Abstract
Plants have emerged as powerful biofactories for producing proteins of interest. Compared to mammalian-based expression system, plant biofactories offer unique advantages in terms of higher scalability, lower production costs, reduced risk of contamination by human pathogens and faster expression rate (Chung et al., 2022; Eidenberger et al., 2023). Over the past three decades, plant-based expression platforms have been successfully used to generate a wide array of recombinant products, including vaccine antigens, therapeutic antibodies and bioactive proteins. The recent pandemic of Ebola and SARS-CoV-2 has further demonstrated the potential of plant biofactories as an alternative protein production system, enabling rapid responses to global health crises and providing high-quality products (Capell et al., 2020; Zeitlin et al., 2011). A critical consideration for plant biofactories is enhancing the yield of the target protein. While transgenic systems are useful for large-scale production of single vaccines or therapeutics, they require a time-consuming process and may suffer from low yields due to RNA silencing. Alternatively, engineered plant viral vectors have been developed to efficiently deliver genes of interest into plants, facilitating rapid expression of vaccines, monoclonal antibodies or other therapeutic proteins (Abrahamian et al., 2020). However, plant antiviral responses, which manifest at the DNA, RNA and protein levels can significantly impact the yield of target proteins. It is speculated that modulating antiviral host factors could enhance plant viral vector infection and, consequently, improve overall protein expression efficiently. Previously, key components of the conserved antiviral RNA silencing pathway were attractive targets that had been knocked out to improve the productivity of transient expression. However, CRISPR-based knockout of RDR6 in Nicotiana benthamiana resulted in abnormal and sterile flowers similar to Arabidopsis ∆rdr6 mutant, limiting the utility of these engineered plants (Matsuo and Atsumi, 2019). Therefore, identification of antiviral host factors whose knockout can facilitate plant viral vector infection without imposing growth penalties would be ideal for improving target protein production. Cell wall is a rigid outer layer that provides mechanical support and acts as a physical barrier against biotic and abiotic stresses. In this study, we found that the mRNA level of a glycine-rich cell wall structural protein (NbGRP) was upregulated by threefold in response to tomato brown rugose fruit virus (ToBRFV) infection (Figure S1a, Table S1). Sequence analysis showed that NbGRP encodes 132 amino acids, including a potential signal peptide (SP) from residues 1 to 25 at the N-terminus and a glycine-rich domain conserved in the class II GRP (Figure S1b). Confocal microscopy revealed that the NbGRP protein fused to yellow fluorescent protein (NbGRP-YFP) displayed a punctate distribution pattern at the cell wall boundaries (Figure 1a), suggesting localization of NbGRP at plasmodesmata (PD). Furthermore, NbGRP-YFP colocalized with the cell wall marker AtPDLP1 tagged with cyan fluorescent protein (AtPDLP1-CFP) at punctate spots along the cell wall (Figure 1b). After plasmolyzing the cells with 10% NaCl, the fluorescence of NbGRP-YFP remained in the cell wall (Figure 1c), confirming that NbGRP is a PD-localized protein. Next, we investigated whether NbGRP affects ToBRFV infection. We co-infiltrated a ToBRFV infectious clone expressing green fluorescent protein (ToBRFV-GFP) along with an NbGRP expression vector (myc-NbGRP) or a control vector (myc-GUS) in N. benthamiana. At 7 days post infiltration (dpi), GFP fluorescence in the upper systemic leaves of N. benthamiana plants co-infiltrated with ToBRFV-GFP and myc-NbGRP was reduced compared to those co-infiltrated with ToBRFV-GFP and myc-GUS (Figure 1d). Quantitative RT-PCR and western blot analysis confirmed that transient overexpression of NbGRP significantly inhibited both the RNA and protein levels of ToBRFV (Figure S1c,d). We then employed the tobacco rattle virus (TRV)-induced gene silencing system (Data S1) to silence NbGRP in N. benthamiana. After determining the silencing efficacy, we inoculated the upper systemic leaves with ToBRFV-GFP. As shown in Figure S1e–h, GFP fluorescence, viral genomic RNA and ToBRFV coat protein levels were significantly higher in TRV-NbGRP plants compared to TRV-GUS plants, indicating that NbGRP functions as an antiviral host factor against ToBRFV. To generate engineered N. benthamiana plants for enhancing the yield of plant virus-based expression, we designed single-guide RNAs (sgRNAs) targeting NbGRP and obtained NbGRP gene-edited plants (Figure S1i). Interestingly, the NbGRP mutants had enlarged leaves during germination and growth stages, resulting in a 100% increase in biomass compared to wild-type plants (Figure 1f,g). When infiltrated with ToBRFV-GFP, the NbGRP-edited plants showed significantly higher levels of ToBRFV RNA and protein, as well as increased GFP expression (Figure 1h,i, Figure S1j). To test whether the NbGRP-edited N. benthamiana plants also facilitate infection by other commonly used plant viral vectors, we infiltrated them with TMV-GFP and PVX-GFP individually. Notably, the NbGRP mutants exhibited enhanced infection by both TMV-GFP and PVX-GFP, with GFP accumulation increasing by approximately twofold compared to wild-type plants (Figure 1j–m, Figure S1k,l). These results demonstrated that CRISPR/Cas9-mediated editing of NbGRP enhances the spread of the three tested viruses, thereby increasing the yield of plant viral vector-mediated GFP expression. To further evaluate whether the NbGRP mutant can enhance the accumulation of other heterologous proteins, we generated a recombinant TMV construct expressing a GFP nanobody tagged with N-terminal 6 × His epitopes (TMV-GFPNano) (Figure 1n). After confirming the expression of the GFP nanobody in the upper systemically infected leaves of N. benthamiana infiltrated with TMV-GFPNano (Figure 1o), we compared GFP nanobody accumulation in wild-type and NbGRP mutant plants. Immunoblot using anti-His tag antiserum revealed an approximately twofold increase in the NbGRP mutant compared to wild-type plants (Figure 1p). We then purified the GFP nanobody from these plants (Figure S2) and showed that the purified GFP nanobody can be used to detect GFP (Figure 1q). Quantification of the recovered GFP nanobody also revealed a significant increase in the NbGRP mutant compared to the wild-type plants (Figure 1r). These findings highlight the potential of the NbGRP mutant for improving heterologous protein expression in N. benthamiana. In conclusion, we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana to enhance plant viral vector-mediated heterologous protein production without compromising plant growth (Figure 1s). Although it remains to investigate whether the engineered plants can improve the yield of other plant viral vector-mediated target protein expression and whether there is any side effect when expressing other target proteins, our proof-of-concept results demonstrate how targeting antiviral host factors can improve the yield of recombinant proteins, as evidenced by the enhanced expression of a reporter protein and a GFP nanobody. This approach holds significant promise for increasing the production of heterologous proteins and could be extended to other pharmaceutical and non-pharmaceutical targets. This work was supported by grants from the National Key Research and Development Program of China (2022YFD1401200), Key Research and Development Program of Ningxia (2023BCF01053) and the National Natural Science Foundation of China (32272518). X.Y., X.Z. (Xueping Zhou) and W.G. designed the experiments. Z.F., X.Z. (Xinru Zhao), M.D., X.X. and H.Z. conducted the experiments and analysed the data. Z.F. and X.Y. drafted the manuscript with the input of all other authors. X.Y., X.Z. (Xueping Zhou) and W.G. revised the manuscript. The authors have declared no conflict of interest. The data that supports the findings of this study are available in the supplementary material of this article. Figure S1 Editing of NbGRP facilitates virus infection. Figure S2 SDS-PAGE showing the purified GFP nanobody from wild-type and NbGRP knockout plants, respectively. Table S1 Primers used in this study. Data S1 Methods. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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Claims 12
Claim · model-written summary
NbGRP mRNA was upregulated threefold in response to ToBRFV infection.
- Taxon
- N. benthamiana
Nicotiana benthamianae2 - Interactor
- tomato brown rugose fruit virus (ToBRFV)Tomato brown rugose fruit viruse1pathogen
- Gene
- NbGRPe1
- Outcome
- mRNA levele1
- Direction
- upregulatede1
- Value
- threefolde1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“we found that the mRNA level of a glycine-rich cell wall structural protein (NbGRP) was upregulated by threefold in response to tomato brown rugose fruit virus (ToBRFV) infection”
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
Claim · model-written summary
NbGRP-YFP shows a punctate distribution at the cell wall boundaries.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Gene
- NbGRPe3
- Outcome
- punctate distribution pattern at the cell wall boundariese3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e3“Confocal microscopy revealed that the NbGRP protein fused to yellow fluorescent protein (NbGRP-YFP) displayed a punctate distribution pattern at the cell wall boundaries”
Claim · model-written summary
NbGRP is a plasmodesmata-localized protein, as its fluorescence remained in the cell wall after plasmolysis.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Gene
- NbGRPe4
- Outcome
- PD-localized proteine4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e4“After plasmolyzing the cells with 10% NaCl, the fluorescence of NbGRP-YFP remained in the cell wall (Figure 1c), confirming that NbGRP is a PD-localized protein”
Claim · model-written summary
Transient overexpression of NbGRP significantly inhibited the RNA and protein levels of ToBRFV.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Treatment
- transient overexpression of NbGRPe5
- Interactor
- ToBRFVe5pathogen
- Gene
- NbGRPe5
- Outcome
- RNA and protein levels of ToBRFVe5
- Direction
- inhibitede5
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e5“Quantitative RT-PCR and western blot analysis confirmed that transient overexpression of NbGRP significantly inhibited both the RNA and protein levels of ToBRFV”
Claim · model-written summary
TRV-induced silencing of NbGRP raised GFP fluorescence, viral genomic RNA and ToBRFV coat protein levels relative to TRV-GUS plants.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Treatment
- tobacco rattle virus (TRV)-induced gene silencinge6
- Interactor
- ToBRFVe7pathogen
- Gene
- NbGRPe6
- Outcome
- GFP fluorescence, viral genomic RNA and ToBRFV coat protein levelse7
- Direction
- highere7
- Comparator
- TRV-GUS plantse7
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e6“We then employed the tobacco rattle virus (TRV)-induced gene silencing system (Data S1) to silence NbGRP in N. benthamiana”
e7“GFP fluorescence, viral genomic RNA and ToBRFV coat protein levels were significantly higher in TRV-NbGRP plants compared to TRV-GUS plants”
Claim · model-written summary
NbGRP mutants have enlarged leaves and a 100% increase in biomass compared with wild-type plants.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Material
- NbGRP mutantse1
- Organ / stage
- leavese1
- Treatment
- NbGRP mutantse1
- Gene
- NbGRPe1
- Outcome
- biomasse1
- Direction
- increasee1
- Value
- 100%e1
- Comparator
- wild-type plantse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e1“the NbGRP mutants had enlarged leaves during germination and growth stages, resulting in a 100% increase in biomass compared to wild-type plants”
Claim · model-written summary
When infiltrated with ToBRFV-GFP, NbGRP-edited plants show significantly higher ToBRFV RNA and protein levels.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Material
- NbGRP-edited plantse1
- Treatment
- infiltrated with ToBRFV-GFPe1
- Interactor
- ToBRFVe1pathogen
- Gene
- NbGRPe1
- Outcome
- ToBRFV RNA and proteine1
- Direction
- highere1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e1“When infiltrated with ToBRFV-GFP, the NbGRP-edited plants showed significantly higher levels of ToBRFV RNA and protein, as well as increased GFP expression”
Claim · model-written summary
When infiltrated with ToBRFV-GFP, NbGRP-edited plants show increased GFP expression.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Material
- NbGRP-edited plantse1
- Treatment
- infiltrated with ToBRFV-GFPe1
- Interactor
- ToBRFVe1pathogen
- Gene
- NbGRPe1
- Outcome
- GFP expressione1
- Direction
- increasede1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e1“When infiltrated with ToBRFV-GFP, the NbGRP-edited plants showed significantly higher levels of ToBRFV RNA and protein, as well as increased GFP expression”
Claim · model-written summary
NbGRP mutants show enhanced infection by TMV-GFP and PVX-GFP, with GFP accumulation increasing approximately twofold compared with wild-type plants.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Material
- NbGRP mutantse1
- Treatment
- TMV-GFP and PVX-GFPe1
- Interactor
- TMV-GFPe1other
- Second interactor
- PVX-GFPe1other
- Outcome
- GFP accumulatione1
- Direction
- increasinge1
- Value
- approximately twofolde1
- Comparator
- wild-type plantse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e1“the NbGRP mutants exhibited enhanced infection by both TMV-GFP and PVX-GFP, with GFP accumulation increasing by approximately twofold compared to wild-type plants”
Claim · model-written summary
The NbGRP mutant shows an approximately twofold increase in GFP nanobody accumulation from TMV-GFPNano compared with wild-type plants.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Material
- NbGRP mutante1
- Treatment
- TMV-GFPNanoe4
- Outcome
- GFP nanobody accumulatione3
- Direction
- increasee1
- Value
- approximately twofolde1
- Comparator
- wild-type plantse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e1“Immunoblot using anti-His tag antiserum revealed an approximately twofold increase in the NbGRP mutant compared to wild-type plants”
e3“we compared GFP nanobody accumulation in wild-type and NbGRP mutant plants”
e4“we generated a recombinant TMV construct expressing a GFP nanobody tagged with N-terminal 6 × His epitopes (TMV-GFPNano)”
Claim · model-written summary
The recovered GFP nanobody is significantly increased in the NbGRP mutant compared with wild-type plants.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Material
- NbGRP mutante1
- Treatment
- NbGRP mutante1
- Outcome
- recovered GFP nanobodye1
- Direction
- increasee1
- Comparator
- wild-type plantse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e1“Quantification of the recovered GFP nanobody also revealed a significant increase in the NbGRP mutant compared to the wild-type plants”
Claim · model-written summary
The purified GFP nanobody can be used to detect GFP.
- Taxon
- N. benthamianaNicotiana benthamianae2
- Outcome
- detect GFPe1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“we present an experimental strategy involving the genetic editing of an antiviral cell wall component in N. benthamiana”
e1“showed that the purified GFP nanobody can be used to detect GFP”
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