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 demonstrates that calcium-dependent protein kinases NbCDPK4 and NbCDPK5 directly phosphorylate the NADPH oxidase NbRBOHB at Ser‑123, enhancing sustained ROS production during effector-triggered immunity in Nicotiana benthamiana. Constitutively active CDPKs also upregulate NbRBOHB transcription, and phosphorylation of Ser‑123 is amplified by Ca2+ influx triggered by an autoactive helper NLR (NRC4). These results define a NbCDPK‑NbRBOHB signaling module that links NLR activation to prolonged ROS bursts in ETI.

The study establishes a transient Agrobacterium-mediated expression platform in Nicotiana benthamiana to produce glycosylated irregular monoterpenes by enhancing DMAPP biosynthesis through co‑expression of DXS, IDI, and HMGR. Engineering of plastidial and cytoplasmic pathways, including a bacterial cyclolavandulyl diphosphate synthase, led to the accumulation of six novel glucoside derivatives, reaching up to 6.6 µmol g⁻¹ fresh weight, the highest reported for plant‑based production.

The authors present a protocol for the large‑scale isolation of RNA‑binding proteins cross‑linked to RNA, involving in vivo UV‑crosslinking, tissue lysis, fractionation, and organic‑solvent purification of RNA‑protein adducts for downstream proteomics analysis. Although developed with Nicotiana benthamiana leaves, the method can be adapted to other plant species and tissues.

The study used Nicotiana benthamiana lines expressing fluorescent biosensors for PI4P and PI(4,5)P2 to visualize root colonisation by the pathogen Phytophthora palmivora and the mutualist fungus Funneliformis mosseae. Distinct phosphoinositide patterns distinguished the two interactions, and co‑colonisation induced recruitment of PI4P to pathogen haustoria, correlating with increased resistance, indicating dynamic remodeling of host membrane identity.