Transient expression

Nicotiana benthamiana, a relative of tobacco, plants were cultivated in a growth chamber at 16 h under light at 24 °C and 40% relative humidity under a 16 h:8 h, light: dark photoperiod with 150 µmol m⁻² s⁻¹ light. Three-week-old plants were transplanted into pots and left to grow in the growth chamber under the same conditions for three additional weeks. The overexpression constructs, pBYR2fp-M_IRESE and pBYR2fp-N_FLAG, were separately introduced into A. tumefaciens strain GV3101 by the freeze–thaw method. The integrity of the plasmids in A. tumefaciens was confirmed by restriction mapping. For transient expression, the Agrobacterium pellet containing pBYR2fp-M_IRESE and pBYR2fp-N_FLAG, separately, was resuspended and diluted in 1 × infiltration buffer [10 mM 2-(N-morpholino] ethanesulfonic acid (MES), 10 mM MgSO₄, 200 μM acetosyringone, at pH 5.7) to an OD₆₀₀ of 1.0. Both A. tumefaciens strains were mixed together with same ratio, and bacterial suspensions were syringe-infiltrated into the abaxial side of 6-week-old N. benthamiana plant leaves which located at 4 and 6 numbered from the bottom and maintained at 24 °C growth chamber (Fig. 1b). Leaf tissues was harvested at 3 days post infiltration (dpi) for protein expression analysis and self-assembly of virus-like particles using western blot and TEM.