Immunity gene silencing increases transient protein
expression in Nicotiana benthamiana

Isobel L Dodds, Emma C Watts, Mariana Schuster, Pierre Buscaill, Yasin Tumas, Nicholas J Holton, Shijian Song, Johannes Stuttmann, Matthieu H a J Joosten, Tolga Bozkurt, et al.

► To cite this version:

Isobel L Dodds, Emma C Watts, Mariana Schuster, Pierre Buscaill, Yasin Tumas, et al.. Immunity gene silencing increases transient protein expression in Nicotiana benthamiana. Plant Biotechnology Journal, 2025, 23, pp.1768 - 1770. 10.1111/pbi.70005 . cea-05405367

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Distributed under a Creative Commons Attribution 4.0 International License
Plant Biotechnology Journal (2025) **23**, pp. 1768–1770

doi: 10.1111/pbi.70005

## Brief Communication

# Immunity gene silencing increases transient protein expression in *Nicotiana benthamiana*

Isobel L. Dodds<fnref n="1" /> ID, Emma C. Watts<fnref n="1" /> ID, Mariana Schuster<fnref n="1" /> ID, Pierre Buscaill<fnref n="1" /> ID, Yasin Tumas<fnref n="2" /> ID, Nicholas J. Holton<fnref n="3" /> ID, Shijian Song<fnref n="1" /> ID, Johannes Stuttmann<fnref n="4" /> ID, Matthieu H. A. J. Joosten<fnref n="5" /> ID, Tolga Bozkurt<fnref n="2" /> ID and Renier A. L. van der Hoorn<fnref n="1" />,* ID

Received 18 July 2024;
revised 26 January 2025;
accepted 30 January 2025.

*Correspondence (Tel +44 1865 275077; email renier.vanderhoorn@biology.ox.ac.uk)

**Keywords:** transient expression, agroinfiltration, *Nicotiana benthamiana*, *Agrobacterium tumefaciens*, GFP fluorescence, virus-induced gene silencing.

The infiltration of *Nicotiana benthamiana* with *Agrobacterium tumefaciens* (agroinfiltration) has become a routine expression platform for plant science and molecular pharming, yet this platform remains to be further optimized. We recently showed that *N. benthamiana* silenced for the cold shock protein (CSP) receptor (CORE) enables 8-fold more GFP production in older, 6–8-week-old plants, which are normally not used because of low transient expression efficiencies (Dodds et al., 2023). Here, we investigated whether we can also increase transient protein expression levels in routinely used younger, 5-week-old juvenile plants, by silencing immunity-related genes.

We selected 21 immunity-related genes encoding proteins that act at different levels in the plant immune system (Table S1). Besides CORE, we silenced receptor-encoding genes WAK1 (Wall-associated Protein Kinase), CERK1 (Chitin Elicitor Receptor Kinase-1), BAK1 (BRI1-associated Receptor Kinase-1), SOBIR1 (Suppressor of BIR1-1) and REO2 (Receptor of SCPs). We also tested silencing of immune signaling components such as F-box protein ACIF1 (Avr/Cf-induced F-box-1); lipase-like proteins EDS1 (Enhanced Disease Susceptibility-1) and SAG101 (Senescence-associated Gene-101); CDPK (Calcium-dependent Protein Kinase), MPK3/6 (MAP protein kinases-3 and -6), Nod-like helper receptors NRC2/3/4 (NLR Required for Cf Signaling) and chaperone CRT3a (Calreticulin-3a). We also included genes required for stress hormone signaling, including PAL (Phenylalanine Ammonia Lyase), ICS (Isochorismate Synthase), NPR1 (Nonexpressor of PR genes-1), EIN2 (Ethylene-insensitive-2) and WRKY transcription factors. Finally, we included genes encoding AHA2 (Arabidopsis H⁺-ATPase 2) and RBOHB (Respiratory Burst Oxidase Homolog B). Genes encoding phytoene desaturase (PDS) and β-glucuronidase (GUS) were included as positive and negative controls for silencing, respectively. We resynthesized

the silencing fragments as published previously (Table S1) and selected novel fragments targeting ACIF1, CDPK, CORE, ICS and AHA2 (Dodds et al., 2023, Tables S2 and S3).

Tobacco Rattle Virus (TRV) vectors, each carrying a fragment of these 21 immunity genes and the controls were agroinfiltrated into 2-week-old seedlings and plants were tested for transient expression three weeks later. Transcript levels of the targeted genes were downregulated with novel silencing fragments (Figure S1). At that stage, no strong phenotypes were observed in TRV-inoculated plants, except for photobleaching in TRV::PDS plants, dwarfed TRV::BAK1 and TRV::CDPK plants, and small, chlorotic TRV::ICS plants (Figure S2). Leaf discs of TRV::RBOHB and TRV::BAK1 plants showed a reduced oxidative burst upon flg22 treatment (Figure S3), consistent with the effective silencing of these genes.

Silenced plants were agroinfiltrated with a 1:1:1 mixture of Agrobacteria delivering three 'traffic light' reporters: bioluminescent AgroLux bacteria (Jutras et al., 2021, orange), mixed with Agrobacteria delivering expression cassettes for cytonuclear GFP (cGFP, green) and secreted RFP (sRFP, red). GFP fluorescence in leaf discs taken at 5 days post-infiltration (5 dpi) was significantly higher in TRV::ICS, TRV::CERK1, TRV::NPR1 and TRV::EIN2 plants, when compared to TRV::GUS plants (Figure 1a). The same silenced plants also showed more RFP fluorescence when compared to TRV::GUS control plants (Figure S4a). No altered fluorescence was detected in TRV::CORE plants, consistent with low CORE expression in juvenile plants (Wang et al., 2016). AgroLux bioluminescence was similar between these silenced plants (Figure S4b), indicating that silencing these immunity genes does not increase Agrobacterium population levels. Scanning of agroinfiltrated leaves in subsequent experiments confirmed that silencing CERK1 or NPR1 increased GFP-fluorescence by >4-fold and ICS silencing even >10-fold, when compared to the GUS silencing control (Figure 1b,c). Also, a significant, 1.7-fold increased GFP fluorescence was detected in TRV::EIN2, when compared to TRV::GUS plants (Figure S5), consistent with the initial screen.

The observed effects imply that immunity genes CERK1, NPR1 and ICS encode important barriers for transient expression (Figure 1d). CERK1 is a receptor-like kinase involved in the perception of chitin (Miya et al., 2007) and peptidoglycan (Willmann et al., 2011). NPR1 is a transcriptional regulator of responses induced by salicylic acid (SA) (Spoel and Dong, 2024). ICS is a chloroplastic enzyme producing isochorismate, a

<footnote n="1">¹The Plant Chemetics Laboratory, Department of Biology, University of Oxford, Oxford, UK</footnote>

<footnote n="2">²Department of Life Sciences, Imperial College London, London, UK</footnote>

<footnote n="3">³Leaf Expression Systems, Norwich Research Park, Norwich, UK</footnote>

<footnote n="4">⁴Aix Marseille University, CEA, CNRS, BIAM, UMR7265, LEMIRE (Microbial Ecology of the Rhizosphere), Saint-Paul lez Durance, France</footnote>

<footnote n="5">⁵Laboratory of Phytopathology, Wageningen University and Research, Wageningen, The Netherlands</footnote>
<figure><img src="image_2.png" /><figcaption>**Figure 1** Immunity gene silencing increases transient expression in *Nicotiana benthamiana*. (a) Two-week-old *N. benthamiana* plants were inoculated with Tobacco Rattle Virus (TRV) carrying fragments of 21 immunity genes in two different experiments. Systemic leaves were agroinfiltrated 3 weeks later with a 1:1:1 mixture of Agrobacteria carrying binary vectors for expressing cytoplasmic GFP and secreted RFP and bioluminescent AgroLux bacteria. Leaf discs were taken at 5 dpi and analyzed for GFP fluorescence. Error bars represent SE of *n* = 4 (top) and *n* = 12 (bottom) replicates. Data were analyzed by ANOVA with Dunnett's post hoc test. \*P* < 0.05; \*\*\*P* < 0.01; \*\*\*\*P* < 0.0001. (b) Silencing of *CERK1*, *NPR1* and *ICS* increases transient GFP expression. 2-week-old *N. benthamiana* plants were inoculated with Tobacco Rattle Virus (TRV) carrying fragments of various genes to be targeted. Plants were agroinfiltrated 3 weeks later with Agrobacterium carrying a binary vector for GFP expression, and images were taken at 5 dpi and quantified. (c) Quantification of GFP fluorescence detected in (b). Error bars represent SE of *n* = 4 replicates. ANOVA with a Dunnett's Multiple Comparison test. (d) Likely roles of *CERK1*, *ICS* and *NPR1* in suppressing transient gene expression. *CERK1* is a receptor-like kinase that might perceive peptidoglycan (PGN). Isochorismate synthase (ICS) is a metabolic enzyme producing a precursor for salicylic acid (SA) in the chloroplast. Non-expressor of *PR1* (*NPR1*) regulates transcription factors (TFs) that activate immunity genes. (e) Increased transient GFP expression in the *npr1a/npr1b* double mutant. GFP was transiently expressed without P19 and fluorescence was imaged at 5 dpi (left), quantified (middle) and leaf extracts were analysed for anti-GFP western blot (right). Error bars represent the SD of *n* = 18 replicates.</figcaption></figure>

precursor of SA (Spoel and Dong, 2024). The unaltered GFP fluorescence in *TRV::PAL* plants indicates that SA restricting transient expression is not produced via phenylalanine, but through isochorismate in agroinfiltrated leaves (Spoel and Dong, 2024). The effect of *ICS* silencing is twice as strong compared to *NPR1* silencing might be because *ICS* depletion would not only reduce immune signalling, but also reduce SA levels that negatively regulate *vir* gene expression in Agrobacterium (Yuan *et al.*, 2007).

To enhance transient expression without relying on VIGS, we are disrupting the open reading frames of these immunity-related genes using genome editing and stacking mutant genes. So far, the deletion of the two silenced *NPR1* genes (Figure S6), has indeed caused increased GFP fluorescence and accumulation (Figure 1e)

and enhanced IgG accumulation (Figure S7). These genome-edited lines hold important potential for boosting transient gene expression for protein production in *N. benthamiana*.

## Acknowledgements

We thank Urszula Pyzio for excellent plant care, and Sarah Rodgers, Caroline O'Brien and Patricia Bowman for technical support.

## Funding

This project was financially supported by the Interdisciplinary DTC project DDT00060 (ID) and DDT00230 (EW); BBSRC projects
BB/R017913/1 (PB) and BB/S003193/1 (MS); and ERC project
101019324 (RH).

Author contributions

RALH conceived the project; ILD and ECW performed most
experiments with the help of MS and PB and SS; YT, MHAJJ and
TB provided VIGS constructs and JS provided the *npr11/npr1b*
mutant; ILD, ECW and RALH wrote the manuscript with all
authors.

Conflict of interest

None declared.

Data availability statement

The data that supports the findings of this study are available in
the supplementary material of this article.

REFERENCES

Dodds, I., Chen, C., Buscaill, P. and van der Hoorn, R.A.L. (2023) Depletion of the NbCORE receptor drastically improves agroinfiltration productivity in older Nicotiana benthamiana plants. *Plant Biotechnol. J.* **21**, 1103–1105.

Jutras, P.V., Soldan, R., Dodds, I., Schuster, M., Preston, G.M. and van der Hoorn, R.A.L. (2021) AgroLux: bioluminescent Agrobacterium to improve molecular pharming and study plant immunity. *Plant J.* **108**, 600–612.

Miya, A., Albert, P., Shinya, T., Desaki, Y., Ichimura, K., Shirasu, K., Narusaka, Y. *et al.* (2007) CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. *Proc. Natl. Acad. Sci. USA*, **104**, 19613–19618.

Spoel, S.H. and Dong, X. (2024) Salicylic acid in plant immunity and beyond. *Plant Cell*, **36**, 1451–1464.

Wang, L., Albert, M., Einig, E., Fürst, U., Krust, D. and Felix, G. (2016) The pattern-recognition receptor CORE of Solanaceae detects bacterial cold-shock protein. *Nat. Plants*, **2**, 16185.

Willmann, R., Lajunen, H.M., Erbs, G., Newman, M.A., Kolb, D., Tsuda, K., Katagiri, F. *et al.* (2011) Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection. *Proc. Natl. Acad. Sci. USA*, **108**, 19824–19829.

Yuan, Z.C., Edlind, M.P., Liu, P., Saenkham, P., Banta, L.M., Wise, A.A., Ronzone, E. *et al.* (2007) The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium. *Proc. Natl. Acad. Sci. USA*, **104**, 11790–11795.

**Supporting information**

Additional supporting information may be found online in the
Supporting Information section at the end of the article.

Appendix S1 Materials and methods.

Figure S1 Novel TRV constructs silence target genes.

Figure S2 Phenotypes of VIGSed plants.

**Figure S3** Suppressed flg22-induced oxidative burst in *TRV::BAK1* and *TRV::RBOHB* plants.

**Figure S4** Impact of immunity gene silencing on sRFP fluores-
cence and AgroLux luminescence.

**Figure S5** EIN2 silencing increases transient expression levels.

Figure S6 The *npr1* double mutant of *N. benthamiana*.

**Figure S7** Increased IgG accumulation in the *npr1a/npr1b* mutant plants.

Table S1 Genes targeted by VIGS.

**Table S2** Used plasmids.

Table S3 Used fragments for gene silencing.

Table S4 Used oligonucleotides.