Genetius

The study established a transient expression system in Nicotiana benthamiana to boost DMAPP production and enable biosynthesis of glycosylated irregular monoterpenes using three IDS enzymes. Co‑expression of pathway bottleneck enzymes significantly increased yields of chrysanthemyl, lavandulyl, and cyclolavandulyl glucosides, achieving the highest reported concentrations of irregular monoterpene glycosides in a plant system.

The authors present a protocol to isolate RNA-binding proteins (RBPs) cross-linked to RNA in bulk, detailing in vivo UV crosslinking, tissue lysis, fractionation, and organic solvent purification of RNA‑protein adducts for proteomic analysis. While optimized for Nicotiana benthamiana leaves, the method is adaptable to other plant species and tissues.

The study reveals that the Phytophthora infestans RXLR-LWY effector AVRcap1b binds the host ESCRT component NbTOL9a via a novel L-shaped fold, forming a complex that is essential for suppressing immunity through a helper NLR, NbNRC2. Structural analysis and mutagenesis show that disruption of the NbTOL9a‑AVRcap1b interface impairs virulence, indicating the effector acts as a bridge linking NbTOL9a to activated NbNRC2.

Using integrated metabolomics, fluxomics, and proteomics, the study shows that Bamboo mosaic virus infection in Nicotiana benthamiana redirects carbon flux toward glycolysis and the Krebs cycle, increasing mitochondrial enzyme abundance. Silencing the mitochondrial NAD⁺-dependent malic enzyme 1 elevates viral accumulation and disrupts cytoplasmic NADH/NAD⁺ balance, highlighting mitochondrial metabolism’s role in modulating redox status and antiviral defense.

Using Nicotiana benthamiana plants expressing PI4P and PI(4,5)P2 biosensors, the study visualized root colonisation by the pathogen Phytophthora palmivora and the mutualist fungus Funneliformis mosseae. Distinct phosphoinositide patterns were observed—PI(4,5)P2 enriched at mutualist interfaces and PI4P absent from pathogen membranes—but co‑colonisation triggered recruitment of PI4P to pathogen haustoria and increased resistance. These results show that phosphoinositide signatures differentiate pathogenic and mutualistic interfaces and can be dynamically remodelled during simultaneous colonisation.

The study screened 20 YopJ family effectors from diverse phytopathogenic bacteria in Nicotiana benthamiana and found that 12 are recognized by the NLRs NbZAR1 and/or NbPtr1, triggering hypersensitive cell death. The effector‑induced HR can be differentially suppressed by the deacetylase SUPPRESSOR OF AVRBST‑ELICITED RESISTANCE 1, indicating multiple recognition mechanisms. These findings establish the genetic basis for YopJ effector detection in N. benthamiana and set the stage for mechanistic analyses of NbZAR1/JIM2 and NbPtr1 activation.

The study engineered virus-delivered short RNA inserts (vsRNAi) of 24‑32 nt to simultaneously silence homeologous gene pairs in Nicotiana benthamiana, achieving quantitative phenotypes and transcriptome-wide changes linked to 21‑ and 22‑nt sRNA production. The approach was also validated in tomato and scarlet eggplant, demonstrating its utility for functional genomics and trait reprogramming in both the model species and crops.

The study demonstrates that the aphid effectors Mp1 and Mp58 from Myzus persicae interact both in planta and in vitro, forming an oligomeric complex that also associates with the plant host protein VPS52 and localizes to vesicle-like structures in Nicotiana benthamiana. Using ectopic expression, co‑immunoprecipitation, confocal microscopy, recombinant protein assays, and computational modeling, the authors reveal a novel effector‑effector‑target complex, highlighting added complexity in plant‑insect molecular interactions.

The study demonstrates that vesicle trafficking components have opposing impacts on geminivirus infection in Nicotiana benthamiana: silencing NbSAR1 or NbAP-1γ enhances systemic viral DNA accumulation, whereas silencing Nbδ‑COP, NbARF1, or clathrin genes nearly abolishes infection without affecting replication. These results indicate that endocytosis and retrograde transport are essential for viral movement, while vacuolar and autophagy pathways may contribute to antiviral defense, and the effects are specific to geminiviruses.

Silencing polyphenol oxidase (PPO) in Nicotiana benthamiana prevents oxidative browning and protein cross‑linking in agroinfiltrated leaf extracts without affecting plant growth, and it may also boost transient gene expression. These findings suggest that reducing PPO activity can improve plant science techniques and molecular pharming.