Fig. 2

Generation of NanoLUC vectors and characterisation in transient expression systems. a Structure of pGWB vectors extended for NL3F10H translational fusions. The Gateway NL3F10H cassette was generated by Gibson Assembly of PCR products A and B, designed with overlapping regions between them and pGWB601 vector. The vector was digested with Nco/SacI restriction enzymes to insert the new cassette. T-DNA Left Border (LB) and right border (RB). NOPALINE SYNTHASE promoter and terminator (NOS pro, NOS Term). Gateway cassettes of type attR1-attR2 can be used as destination in a LR Gateway cloning reaction. The pGWB vectors have a common Scp^r marker for selection in bacteria, with a pPZP vector backbone. The variants used here were pGW401 (Kan^r), pGWB501 (Hyg^r), pGWB601 (BASTA^r) and pGWB701 (Tun^r). NL3F10H was introduced in each vector by NcoI/SacI sub-cloning. b Plate reader set up for measuring bioluminescence signal on a 96-well plate format using a Tristar luminometer plate reader (Berthold). Light was a mix of red:blue 2:1 LEDs, total intensity 50 µmol m⁻² s⁻¹. c Activity of pGWB601::35S:NL3F10H and pGWB635::35S:LUC in protoplasts over several days. Protoplasts were isolated and transformed with equimolar amount of plasmid by the method of Hanse et al. [29] and followed in a 96-well black plate for 3 days in constant light at 21 °C. Furimazine was used as substrate for NL3F10H 1:50 Furimazine:W5 buffer and for LUC 5 mM luciferin. Mean of three technical replicates error bars show S.E.M. d Transient transformation of Arabidopsis efr-1 seedlings with NanoLUC. Seedlings were transformed by the AGROBEST method using either promoterless pGWB601NL3F10H or pGWB601::35S:NL3F10H. Seedlings were transferred to a 96-well flat white plate with 50 µl of 1:50 Furimazine:0.01% Triton X-100 and tracked for 48 h in constant light. Bars average signal over the time interval for a single pool of 10 seedlings. Error bars S.D