Rhizogenic Agrobacterium protein RolB interacts with the TOPLESS repressor proteins to reprogram plant immunity and development.

Rhizogenic Agrobacterium strains comprise biotrophic pathogens that cause hairy root disease (HRD) on hydroponically grown Solanaceae and Cucurbitaceae crops, besides being widely explored agents for the creation of hairy root cultures for the sustainable production of plant-specialized metabolites. Hairy root formation is mediated through the expression of genes encoded on the T-DNA of the root-inducing (Ri) plasmid, of which several, including root oncogenic locus B (rolB), play a major role in hairy root development. Despite decades of research, the exact molecular function of the proteins encoded by the rol genes remains enigmatic. Here, by means of TurboID-mediated proximity labeling in tomato (Solanum lycopersicum) hairy roots, we identified the repressor proteins TOPLESS (TPL) and Novel Interactor of JAZ (NINJA) as direct interactors of RolB. Although these interactions allow RolB to act as a transcriptional repressor, our data hint at another in planta function of the RolB oncoprotein. Hence, by a series of plant bioassays, transcriptomic and DNA-binding site enrichment analyses, we conclude that RolB can mitigate the TPL functioning so that it leads to a specific and partial reprogramming of phytohormone signaling, immunity, growth, and developmental processes. Our data support a model in which RolB manipulates host transcription, at least in part, through interaction with TPL, to facilitate hairy root development. Thereby, we provide important mechanistic insights into this renowned oncoprotein in HRD.

The saponin bomb: a nucleolar-localized ß-glucosidase hydrolyzes triterpene saponins in Medicago truncatula.

Plants often protect themselves from their own bioactive defense metabolites by storing them in less active forms. Consequently, plants also need systems allowing correct spatiotemporal reactivation of such metabolites, for instance under pathogen or herbivore attack. Via co-expression analysis with public transcriptomes, we determined that the model legume Medicago truncatula has evolved a two-component system composed of a ß-glucosidase, denominated G1, and triterpene saponins, which are physically separated from each other in intact cells. G1 expression is root-specific, stress-inducible, and coregulated with that of the genes encoding the triterpene saponin biosynthetic enzymes. However, the G1 protein is stored in the nucleolus and is released and united with its typically vacuolar-stored substrates only upon tissue damage, partly mediated by the surfactant action of the saponins themselves. Subsequently, enzymatic removal of carbohydrate groups from the saponins creates a pool of metabolites with an increased broad-spectrum antimicrobial activity. The evolution of this defense system benefited from both the intrinsic condensation abilities of the enzyme and the bioactivity properties of its substrates. We dub this two-component system the saponin bomb, in analogy with the mustard oil and cyanide bombs, commonly used to describe the renowned ß-glucosidase-dependent defense systems for glucosinolates and cyanogenic glucosides.

The basic helix-loop-helix transcription factors MYC1 and MYC2 have a dual role in the regulation of constitutive and stress-inducible specialized metabolism in tomato.

Plants produce specialized metabolites to protect themselves from biotic enemies. Members of the Solanaceae family accumulate phenylpropanoid-polyamine conjugates (PPCs) in response to attackers while also maintaining a chemical barrier of steroidal glycoalkaloids (SGAs). Across the plant kingdom, biosynthesis of such defense compounds is promoted by jasmonate signaling in which clade IIIe basic helix-loop-helix (bHLH) transcription factors play a central role. By characterizing hairy root mutants obtained through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) genome editing, we show that the tomato clade IIIe bHLH transcription factors, MYC1 and MYC2, redundantly control jasmonate-inducible PPC and SGA production, and are also essential for constitutive SGA biosynthesis. Double myc1 myc2 loss-of-function tomato hairy roots displayed suppressed constitutive expression of SGA biosynthesis genes, and severely reduced levels of the main tomato SGAs α-tomatine and dehydrotomatine. In contrast, basal expression of genes involved in PPC biosynthesis was not affected. CRISPR-Cas9(VQR) genome editing of a specific cis-regulatory element, targeted by MYC1/2, in the promoter of a SGA precursor biosynthesis gene led to decreased constitutive expression of this gene, but did not affect its jasmonate inducibility. Our results demonstrate that clade IIIe bHLH transcriptional regulators have evolved under the control of distinct regulatory cues to specifically steer constitutive and stress-inducible specialized metabolism.

Involvement of sweet pepper CaLOX2 in jasmonate-dependent induced defence against Western flower thrips.

Insect herbivory can seriously hinder plant performance and reduce crop yield. Thrips are minute cell-content-feeding insects that are important vectors of viral plant pathogens, and are serious crop pests. We investigated the role of a sweet pepper (Capsicum annuum) lipoxygenase gene, CaLOX2, in the defense of pepper plants against Western flower thrips (Frankliniella occidentalis). This was done through a combination of in-silico, transcriptional, behavioral and chemical analyses. Our data show that CaLOX2 is involved in jasmonic acid (JA) biosynthesis and mediates plant resistance. Expression of the JA-related marker genes, CaLOX2 and CaPIN II, was induced by thrips feeding. Silencing of CaLOX2 in pepper plants through virus-induced gene silencing (VIGS) resulted in low levels of CaLOX2 transcripts, as well as significant reduction in the accumulation of JA, and its derivatives, upon thrips feeding compared to control plants. CaLOX2-silenced pepper plants exhibited enhanced susceptibility to thrips. This indicates that CaLOX2 mediates JA-dependent signaling, resulting in defense against thrips. Furthermore, exogenous application of JA to pepper plants increased plant resistance to thrips, constrained thrips population development and made plants less attractive to thrips. Thus, a multidisciplinary approach shows that an intact lipoxygenase pathway mediates various components of sweet pepper defense against F. occidentalis.

Identification of Plant Protein-Metabolite Interactions by Limited Proteolysis-Coupled Mass Spectrometry (LiP-MS).

The interactions between metabolites and proteins constitute crucial events in cell signaling and metabolism. In recent years, large-scale proteomics techniques have emerged to identify and characterize protein-metabolite interactions. However, their implementation in plants is generally lagging behind, preventing a complete understanding of the regulatory mechanisms governing plant physiology. Recently, a novel approach to identify metabolite-binding proteins, namely, limited proteolysis-coupled mass spectrometry (LiP-MS), was developed originally for microbial proteomes. Here, we present an adapted and accessible version of the LiP-MS protocol for use in plants. Plant proteomes are extracted and incubated with the metabolite of interest or control treatment, followed by a limited digestion by a nonspecific/promiscuous protease. Subsequently, a conventional shotgun proteomics sample preparation is performed including a complete digestion with the sequence-specific protease trypsin. Finally, label-free proteomics analysis is applied to identify structure-dependent proteolytic patterns corresponding to protein targets of the specific metabolite and their binding sites. Given its amenability to relatively high throughput, the LiP-MS approach may open a potent avenue for the discovery of novel regulatory mechanisms in plant species.

Structural and functional diversity in plant specialized metabolism signals and products: The case of oxylipins and triterpenes.

Metabolic enzymes tend to evolve towards catalytic efficacy, precision and speed. This seems particularly true for ancient and conserved enzymes involved in fundamental cellular processes that are present virtually in every cell and organism and converting and producing relatively limited metabolite numbers. Nevertheless, sessile organisms like plants have an astonishing repertoire of specific (specialized) metabolites that, by numbers and chemical complexity, by far exceed primary metabolites. Most theories agree that early gene duplication, subsequent positive selection and diversifying evolution have allowed relaxed selection of duplicated metabolic genes, thus facilitating the accumulation of mutations that could broaden substrate/product specificity and lower activation barriers and kinetics. Here, we use oxylipins, oxygenated fatty acids of plastidial origin to which the phytohormone jasmonate belongs, and triterpenes, a large group of specialized metabolites whose biosynthesis is often elicited by jasmonates, to showcase the structural and functional diversity of chemical signals and products in plant metabolism.

Why and How to Dig into Plant Metabolite-Protein Interactions.

Interaction between metabolites and proteins drives cellular regulatory processes within and between organisms. Recent reports highlight that numerous plant metabolites embrace multiple biological activities, beyond a sole role as substrates, products, or cofactors of enzymes, or as defense or growth-regulatory compounds. Though several technologies have been developed to identify and characterize metabolite-protein interactions, the systematic implementation of such methods in the plant field remains limited. Here, we discuss the plant metabolic space, with a specific focus on specialized metabolites and their roles, and review the technologies to study their interaction with proteins. We approach it both from a plant's perspective, to increase our understanding of plant metabolite-dependent regulatory networks, and from a human perspective, to empower agrochemical and drug discoveries.

Induced tolerance to abiotic and biotic stresses of broccoli and Arabidopsis after treatment with elicitor molecules.

The plant hormones salicylic acid (SA) and jasmonic acid (JA) regulate defense mechanisms capable of overcoming different plant stress conditions and constitute distinct but interconnected signaling pathways. Interestingly, several other molecules are reported to trigger stress-specific defense responses to biotic and abiotic stresses. In this study, we investigated the effect of 14 elicitors against diverse but pivotal types of abiotic (drought) and biotic (the chewing insect Ascia monuste, the hemibiotrophic bacterium Pseudomonas syringae DC 3000 and the necrotrophic fungus Alternaria alternata) stresses on broccoli and Arabidopsis. Among the main findings, broccoli pre-treated with SA and chitosan showed the highest drought stress recovery in a dose-dependent manner. Several molecules led to increased drought tolerance over a period of three weeks. The enhanced drought tolerance after triggering the SA pathway was associated with stomata control. Moreover, methyl jasmonate (MeJA) reduced A. monuste insect development and plant damage, but unexpectedly, other elicitors increased both parameters. GUS reporter assays indicated expression of the SA-dependent PR1 gene in plants treated with nine elicitors, whereas the JA-dependent LOX2 gene was only expressed upon MeJA treatment. Overall, elicitors capable of tackling drought and biotrophic pathogens mainly triggered the SA pathway, but adversely also induced systemic susceptibility to chewing insects. These findings provide directions for potential future in-depth characterization and utilization of elicitors and induced resistance in plant protection.