*4.4. Y2H and BiFC*

The full-length ORF sequence of *IlAP2* was inserted into the PGADT7 vector using a ClonExpress II One Step Cloning Kit (Vazyme) to form the recombinant vector PGADT7– *IlAP2*. According to the Yeast Protocols Handbook (PT3024-1, Clontech, USA), the negative control (PGBKT7-LAM + PGADT7-T), positive control (PGBKT7-53 + PGADT7-T) and recombinant vector (PGADT7–*IlAP2* + PGBKT7–*IlMT2a*) were transformed into YH2GOLD yeast strain using the Yeastmaker™ Yeast Transformation System 2 (Takara). After growing in SD-Trp-Leu and SD-Trp-Leu/x-α-Gal medium (Takara) for 3 days, the monoclones were inoculated into YPDA liquid medium for 24 h of dark culture. Ten-fold serial dilutions (1, 1:10, 1:102, 1:103 and 1:104) were spotted onto SD-Trp-Leu and SD-Trp-Leu /x-α-Gal solid media for 3 days to observe the phenotype.

The full-length ORF sequence of *IlAP2* was inserted into the PSM35S–NYFP vector using the ClonExpress II One Step Cloning Kit (Vazyme). The full-length *IlMT2a* ORF sequence was inserted into the PSM35S–CyFP vector to form the recombinant vector PSM35S–*IlMT2a*–CyFP. The constructed vector plasmids were transferred into *Agrobacterium tumefaciens GV3101* using electro-transformation method and cultured for 2 days at 30 ◦C. Subsequently, the constructed vector plasmids were mixed in a 1:1 ratio and injected into the lower epidermis of tobacco leaves. The results were observed with confocal laser scanning microscopy (Zeiss LSM 710 META) after 2 days under low light conditions with BRZ1-nTFP + HLH40-cYFP as positive control [47–49].
