Genetius

The study demonstrates that the Nicotiana benthamiana NLR Roq1, previously known to recognize the XopQ/HopQ1/RipB effector family, also detects the structurally distinct HopAG1 effector, leading to reduced bacterial growth and disease symptoms. Roq1-HopAG1 interaction was confirmed by co‑immunoprecipitation and attributed to the Nudix domain of HopAG1 binding a similar receptor interface as XopQ, suggesting broader effector recognition potential for Roq1 and other TNLs.

The study demonstrates that infiltrating Nicotiana benthamiana leaves with specific nopaline-type Agrobacterium tumefaciens strains dramatically increases local glandular trichome density within 15 days, an effect linked to the bacterial trans-zeatin synthase (tzs) gene that produces the cytokine trans‑zeatin. This simple Agrobacterium‑mediated approach enables direct comparison of high‑density trichome regions with adjacent isogenic tissue on the same leaf.

Infiltration of Nicotiana benthamiana leaves with Agrobacterium tumefaciens strain GV3101 carrying the pMP90 Ti plasmid triggers de novo formation of capitate glandular trichomes and elevates acyl‑sugar production, an effect absent with other strains. The responsible factor is the trans‑zeatin synthase (tzs) gene on pMP90, and exogenous application of cytokinins (trans‑zeatin or benzylaminopurine) alone can reproduce trichome induction, linking cytokinin signaling to trichome development. The study highlights that Agrobacterium-mediated transient assays can have unintended developmental and biochemical impacts, recommending strain testing to mitigate such effects.

The study systematically examines sources of variability in Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana, analyzing a large dataset of 1,915 plants collected over three years. It demonstrates that normalization methods must be validated for each experimental context and provides a statistical model and power analysis framework to determine appropriate sample sizes for detecting specific effect sizes, offering practical guidelines to improve reproducibility in quantitative plant and synthetic biology studies.

Using CRISPR‑Cas9, researchers knocked down CCD7 or CCD8 in Nicotiana benthamiana to suppress strigolactone synthesis, producing compact plants with a 45%–50% smaller spatial footprint while preserving recombinant protein yields (GFP and rituximab). The mutants showed altered leaf proteome, auxin/cytokinin balance, and metabolic fluxes without affecting overall growth rate, demonstrating suitability for indoor vertical farming biopharma production.

The authors created two Nicotiana benthamiana lines with CRISPR-mediated knockouts of two polyphenol oxidase genes, which exhibited slightly accelerated growth and retained normal transient GFP expression. These ppo-deficient plants produced leaf extracts that remained greener with markedly reduced enzymatic browning and protein crosslinking, leading to a nearly fourfold increase in yield and improved purity of a transiently expressed His‑tagged tomato protease. The study demonstrates that PPO depletion can enhance recombinant protein recovery from plant tissue.

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.

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.