Yeast Two-Hybrid Screen and Assays

In order to find UBAC2a-interacting proteins, a Gal4-based yeast two-hybrid system was utilized as previously described (Zhou J et al., 2018). In brief, UBAC2a was PCR amplified using gene-specific primers (5′-agc​gaa​ttc​atg​aac​ggc​ggt​ccc​tcc-3′ and 5′-agc​gtc​gac​tta​gtt​tct​gtc​gaa​tcc​cat​t-3′) and cloned into pBD-GAL4 vector to generate the bait vector. The Arabidopsis HybridZAP-2.1 two-hybrid cDNA library was prepared from Arabidopsis plants as previously described (Xu et al., 2006). The bait plasmid and the cDNA library were used to transform yeast strain YRG-2. Yeast transformants were plated onto selection medium lacking Trp, Leu, and His and confirmed by β-galactosidase activity assays using o-nitrophenyl-β-d-galactopyranose as substrate.

To identify NAIP1-interacting proteins, full-length NAIP1 was PCR amplified using gene-specific primers (5′-agc​gag​ctc​atg​tca​gag​ata​gaa​gaa​gaa​gaa​ga-3′ and 5′-agc​gga​tcc​tta​gtg​agc​gtt​gcg​tgt​g-3′) and cloned into pBD-GAL4 vector as bait vector in screening the Arabidopsis HybridZAP-2.1 two-hybrid cDNA library for interacting proteins. For assays of interactions among NAIP2 and NAIP3 with NAI2 and NAIP1, full-length NAIP2 was PCR amplified using gene-specific primers (NAIP2, 5′-agc​act​agt​atg​gga​gac​gac​cag​cta​ga-3′ and 5′-agc​gga​tcc​tta​acg​cat​gtt​tct​gtt​aag​g-3′; NAIP3, 5′-agc​gag​ctc​atg​tct​caa​agc​ggc​ggt-3′ and 5′-agc​act​agt​tca​acg​ccc​act​tgt​gag​aag​tc-3′) and cloned into the bait vector pBD-GAL4. The DNA fragment for the C-terminal HHD domain of NAIP1 was PCR amplified using the primers 5′-agc​gga​tcc​tta​gtg​agc​gtt​gcg​tgt​g-3′ and 5′-agc​gaa​ttc​tct​cct​cga​cgc​cat​tct​gt-3′, and the PCR product was inserted into the pBD-GAL4 vector. The DNA fragment for the N-terminal domain of NAIP1 was generated by removing the C-terminal BglII-BamHI fragment from the NAIP1 coding sequence in the pBD-NAIP1 vector. Various combinations of bait and prey constructs were cotransformed into yeast cells, and interactions were analyzed through assays of the LacZ β-galactosidase reporter gene activity.