Depletion of the NbCORE receptor drastically improves agroinfiltration productivity in older Nicotiana benthamiana plants.

Nicotiana benthamiana is increasingly used for transient gene expression to produce antibodies, vaccines, and other pharmaceutical proteins but transient gene expression is low in fully developed, 6-8-week old plants. This low gene expression is thought to be caused by the perception of the cold shock protein (CSP) of Agrobacterium tumefaciens. The CSP receptor is contested because both NbCSPR and NbCORE have been claimed to perceive CSP. Here, we demonstrate that CSP perception is abolished in 6-week-old plants silenced for NbCORE but not NbCSPR. Importantly, older NbCORE-silenced plants support a highly increased level of GFP fluorescence and protein upon agroinfiltration. The drastic increase in transient protein production in NbCORE-depleted plants offers new opportunities for molecular farming, where older plants with larger biomass can now be used for efficient protein expression.

Comparative validity of three simulation platforms for objective assessment of otoscopy skills.

OBJECTIVES: To assess the face, construct and content validity of three different platforms for otoscopy skills assessment, using a traditional otoscope with manikin, digital otoscope (Tympahealth) with manikin, and traditional otoscope with a low-cost model ear (SimEar). DESIGN: Prospective mixed methods study. SETTING: Tertiary hospital. PARTICIPANTS: Postgraduate trainees and expert assessors. MAIN OUTCOME MEASURES: Face and Content validity based on expert assessor ranking on each model and their feedback from semi-structured interviews. Construct validity based on Objective Structured Clinical Examination scores. RESULTS: Each platform differed in face, construct and content validity scores, with no one platform consistently outperforming others. Three main themes were identified during thematic analysis of expert assessor interviews: ability to assess what is seen, anatomical reality, and ease of use. The low-cost model showed greatest potential, where modification to include a silicone ear could lead to high validity with marginal increase in cost. CONCLUSION: Several modalities for assessing otoscopy skills exist, each with advantages and disadvantages. Modifications to a low-cost model, for use with either a traditional or digital otoscope, could prove to be the best model.

AgroLux: bioluminescent Agrobacterium to improve molecular pharming and study plant immunity.

Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration. By integrating a single copy of the lux operon into the genome, we generated a stable 'AgroLux' strain, which is bioluminescent without affecting Agrobacterium growth in vitro and in planta. To illustrate its versatility, we used AgroLux to demonstrate that high light intensity post infiltration suppresses both Agrobacterium luminescence and protein expression. We also discovered that AgroLux can detect Avr/Cf-induced immune responses before tissue collapse, establishing a robust and rapid quantitative assay for the hypersensitive response (HR). Thus, AgroLux provides a non-destructive, versatile and easy-to-use imaging tool to monitor both Agrobacterium and plant responses.

Chemoproteomics Reveals the Pan-HER Kinase Inhibitor Neratinib To Target an Arabidopsis Epoxide Hydrolase Related to Phytohormone Signaling.

Plant phytohormone pathways are regulated by an intricate network of signaling components and modulators, many of which still remain unknown. Here, we report a forward chemical genetics approach for the identification of functional SA agonists in Arabidopsis thaliana that revealed Neratinib (Ner), a covalent pan-HER kinase inhibitor drug in humans, as a modulator of SA signaling. Instead of a protein kinase, chemoproteomics unveiled that Ner covalently modifies a surface-exposed cysteine residue of Arabidopsis epoxide hydrolase isoform 7 (AtEH7), thereby triggering its allosteric inhibition. Physiologically, the Ner application induces jasmonate metabolism in an AtEH7-dependent manner as an early response. In addition, it modulates PATHOGENESIS RELATED 1 (PR1) expression as a hallmark of SA signaling activation as a later effect. AtEH7, however, is not the exclusive target for this physiological readout induced by Ner. Although the underlying molecular mechanisms of AtEH7-dependent modulation of jasmonate signaling and Ner-induced PR1-dependent activation of SA signaling and thus defense response regulation remain unknown, our present work illustrates the powerful combination of forward chemical genetics and chemical proteomics for identifying novel phytohormone signaling modulatory factors. It also suggests that marginally explored metabolic enzymes such as epoxide hydrolases may have further physiological roles in modulating signaling.

A Bioluminescent Agrobacterium tumefaciens for Imaging Bacterial Metabolic Activity in Planta.

Bioluminescence enables the monitoring of spatiotemporal dynamics and activity of bacterial populations in planta. We here describe a procedure to use AgroLux, a bioluminescent Agrobacterium tumefaciens, as a tool to study bacterial responses upon agroinfiltration. The first method details how to transform bioluminescent AgroLux to carry binary plasmids of interests. Then, a simple agroinfiltration assay for in planta imaging of bioluminescence signals is presented. AgroLux assays will increase our understanding of plant-Agrobacterium interactions and plant immunity and improve molecular farming.

Proteases of Nicotiana benthamiana: an emerging battle for molecular farming.

Molecular farming increasingly uses the tobacco relative Nicotiana benthamiana for production of recombinant proteins through transient expression. Several proteins are produced efficiently with this expression platform, but yields for other proteins are often very low. These low yields are frequently due to endogenous proteases. The latest genome annotations indicate that N. benthamiana encodes for at least 1243 putative proteases that probably act redundantly and consecutively on substrates in different subcellular compartments. Here, we discuss the N. benthamiana protease repertoire that may affect recombinant protein production and recent advances in protease depletion strategies to increase recombinant protein production in N. benthamiana.