solanum lycopersicum plants responding to phytophthora capsici

Pathogenic microorganisms account for large production losses in the agricultural sector. Phytophthora capsici is an oomycete that causes blight and fruit rot in important crops, especially those in the Solanaceae family. P. capsici infection is di cult to control due to genetic diversity, arising from sexual reproduction, and resistant spores that remain dormant in soil. In this study, the metabolomics of tomato plants responding to infection by P. capsici were investigated. Non-targeted metabolomics, based on liquid chromatography coupled to mass spectrometry (LC-MS), were used with multivariate data analyses to investigate time-dependent metabolic reprogramming in the roots, stems, and leaves of stem-infected plants, over an 8 day period. In addition, phytohormones and amino acids were determined using quantitative LC-MS. Methyl salicylate and 1-aminocyclopropane-1-carboxylate were detected as major signalling molecules in the defensive response to P. capsici. As aromatic amino acid precursors of secondary metabolic pathways, both phenylalanine and tryptophan showed a continuous increase over time in all tissues, whereas tyrosine peaked at day 4. Non-targeted metabolomic analysis revealed phenylpropanoids, benzoic acids, glycoalkaloids, flavonoids, amino acids, organic acids, and fatty acids as the major classes of reprogrammed metabolites. Correlation analysis showed that metabolites derived from the same pathway, or synthesised by di erent pathways, could either have a positive or negative correlation. Furthermore, roots, stems, and leaves showed contrasting time-dependent metabolic reprogramming, possibly related to the biotrophic vs. necrotrophic life-stages of the pathogen, and overlapping biotic and abiotic stress signaling. As such, the targeted and untargeted approaches complemented each other, to provide a detailed view of key time-dependent metabolic changes, occurring in both the asymptomatic and symptomatic stages of infection.http://www.mdpi.com/journal/metabolitesam2021Plant Production and Soil Scienc

Metabolomic evaluation of tissue specific defense responses in tomato plants modulated by PGPR-priming against Phytophthora capsici infection

Plant growth-promoting rhizobacteria (PGPR) can stimulate disease suppression through the induction of an enhanced state of defense readiness. Here, untargeted ultra-high performance liquid chromatography–mass spectrometry (UHPLC–MS) and targeted ultra-high performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC–QqQ-MS) were used to investigate metabolic reprogramming in tomato plant tissues in response to priming by Pseudomonas fluorescens N04 and Paenibacillus alvei T22 against Phytophthora capsici. Roots were treated with the two PGPR strains prior to stem inoculation with Ph. capsici. Metabolites were methanol-extracted from roots, stems and leaves at two–eight days post-inoculation. Targeted analysis by UHPLC–QqQMS allowed quantification of aromatic amino acids and phytohormones. For untargeted analysis, UHPLC–MS data were chemometrically processed to determine signatory biomarkers related to priming against Ph. capsici. The aromatic amino acid content was differentially reprogrammed in Ps. fluorescens and Pa. alvei primed plants responding to Ph. capsici. Furthermore, abscisic acid and methyl salicylic acid were found to be major signaling molecules in the tripartite interaction. LC– MS metabolomics analysis showed time-dependent metabolic changes in the primed-unchallenged vs. primed-challenged tissues. The annotated metabolites included phenylpropanoids, benzoic acids, glycoalkaloids, flavonoids, amino acids, organic acids, as well as oxygenated fatty acids. Tissue-specific reprogramming across diverse metabolic networks in roots, stems and leaves was also observed, which demonstrated that PGPR priming resulted in modulation of the defense response to Ph. capsici infection.The South African National Research Foundationhttp://www.mdpi.com/journal/plantspm2022Plant Production and Soil Scienc