Abstract
Deconstructed viral vector systems for large-scale production of recombinant proteins in Nicotiana benthamiana plants require Agrobacterium tumefaciens-assisted delivery into mesophyll cells by vacuum infiltration of leaves. To clarify the importance of uniform infiltration over the leaf surface and to propose a possible method for uniform infiltration, we quantified the extent of leaf infiltration and evaluated the potential effect of uniform infiltration on recombinant protein yield. We also investigated the effects of plant characteristics (e.g., plant age, leaf dry mass per area) and leaf detachment treatment on the extent of infiltration. First, a simple method was developed to measure the extent of leaf infiltration using a red dye solution. The quantitative results showed that the extent of infiltration in young and old leaves was substantially lower than in mature leaves. However, recombinant hemagglutinin (HA), an influenza vaccine antigen, accumulated in the infiltrated area of young and old leaves, indicating that they can synthesize and accumulate HA at detectable levels. The extent of infiltration was affected by the plant age but not by leaf dry mass per area. Improving the extent of infiltration by supplemental syringe infiltration significantly increased total HA content in leaves. Thus, increasing the infiltrated leaf area represents a potential strategy for increasing the recombinant protein yield in deconstructed viral vector-based transient gene expression systems with A. tumefaciens. The extent of infiltration was also improved without the need for time-consuming syringe infiltration when detached leaves were subjected to vacuum infiltration, suggesting that this may be a potential method to increase the extent of infiltration.
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Acknowledgements
The authors would like to thank Shoko Miyagi and Akiko Ikeno for their technical assistance in experiments and Dr. Nobuyuki Matoba for providing invaluable suggestions. The magnICON vector was provided by Icon Genetics GmbH, Halle (Saale), Germany. The pNM216 and N. benthamiana seeds were provided by Kentucky BioProcessing, Inc., Owensboro, KY, USA. This work was financially supported in part by JSPS KAKENHI (Grant Numbers JP23780255 and JP17H03893 to RM) and by Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry (Grant Number 25025A) from the Ministry of Agriculture, Forestry, and Fisheries, Japan, to RM.
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Matsuda, R., Kushibiki, T., Fujiuchi, N. et al. Agroinfiltration of leaves for deconstructed viral vector-based transient gene expression: infiltrated leaf area affects recombinant hemagglutinin yield. Hortic. Environ. Biotechnol. 59, 547–555 (2018). https://doi.org/10.1007/s13580-018-0047-6
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DOI: https://doi.org/10.1007/s13580-018-0047-6