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

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.

Nicotiana benthamiana is frequently used for transient gene expression (Bally et al., 2018). In addition to studies on subcellular localization, protein-protein interaction and enzymatic activities, transient gene expression is commercially used to produce antibodies, vaccines, and other pharmaceutical proteins (Sainsbury, 2020; Schillberg and Spiegel, 2022). Transient expression is achieved by infiltrating leaves or whole plants with disarmed Agrobacterium tumefaciens harbouring a binary vector that carries genes-of-interest on the transfer DNA (T-DNA). A. tumefaciens transfers this T-DNA into the plant cell, where it is expressed.
The success of transient gene expression decreases with the age of the N. benthamiana plants, despite having more biomass and large leaves that are easy to infiltrate (Lai and Chen, 2012;Saur et al., 2016). Best expression is achieved in 3-5 week-old plants and poor expression in older, 6-8-week-old plants that start flowering. The poor gene expression is thought to be caused by the perception of cold shock protein (CSP) of A. tumefaciens (Saur et al., 2016). A 22 amino acid fragment of CSP called csp22 is sufficient to trigger immune responses including a burst of reactive oxygen species (ROS) (Felix and Boller, 2003;Saur et al., 2016). The csp22-induced ROS burst is observed from leaf discs from old plants, but not from young plants (Saur et al., 2016), implicating that CSP recognition might indeed underpin the success of transient gene expression in older plants.
Two distinct receptors, the receptor-like protein NbCSPR (Saur et al., 2016) and the receptor-like kinase NbCORE (Wang et al., 2016), respectively, have been proposed to act as CSP receptors. Transcripts of both receptors, encoding proteins with only 29.9% amino acid identity, are detectable only in older N. benthamiana plants. NbCSPR was reported to interact with csp22 and to be required for its perception because depletion of NbCSPR by virus-induced gene silencing (VIGS) suppressed the csp22-induced ROS response. The silencing of NbCSPR also resulted in higher transient expression in older plants after Agroinfiltration of a reporter gene. In disagreement with this report, Wang et al. (2016) could not confirm the role of NbCSPR in csp22 perception. Rather, they report that CORE tomato (SlCORE) forms the specific, high-affinity receptor binding site that is required and sufficient for csp22 perception (Wang et al., 2016). They also found that its ortholog in N. benthamiana, NbCORE, but not NbCSPR, confers csp22 responsiveness when transformed into Arabidopsis thaliana, which is otherwise insensitive to csp22 (Wang et al., 2016).
Here, we depleted NbCSPR and NbCORE by VIGS to investigate if CSP perception hampers recombinant protein production in older plants. To silence NbCSPR, we used the same 299 bp gene fragment of NbCSPR used earlier (Saur et al., 2016; Tables S1 and S2), and cloned this into a vector expressing RNA2 of tobacco rattle virus (TRV2gg). Similarly, a 300 bp fragment specific to NbCORE was cloned into TRV2gg. Alignments of the used silencing fragments with the coding sequences of NbCORE and NbCSPR show that cross-silencing is unlikely ( Figure S1). TRV carrying a fragment of betaglucuronidase (TRV::GUS) was included as a negative control. 2-week-old N. benthamiana plants were infected with TRV carrying the silencing fragments. The TRV::NbCSPR and TRV:: NbCORE plants have no developmental phenotypes compared to TRV::GUS plants (Figure 1a).
Leaf discs from 4-week and 6-week-old VIGS plants were tested for a csp22-induced oxidative burst. Importantly, the csp22-induced ROS burst is absent from 6-week-old TRV:: NbCORE plants and is present in TRV::NbCSPR plants, comparable to TRV::GUS control plants (Figure 1b and Figure S2). As reported before, younger, 4-week-old plants, only have very weak csp22-induced responses that can vary per batch of plants (Figure 1b and Figure S2). These data demonstrate that NbCORE is essential for the csp22-induced oxidative burst in older N. benthamiana plants. Our data indicate that NbCSPR is not necessary for csp22 perception, consistent with the findings of Wang et al. (2016) that NbCSPR is not sufficient for csp22 perception.
To investigate to what level the depletion of NbCORE promotes transient gene expression, we agroinfiltrated 6w-old TRV::GUS and TRV::NbCORE plants with Agrobacterium delivering enhanced GFP driven by a strong 35S promoter (35S:eGFP, Kourelis et al., 2021), and scanned the agroinfiltrated leaves for fluorescence five days later. Bright GFP fluorescence was detected in TRV::NbCORE plants, whereas hardly any fluorescence was detected in TRV::GUS plants (Figure 1c), corresponding to a nearly eight-fold increased GFP fluorescence (Figure 1d). A similar increased fluorescence was observed upon infiltrating 8-week-old plants (Figure 1d). Western blot analysis confirmed a drastically increased GFP protein level in TRV::NbCORE plants compared to the TRV::GUS control plants (Figure 1e).
Our data showing that NbCORE is required for csp22-induced oxidative burst is consistent with reports that NbCORE binds csp22 with high affinity (Kd = 6 nM, Wang et al., 2016) and that transient expression of NbCORE confers csp22-responsiveness to leaves of young plants (Wei et al., 2018). The csp22-induced ROS burst in TRV::NbCSPR plants was similar to the TRV::GUS control, which contradicts earlier work (Saur et al., 2016). Our data is, however, consistent with experiments that NbCSPR is unable to confer csp22-responsiveness (Wang et al., 2016). A more recent report also shows that NbCSPR is identical to RE02, the receptor for VmE02, a conserved Cys-rich protein secreted by diverse microbes (Nie et al., 2021). The csp22-induced oxidative burst is absent from 6-week-old TRV::NbCORE plants but present in TRV::GUS and TRV::NbCSPR plants. Error shades represent the standard error of n = 6 leaf discs. (c) NbCORE depletion causes bright GFP fluorescence upon agroinfiltration of 6-week-old plants. The image was taken 5 days agroinfiltration with 35S:eGFP. Scale bar, 1 cm. (d) Significant increase in GFP fluorescence upon NbCORE depletion. GFP fluorescence was quantified from images of n = 4 biological replicates of 6-week and 8-week-old VIGS plants agroinfiltrated with 35S:eGFP 5 days before fluorescence scanning. Fluorescence was quantified using ImageJ and normalized by leaf area. ****, P value = 0.0000084 (t-test). (e) TRV::NbCORE plants accumulate much more GFP protein upon agroinfiltration than TRV::GUS plants. Total leaf proteins were extracted from VIGS plants, 5 days after agroinfiltration with 35S:eGFP, and analysed by anti-GFP western blot. CBB, Coomassie brilliant blue.
Our results offer new opportunities in molecular farming, where older plants with larger biomass can now be used for efficient transient gene expression. A more durable depletion of NbCORE can be achieved by genome editing, or by engineering A. tumefaciens strains to contain a CSP that is no longer recognized by NbCORE. Both approaches will drastically improve transient protein production in older N. benthamiana plants, without the need for a licence to work with TRV to deplete NbCORE by VIGS.