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

Abstract

Summary 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.

Extracellular plant subtilases dampen cold shock peptide elicitor levels

“…To confirm that Nb CORE is required for detecting csp22, we depleted Nb CORE with Virus-induced Gene Silencing (VIGS) using Tobacco Rattle Virus (TRV) vectors carrying a 300bp fragment of Nb CORE, or of β-glucuronidase (GUS) as a negative control (Dodds et al, 2023). Oxidative burst assays showed that 6wk-old TRV::CORE plants are blind to csp22 of CspD, in contrast to TRV::GUS plants, which show an csp22-induced oxidative burst ( Figure 1E ).…”

“…Virus-induced gene silencing (VIGS) -N. benthamiana plants were silenced for NbCORE, SBT5.2, glucuronidase GUS (negative control) and phytoene desaturase PDS (positive control) were generated using VIGS as previously described (Dodds et al, 2023, Beritza et al, 2024. Briefly, overnight cultures of A. tumefaciens GV3101 were collected and resuspended in agroinfiltration buffer (10 mM MgCl2, 10 mM MES pH 5.0, and 100 mM acetosyringone).…”

“…Residues 5-19 of csp22 (csp15) constitute the active PAMP epitope that can trigger plant immune responses, including a burst of reactive oxygen species (ROS) (Felix and Boller, 2003; Saur et al, 2016). Two distinct receptors, the receptor-like protein CSPR and the receptor-like kinase CORE, were claimed to act as CSP receptors in older N. benthamiana plants (Saur et al, 2016; Wang et al, 2016), but the affinity of csp22 for CORE is much higher than for CSPR (Wang et al, 2016), and only silencing of CORE makes these plants insensitive for csp22 (Dodds et al, 2023). CSPR was later identified as RE02 and is involved in perceiving small Cys-rich proteins from Valsa mali (Nie et al, 2020), and Sclerotinia sclerotiorum (Yang et al, 2023).…”

Improving transient protein expression in agroinfiltrated Nicotiana benthamiana

“…Nb CORE, for instance, is an immune receptor that recognises cold shock proteins of Agrobacterium but is expressed only in older N. benthamiana plants (Wang et al ., 2016). Depletion of Nb CORE with virus‐induced gene silencing caused an eightfold higher transient green fluorescent protein (GFP) expression in older plants (Dodds et al ., 2023). Meanwhile, suppression of immune responses has been achieved with bacterial type‐III effector AvrPto, which inhibits immune‐related kinases (Xing et al ., 2007).…”