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Plant Cell Reports

, Volume 37, Issue 11, pp 1513–1522 | Cite as

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

  • Noriho Fukuzawa
  • Chikara Masuta
  • Takeshi Matsumura
Original Article

Abstract

Key message

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)”.

Keywords

Cucumber mosaic virus Virus vector Agroinfection Transgenic plant Aphid Plant-based platform 

Notes

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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

299_2018_2322_MOESM1_ESM.pdf (114 kb)
Table S1. Primers used in this study (PDF 114 KB)
299_2018_2322_MOESM2_ESM.pdf (129 kb)
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 (225 kb)
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 (136 kb)
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 (57 kb)
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)
299_2018_2322_MOESM6_ESM.pdf (250 kb)
Supplementary references (PDF 251 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Noriho Fukuzawa
    • 1
  • Chikara Masuta
    • 2
  • Takeshi Matsumura
    • 1
  1. 1.Bioproduction Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)SapporoJapan
  2. 2.Graduate School of AgricultureHokkaido UniversitySapporoJapan

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