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 authors present PIVOT, a single‑cell screening platform that combines viral superinfection exclusion with a engineered cell‑surface marker to isolate protoplasts expressing individual transgenes in Nicotiana benthamiana. Using an Arabidopsis ORF library, the method identified known and novel regulators of cytokinin signaling, demonstrating its potential for high‑throughput functional genetics across plants.

The authors optimized a tobacco rattle virus (TRV) vector to deliver engineered ISDra2 TnpB variants (eTnpBc) for plant genome editing, achieving up to 90% somatic editing efficiency in systemic leaves and up to 100% editing in offspring. The eTnpBc editor outperformed wild‑type TnpB and demonstrates potential for transformation‑ and transgene‑free editing in plants.

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.

The study engineered Tobacco rattle virus vectors incorporating distinct RNA secondary structures as mobility factors to improve guide RNA delivery to plant meristems. Using Nicotiana benthamiana plants expressing Cas9, optimal virus constructs were identified that generated both somatic and heritable edits, and these constructs were successfully applied to edit the emerging oilseed crop pennycress (Thlaspi arvense).

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 reveals that calcium-dependent protein kinases NbCDPK4 and NbCDPK5 phosphorylate the NADPH oxidase NbRBOHB at Ser‑123, driving sustained reactive oxygen species production during effector‑triggered immunity. Both direct phosphorylation and transcriptional up‑regulation of NbRBOHB by NbCDPKs link NLR activation to ROS‑mediated hypersensitive cell death, with the autoactive helper NLR NRC4 further enhancing Ser‑123 phosphorylation via its Ca²⁺ channel motif.