Search

The plant kingdom represents the biggest source of feedstock, food, and added-value compounds. Engineering plant metabolic pathways to increase the phytochemical production or improve the nutraceutical value of crops is challenging because of the intricate interaction networks that link multiple genes, enzymatic steps, and metabolites, even when pathways are fully elucidated. The development of clustered regularly interspaced short palindromic repeats - CRISPR-associated (CRISPR-Cas) technologies has helped to overcome limitations in metabolic engineering, providing efficient and versatile tools for multigene editing. CRISPR approaches in plants were shown to have a remarkable efficiency in genome editing of different species to improve agronomic and metabolic traits. Here, we give an overview of the different achievements and perspectives of CRISPR technology in plant metabolic engineering.

Pathogens produce effectors to overcome plant immunity, thereby threatening crop yields and global food security. Large-scale interactomic studies have revealed that pathogens from different kingdoms of life target common plant proteins during infection, the so-called effector hubs. These hubs often play central roles in numerous plant processes through their ability to interact with multiple plant proteins. This ability arises partly from the presence of intrinsically disordered domains (IDDs) in their structure. Here, we highlight the role of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) and JASMONATE-ZIM DOMAIN (JAZ) transcription regulator families as plant signaling and effector hubs. We consider different evolutionary hypotheses to rationalize the existence of diverse effectors sharing common targets and the possible role of IDDs in this interaction.

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.

Specialized metabolites are produced by plants to fend off biotic enemies. Across the plant kingdom, the biosynthesis of these defense compounds is promoted by jasmonate signaling in which clade IIIe basic helix-loop-helix (bHLH) transcription factors take on a central role. Tomato (Solanum lycopersicum) produces cholesterol-derived steroidal glycoalkaloids (SGAs) that act as phytoanticipins against a broad variety of herbivores and pathogens. The biosynthesis of SGAs from cholesterol occurs constitutively in tomato plants and can be further stimulated by jasmonates. Here, we demonstrate that the two tomato clade IIIe bHLH transcription factors, MYC1 and MYC2, redundantly and specifically control the constitutive biosynthesis of SGAs. Double myc1 myc2 loss-of-function tomato hairy roots displayed suppressed constitutive expression of cholesterol and SGA biosynthesis genes, and consequently severely reduced levels of the main tomato SGAs α-tomatine and dehydrotomatine. In contrast, basal expression of genes involved in canonical jasmonate signaling or in the biosynthesis of highly jasmonate-inducible phenylpropanoid-polyamine conjugates was not affected. Furthermore, CRISPR-Cas9(VQR)-mediated genome editing of a specific cis-regulatory element, targeted by MYC1/2, in the promoter of a cholesterol 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 might have evolved to regulate the biosynthesis of specific constitutively accumulating specialized metabolites independent of jasmonate signaling.One sentence summaryThe clade IIIe basic helix-loop-helix transcription factors MYC1 and MYC2 control the constitutive biosynthesis of tomato steroidal glycoalkaloids and might do so independently of jasmonate signaling.

Made available in DSpace on 2020-12-12T02:09:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Plants produce a vast array of defense compounds to protect themselves from pathogen attack or herbivore predation. Saponins are a specific class of defense compounds comprising bioactive glycosides with a steroidal or triterpenoid aglycone backbone. The model legume Medicago truncatula synthesizes two types of saponins, hemolytic saponins and nonhemolytic soyasaponins, which accumulate as specific blends in different plant organs. Here, we report the identification of the seed-specific transcription factor TRITERPENE SAPONIN ACTIVATION REGULATOR3 (TSAR3), which controls hemolytic saponin biosynthesis in developing M. truncatula seeds. Analysis of genes that are coexpressed with TSAR3 in transcriptome data sets from developing M. truncatula seeds led to the identification of CYP88A13, a cytochrome P450 that catalyzes the C-16a hydroxylation of medicagenic acid toward zanhic acid, the final oxidation step of the hemolytic saponin biosynthesis branch in M. truncatula. In addition, two uridine diphosphate glycosyltransferases, UGT73F18 and UGT73F19, which glucosylate hemolytic sapogenins at the C-3 position, were identified. The genes encoding the identified biosynthetic enzymes are present in clusters of duplicated genes in the M. truncatula genome. This appears to be a common theme among saponin biosynthesis genes, especially glycosyltransferases, and may be the driving force of the metabolic evolution of saponins. Ghent University Department of Plant Biotechnology and Bioinformatics VIB Center for Plant Systems Biology Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP) Institut de Recherche en Horticulture et Semences-Unités Mixtes de Recherche Université d’Angers INRAE Institut Agro, SFR 4207 QuaSaV VIB Metabolomics Core Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP)

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.