Transient Expression of GFP-IgBBX in Tobacco

The p35S::GFP-IgBBX constructs were introduced into tobacco epidermal cells via the Agrobacterium-infiltrated tobacco (Nicotiana benthamiana) leaf method [26 (link)]. The plasmid of IgBBX for transient transformation was generated using the Invitrogen Gateway system, according to the manufacturer’s instructions. The IgBBX ORFs, lacking the stop codon, were amplified using a Phusion^®High-Fidelity PCR kit (New England Biolabs, Ipswich, MA, USA) (primers listed in Table S1), then inserted into the pMD19-T easy vector (TaKaRa, Tokyo, Japan) to allow its sequencing-based validation. Each pENTR^TM1A-IgBBX plasmid was previously subjected to the LR Clonase™ II enzyme mix (Invitrogen, Carlsbad, CA, USA) reaction to obtain GFP-fused constructs using the binary vector pMDC43, resulting in the plasmid p35S::GFP-IgBBX. For transient expression, Agrobacterium tumefaciens strain EHA105 carrying the pMDC43 and pMDC43-IgBBX was grown separately to OD600 = 0.8 and coinfiltrated with the p19 strain into leaves of five-week-old N. benthamiana. The YFP fluorescent signals were monitored 48 to 72 h after infiltration using a confocal laser scanning microscopy (LSM 700, Carl Zeiss).