Transient gene expression in whole plants by using viral vectors is promising as a rapid, mass production system for biopharmaceutical proteins. Recent studies have indicated that plant growth conditions such as air temperature markedly influence the accumulation levels of target proteins. Here, we investigated time course of the amount of recombinant hemagglutinin (HA), a vaccine antigen of influenza virus, in leaves of Nicotiana benthamiana plants grown at 20 degrees C or 25 degrees C post viral vector inoculation. The HA content per unit of leaf biomass increased and decreased from 4 to 6 days post inoculation at 20 degrees C and 25 degrees C, respectively, irrespective of the subcellular localization of HA. The overall HA contents were higher when HA was targeted to the endoplasmic reticulum (ER) rather than the apoplast. Necrosis of leaf tissues was specifically observed in plants inoculated with the ER-targeting vector and grown at 25 degrees C. With the ER targeting vector, the maximum HA contents at 20 degrees C and 25 degrees C were recorded at 6 and 4 days post inoculation, respectively, and were comparable to each other. HA contents thereafter decreased at both temperatures; the rate of reduction appeared faster at 25 degrees C than at 20 degrees C. From a practical point of view, our results indicate that the strategy of targeting HA to the ER, growing plants at a lower temperature of 20 degrees C, and harvesting leaves at around a week after vector inoculation should be implemented to obtain a high HA yield stably and efficiently. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.