2.3 Y2H Assay
Y2H was performed using the Matchmaker® Gold Yeast Two-Hybrid System (Clontech, USA), according to the manufacturer’s protocol. To generate a vector for Y2H analysis, full-length cDNAs of PpVIN2 (GenBank Accession No. XM_007210252) and PpINHs (GenBank Accession Nos. XM_007208838, XM_007223513,XM_007209598,XM_007223389,and XM_007217302) were cloned into pGBKT7 (BD) and pGADT7 (AD) vectors, respectively. PpVIN2 was inserted at the BamHI-PstI restriction sites of pGBKT7, yielding BD-PpVIN2 . Each PpINH was inserted at the BamHI-XhoI restriction sites pGADT7, yielding five AD-PpINHs . Primers are shown in Table S1 . The invertase and inhibitor plasmids were co-transformed into the yeast strain Y2H Gold, according to the manufacturer’s instructions (Clontech, USA), including the recommended tests for autoactivation and toxicity. Transformants were plated onto the synthetic dropout nutrient media SD-LT (SD/-Leu/-Trp) containing X-α-Gal, and SD-LTHA (SD/-Leu/-Trp/-His/-Ade). pGADT7-T, pGBKT7-53, and pGBKT7-Lam were also co-transformed and analyzed separately as controls.