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Rapid transient protein production by the coat protein-deficient cucumber mosaic virus vector: non-packaged CMV system, NoPaCS

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Abstract

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We developed a non-packaged CMV system (NoPaCS) for CMV-agroinfection with a virus-inescapable transgenic plant platform, enabling rapid, high production of a large-sequence target protein.

Abstract

For rapidly producing high levels of a desirable protein, many plant virus vectors have been developed. However, there is always a concern that such recombinant viruses may escape into the environment. Especially for insect-transmissible viruses, certain measures must be taken. We here developed a new cucumber mosaic virus (CMV) RNA 3-based vector that is not transmitted by aphids because we deleted the coat protein (CP) gene responsible for aphid transmission and replaced it with a foreign gene. Transgenic Nicotiana benthamiana plants expressing CMV RNA 1 (CR1Tg) were found to be the most suitable platform for producing a recombinant protein using the CMV vector. By agroinfiltrating CR1Tg plants with the RNA 2 construct and the CMV vector harboring the green fluorescence protein (GFP) gene instead of the CP gene, we achieved a high yield of GFP (e.g., ~ 750 mg/kg FW) throughout the bacteria-infiltrated tissues at 2–3 days after infiltration. Furthermore, with this CMV-agroinfection system, a large gene such as the β-glucuronidase (GUS) gene can be expressed because the viral RNAs are not necessarily encapsidated for replication. The system is designated “non-packaged CMV system (NoPaCS)”.

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Acknowledgements

This work was supported in part by grants from the Ministry of Economy, Trade and Industry (METI) of Japan and New Energy and Industrial Technology Development Organization (NEDO).

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Authors and Affiliations

  1. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo, 062-8517, Japan

    Noriho Fukuzawa & Takeshi Matsumura

  2. Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, 060-8589, Japan

    Chikara Masuta

Authors
  1. Noriho Fukuzawa
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  2. Chikara Masuta
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  3. Takeshi Matsumura
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Corresponding author

Correspondence to Takeshi Matsumura.

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The authors declare that they have no conflict of interest.

Additional information

Communicated by Eugenio Benvenuto.

Electronic supplementary material

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Table S1. Primers used in this study (PDF 114 KB)

299_2018_2322_MOESM2_ESM.pdf

Fig. S1 GFP expression in CR2Tg or (CR1+2)Tg after vacuum infiltration with the Agrobacterium suspension harboring either CR1+CR3Δ33GFP (a) or only CR3Δ33GFP (b) at 3 dpi. CR2Tg (No. 24) is the transgenic plant expressing CMV RNA 2. (CR1+2)Tg (No. 6) is the transgenic plant expressing both RNA 1 and RNA 2 (PDF 130 KB)

299_2018_2322_MOESM3_ESM.pdf

Fig. S2 Comparison of protein production in plants by the virus-based vector systems. The expression levels of GFP were compared among 4 representative virus vectors (CMV, AMV, CPMV and TMV) (PDF 225 KB)

299_2018_2322_MOESM4_ESM.pdf

Fig. S3 GFP levels in CR1Tg lines after vacuum infiltration with Agrobacterium suspension harboring CR2+CR3Δ33GFP. Tissues were harvested at 3, 5 or 7 dpi. Agroinfiltrated plants were 7 weeks old. Two lines of CR1Tg (Nos. 30 and 79) were used. To estimate levels, a purchased, recombinant GFP was used (PDF 137 KB)

299_2018_2322_MOESM5_ESM.pdf

Fig. S4 IL1-ra levels in a CR1Tg line after vacuum infiltration with Agrobacterium suspension harboring CR2+CR3Δ33IL1-ra. The IL1-ra gene was prepared according to Fukuzawa et al. (2011). Tissues were harvested at 1, 2 or 3 dpi. Agroinfiltrated plants were 7 weeks old. CR1Tg No. 29 line was used. Note that IL1-ra levels were highest at 2 dpi. To estimate levels, a purchased, recombinant IL1-ra protein was used (PDF 58 KB)

Supplementary references (PDF 251 KB)

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Fukuzawa, N., Masuta, C. & Matsumura, T. Rapid transient protein production by the coat protein-deficient cucumber mosaic virus vector: non-packaged CMV system, NoPaCS. Plant Cell Rep 37, 1513–1522 (2018). https://doi.org/10.1007/s00299-018-2322-5

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  • DOI: https://doi.org/10.1007/s00299-018-2322-5

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