Cataloguing Protein Complexes In Planta Using TurboID-Catalyzed Proximity Labeling.

Mapping protein-protein interactions is crucial to understand protein function. Recent advances in proximity-dependent biotinylation (BioID) coupled to mass spectrometry (MS) allow the characterization of protein complexes in diverse plant models. Here, we describe the use of BioID in hairy root cultures of tomato and provide detailed information on how to analyze the data obtained by MS.

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 Heat Shock Protein 40-Type Chaperone MASH Supports the Endoplasmic Reticulum-Associated Degradation E3 Ubiquitin Ligase MAKIBISHI1 in Medicago truncatula.

Jasmonates (JA) are oxylipin-derived phytohormones that trigger the production of specialized metabolites that often serve in defense against biotic stresses. In Medicago truncatula, a JA-induced endoplasmic reticulum-associated degradation (ERAD)-type machinery manages the production of bioactive triterpenes and thereby secures correct plant metabolism, growth, and development. This machinery involves the conserved RING membrane-anchor (RMA)-type E3 ubiquitin ligase MAKIBISHI1 (MKB1). Here, we discovered two additional members of this protein control apparatus via a yeast-based protein-protein interaction screen and characterized their function. First, a cognate E2 ubiquitin-conjugating enzyme was identified that interacts with MKB1 to deliver activated ubiquitin and to mediate its ubiquitination activity. Second, we identified a heat shock protein 40 (HSP40) that interacts with MKB1 to support its activity and was therefore designated MKB1-supporting HSP40 (MASH). MASH expression was found to be co-regulated with that of MKB1. The presence of MASH is critical for MKB1 and ERAD functioning because the dramatic morphological, transcriptional, and metabolic phenotype of MKB1 knock-down M. truncatula hairy roots was phenocopied by silencing of MASH. Interaction was also observed between the Arabidopsis thaliana (Arabidopsis) homologs of MASH and MKB1, suggesting that MASH represents an essential and plant-specific component of this vital and conserved eukaryotic protein quality control machinery.

A Seed-Specific Regulator of Triterpene Saponin Biosynthesis in Medicago truncatula.

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-16α 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.