Genetius

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 used CRISPR/Cas9 to edit the downstream region of the Arabidopsis thaliana FLOWERING LOCUS T (FT) gene, identifying a 2.3‑kb segment containing the Block E enhancer as crucial for normal FT expression and flowering. Fine‑scale deletions pinpointed a 63‑bp core module with CCAAT‑ and G‑boxes, and revealed a cryptic CCAAT‑box that becomes active when repositioned, highlighting the importance of local chromatin context and motif arrangement for enhancer function.

The study optimized three wheat transformation methods—immature embryo, callus, and in planta injection—by systematically adjusting Agrobacterium strain, bacterial density, acetosyringone concentration, and incubation conditions, achieving transformation efficiencies up to 66.84%. Using these protocols, CRISPR/Cas9 knockout of the negative regulator TaARE1-D produced mutants with increased grain number, spike length, grain size, and a stay‑green phenotype, demonstrating the platform’s potential to accelerate yield and stress‑tolerance improvements in wheat.

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 identified two subclades of Arabidopsis NUDIX hydrolases that selectively hydrolyze distinct inositol pyrophosphate isomers, with subclade I targeting 4-InsP7 and subclade II targeting 3-InsP7 in a Mg2+-dependent manner. Loss-of-function mutants of subclade II NUDTs displayed disrupted phosphate and iron homeostasis, elevated 1/3-InsP7 levels, and increased resistance to Pseudomonas syringae, revealing roles in nutrient signaling and plant immunity, while cross-kingdom analyses showed conserved PP-InsP‑metabolizing activities.

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 study used single‑cell transcriptomics to compare Arabidopsis thaliana leaf cell responses during pattern‑triggered and effector‑triggered immunity, revealing that core defense modules are broadly shared but differ in timing, intensity, and cell‑type specific receptor dynamics. Distinct mesophyll subpopulations showed divergent resilience patterns, and gene regulatory network analysis identified WRKY‑regulated and salicylic‑acid biosynthesis modules, with the cue1-6 mutant confirming robustness of core immune responses while exposing cryptic sucrose‑responsive pathways.

The authors introduced a polycistronic tRNA‑gRNA array for CRISPR/Cas9 editing in Physcomitrium patens that doubled the frequency of large, targeted deletions compared with conventional single‑gRNA constructs. Using dual‑gRNA targeting, they achieved simultaneous deletion of two to four genes (katanin and TPX2 families) in a single transformation, reaching up to 42% efficiency per gene, though efficiency depended on gRNA pair design.

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 CRISPR/Cas9 to generate rice snrk1 mutants and performed integrated phenotypic, transcriptomic, proteomic, and phosphoproteomic analyses under normal and starvation conditions, revealing SnRK1’s dual role in promoting growth and mediating stress responses. Findings indicate sub-functionalization of SnRK1 subunits and identify novel phosphorylation targets linked to membrane trafficking, ethylene signaling, and ion transport.