Short title: Seed-specific saponin biosynthesis in Medicago One-sentence summary: The discovery of a seed-specific regulator of hemolytic saponin biosynthesis in Medicago truncatula led to the identification of the missing P450 of the hemolytic saponin biosynthesis branch.

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.

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.