Saccharomyces cerevisiae strain ah109

To verify interactions between SlBIR3 and BAK1, the pGADT7 plasmid (Clontech, Mountain View, CA, USA) containing the cytoplasmic domain of SlBIR3, and pGBKT7 plasmid (Clontech, Mountain View, CA, USA) containing the cytoplasmic domain of AtBAK1 or SlBAK1 were co-transformed into yeast (Saccharomyces cerevisiae) strain AH109 (Clontech, Mountain View, CA, USA). The resulting transformants were grown on SD/-Leu-Trp (DDO) medium at 30 °C for 3 days. Individual colonies were picked from the plates and subcultured in 2 mL of SD/-Leu-Trp liquid medium at 30 °C for 24 h. The cultured cells were spotted on SD/-Leu-Trp, SD/-Leu-Trp-His-Ade (QDO, and SD/-Leu-Trp-His-Ade + X-α-Gal (QDO + X-α-Gal) medium and incubated at 30 °C. Plates were photographed after 4 days of incubation.

Yeast two-hybrid assays were conducted as described previously [43] (link). Saccharomyces cerevisiae strain AH109 (Clontech) were co-transformed and plated on solid media lacking leucine and tryptophan (–L/–W; growth). Liquid cultures of co-transformed yeast were serially diluted (A_{600 nm} = 0.2, 0.02 and 0.002) and spotted (2 µL) on plates that also lacked histidine (−L/−W/−H; low stringency), and included 0.5 mM 3-amino-1,2,4,-triazol (−L/−W/−H + 3-AT; medium stringency), or excluded adenine (−L/−W/−H/–A; high stringency), as well as growth control plates.

Y2H screening was performed as described by Wang et al.^{62 (link)}. The tomato cDNA library constructed in the prey vector pGADT7 (AD) was screened with the SlUBC32 cDNA fragment cloned into the bait vector pGBKT7 (BD) in Saccharomyces cerevisiae strain AH109 (Clontech). The yeast zygote was selected on SD/-Leu-Trp-His-Ade medium (-LWHA) supplemented with α-D-galactoside (X-α-gal) according to the manufacturer’s instructions (Clontech). The positive clones carrying putative SlUBC32-interacting proteins were identified by sequencing.
Y2H analysis was carried out using Matchmaker GAL4 Two-Hybrid System 3 following the manufacturer’s protocols (Clontech). The cDNA fragments of the proteins were cloned into the AD and BD vectors, respectively. The primers used for the vector construction are listed in Supplementary Data 3. The resulting constructs were co-transformed into S. cerevisiae strain, and then plated on SD/-Leu-Trp medium (-LW) and SD/-Leu-Trp-His-Ade medium (-LWHA) containing X-α-gal. The transformants carrying empty vectors (BD or AD) were used as negative controls.

For the yeast two-hybrid assay, the fragments of AtSYP71 (cytosolic region), MIP2 (Sec39 domain) and MIP3 were amplified using cDNA obtained from seedlings with specific primers and ligated into pEASY-Blunt vector (TransGen, #CB101-01), respectively. After Sanger sequencing confirmation, the fragments were transferred into pGADT7 or pGBKT7 vectors, respectively. AtSYP81, AtSEC20 and MAG2 constructs were generated in our previous study (Li et al., 2006 (link); Zhao et al., 2018 (link)). The paired constructs were introduced into Saccharomyces cerevisiae strain AH109 (Clontech) and selected on SD/-Leu/-Trp medium. The interactions were detected on SD/-Leu/-Trp/-His/-Ade medium.

For yeast two‐hybrid assay, the open reading frame of ATX1 was amplified and inserted into NcoI and SmaI sites of the bait vector pGBKT7 (BD). The open reading frame of SEC was cloned into ClaI and SacI sites of the prey vector pGADT7, and the cDNA fragment of SEC‐N (cDNA only encoding TPR domains at N‐terminus) was cloned into NdeI and EcoRI sites of pGADT7, respectively. Primers used for plasmid construction are shown in Appendix Table S3. To confirm protein interactions in yeast cells, BD‐ATX1 was cotransformed with AD‐SEC and AD‐SEC‐N into Saccharomyces cerevisiae strain AH109 (Clontech) according to the manufacturer's instructions. Transformants first were grown on SD/‐Leu/‐Trp (SD/‐2) medium, and then, positive clones were screened on SD/‐Leu/‐Trp/‐His/‐Ade (SD/4) medium. pGBKT7‐53 and pGADT7‐T vectors were cotransformed as positive controls, and pGBKT7‐Lam and pGADT7‐T were cotransformed as a negative control.

For the yeast two-hybrid assay, AtSYP81 plasmids were generated in a previous study (Li et al., 2006 (link)). The cytosolic regions of AtSEC22, AtSYP31, and AtSYP32 were amplified using corresponding specific primers, Sec22NdeI-F/BamHI-R, SYP31/32NdeI-F, and SYP31/32BamHI-R, respectively. Then these fragments were ligated into the pEASY-Blunt vector (TransGen, #CB101-01), respectively. After Sanger sequencing confirmation, the fragments were transferred into pGADT7 or pGBKT7 vectors, respectively. The paired constructs were introduced into Saccharomyces cerevisiae strain AH109 (Clontech) and selected on SD/-Leu/-Trp medium. The interactions were examined on SD/-Leu/-Trp/-His/-Ade medium.