Genetius

The study demonstrates that the NLR receptor Roq1 in Nicotiana benthamiana can recognize the effector HopAG1, in addition to the previously known XopQ/HopQ1/RipB family, leading to reduced bacterial growth. Co‑immunoprecipitation and structural analyses suggest HopAG1’s Nudix domain interacts with the same receptor interface as XopQ, implying Roq1 may detect multiple effector families.

The authors present PIVOT, a single‑cell screening platform that combines viral superinfection exclusion and a surface phenotypic marker to enable pooled library delivery and isolation of individual plant cells. Using Nicotiana benthamiana as a heterologous host, they screened an Arabidopsis ORF library and identified known and novel regulators of cytokinin signaling, demonstrating the method’s potential for high‑throughput functional genetics across plants.

Infiltration of Nicotiana benthamiana leaves with certain nopaline-type Agrobacterium tumefaciens strains containing a trans‑zeatin synthase (tzs) gene triggers extremely dense, localized glandular trichome formation within 15 days. The effect is attributed to bacterial production of the cytokinin trans‑zeatin, providing a simple method to dramatically increase trichome density for comparative biological studies.

Agrobacterium tumefaciens strain GV3101 carrying the pMP90 Ti plasmid induces de novo capitate glandular trichome formation and increased acyl‑sugar production in Nicotiana benthamiana leaves, an effect linked to the trans‑zeatin synthase (tzs) gene and cytokinin activity. Other strains lacking tzs do not trigger this response, highlighting that agroinfiltration can have developmental side‑effects and that multiple strains should be evaluated for transient assays.

The study systematically examines variability in Agrobacterium-mediated transient expression in Nicotiana benthamiana, highlighting that normalization methods must be validated for each experimental context. Using data from 1,915 plants across three years, the authors develop a variation model and perform power analysis to guide the required sample size for detecting specific effect sizes. Practical guidelines are offered to reduce variability and improve reproducibility in quantitative and synthetic biology applications.

The authors engineered Tobacco rattle virus vectors with various RNA secondary-structure mobility factors to enhance guide‑RNA delivery for meristem editing. Using Nicotiana benthamiana plants expressing Cas9, they identified optimal constructs that generated distinct somatic and heritable edits, and then successfully applied the method to edit pennycress (Thlaspi arvense). The results highlight virus‑mediated gRNA delivery as a viable strategy for genome editing in recalcitrant crops.

The study used CRISPR‑Cas9 to knock down CCD7 or CCD8 genes in Nicotiana benthamiana, suppressing strigolactone biosynthesis and creating compact, space‑efficient plants suitable for indoor vertical farming. Despite a 45‑50% reduction in spatial footprint, the mutants retained growth rates and recombinant protein yields comparable to the standard LAB strain, demonstrating their utility for biopharmaceutical production.

The study generated genome‑edited Nicotiana benthamiana lines lacking two polyphenol oxidases, which grow slightly faster and display reduced enzymatic browning and protein cross‑linking in leaf extracts while maintaining transient GFP expression. Loss of PPO activity markedly improves the yield and purity of recombinant proteins, exemplified by a four‑fold increase in purification yield of a His‑tagged tomato protease.

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.

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.