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.

Methyl Jasmonate and Methyl-ß-Cyclodextrin Individually Boost Triterpenoid Biosynthesis in Chlamydomonas Reinhardtii UVM4.

The commercialisation of valuable plant triterpenoids faces major challenges, including low abundance in natural hosts and costly downstream purification procedures. Endeavours to produce these compounds at industrial scale using microbial systems are gaining attention. Here, we report on a strategy to enrich the biomass of the biotechnologically-relevant Chlamydomonas reinhardtii strain UVM4 with valuable triterpenes, such as squalene and (S)-2,3-epoxysqualene. C. reinhardtii UVM4 was subjected to the elicitor compounds methyl jasmonate (MeJA) and methyl-ß-cyclodextrine (MßCD) to increase triterpene yields. MeJA treatment triggered oxidative stress, arrested growth, and altered the photosynthetic activity of the cells, while increasing squalene, (S)-2,3-epoxysqualene, and cycloartenol contents. Applying MßCD to cultures of C. reinhardtii lead to the sequestration of the two main sterols (ergosterol and 7-dehydroporiferasterol) into the growth medium and the intracellular accumulation of the intermediate cycloartenol, without compromising cell growth. When MßCD was applied in combination with MeJA, it counteracted the negative effects of MeJA on cell growth and physiology, but no synergistic effect on triterpene yield was observed. Together, our findings provide strategies for the triterpene enrichment of microalgal biomass and medium.

Overexpression of Key Sterol Pathway Enzymes in Two Model Marine Diatoms Alters Sterol Profiles in Phaeodactylum tricornutum.

Sterols are a class of triterpenoid molecules with diverse functional roles in eukaryotic cells, including intracellular signaling and regulation of cell membrane fluidity. Diatoms are a dominant eukaryotic phytoplankton group that produce a wide diversity of sterol compounds. The enzymes 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR) and squalene epoxidase (SQE) have been reported to be rate-limiting steps in sterol biosynthesis in other model eukaryotes; however, the extent to which these enzymes regulate triterpenoid production in diatoms is not known. To probe the role of these two metabolic nodes in the regulation of sterol metabolic flux in diatoms, we independently over-expressed two versions of the native HMGR and a conventional, heterologous SQE gene in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Overexpression of these key enzymes resulted in significant differential accumulation of downstream sterol pathway intermediates in P. tricornutum. HMGR-mVenus overexpression resulted in the accumulation of squalene, cycloartenol, and obtusifoliol, while cycloartenol and obtusifoliol accumulated in response to heterologous NoSQE-mVenus overexpression. In addition, accumulation of the end-point sterol 24-methylenecholesta-5,24(24')-dien-3ß-ol was observed in all P. tricornutum overexpression lines, and campesterol increased three-fold in P. tricornutum lines expressing NoSQE-mVenus. Minor differences in end-point sterol composition were also found in T. pseudonana, but no accumulation of sterol pathway intermediates was observed. Despite the successful manipulation of pathway intermediates and individual sterols in P. tricornutum, total sterol levels did not change significantly in transformed lines, suggesting the existence of tight pathway regulation to maintain total sterol content.

Extrachromosomal Genetic Engineering of the Marine Diatom Phaeodactylum tricornutum Enables the Heterologous Production of Monoterpenoids.

Geraniol is a commercially relevant plant-derived monoterpenoid that is a main component of rose essential oil and used as insect repellent. Geraniol is also a key intermediate compound in the biosynthesis of the monoterpenoid indole alkaloids (MIAs), a group of over 2000 compounds that include high-value pharmaceuticals. As plants naturally produce extremely small amounts of these molecules and their chemical synthesis is complex, industrially sourcing these compounds is costly and inefficient. Hence, microbial hosts suitable to produce MIA precursors through synthetic biology and metabolic engineering are currently being sought. Here, we evaluated the suitability of a eukaryotic microalga, the marine diatom Phaeodactylum tricornutum, for the heterologous production of monoterpenoids. Profiling of endogenous metabolism revealed that P. tricornutum, unlike other microbes employed for industrial production of terpenoids, accumulates free pools of the precursor geranyl diphosphate. To evaluate the potential for larger synthetic biology applications, we engineered P. tricornutum through extrachromosomal, episome-based expression, for the heterologous biosynthesis of the MIA intermediate geraniol. By profiling the production of geraniol resulting from various genetic and cultivation arrangements, P. tricornutum reached the maximum geraniol titer of 0.309 mg/L in phototrophic conditions. This work provides (i) a detailed analysis of P. tricornutum endogenous terpenoid metabolism, (ii) a successful demonstration of extrachromosomal expression for metabolic pathway engineering with potential gene-stacking applications, and (iii) a convincing proof-of-concept of the suitability of P. tricornutum as a novel production platform for heterologous monoterpenoids, with potential for complex pathway engineering aimed at the heterologous production of MIAs.

Phytosterol biosynthesis and production by diatoms (Bacillariophyceae).

Diatoms are abundant unicellular marine photosynthetic algae that have genetically diversified their physiology and metabolism while adapting to numerous environments. The metabolic repertoire of diatoms presents opportunities to characterise the biosynthesis and production of new and potentially valuable microalgal compounds, including sterols. Sterols of plant origin, known as phytosterols, have been studied for health benefits including demonstrated cholesterol-lowering properties. In this review we summarise sterol diversity, the unique metabolic features of sterol biosynthesis in diatoms, and prospects for the extraction of diatom phytosterols in comparison to existing sources. We also review biotechnological efforts to manipulate diatom biosynthesis, including culture conditions and avenues for the rational engineering of metabolism and cellular regulation.