Y2H protein–protein interaction assay. A, B) Vector maps of bait and prey constructs. The ORF of CrROP2, CrROP3, and CrROP5 were cloned in-frame with AD of pGADT7, and the ORF of CrPGGT-I was cloned in-frame with the DBD of pGBKT7 vector. C, D) Mating-based Y2H screening. CrROP2 or CrROP3 or CrROP5 were separately transformed into AH109 (MATa) yeast strain, which were then co-cultivated with the compatible mating type Y187 (MATα) yeast strain having CrPGGT-I or empty vector pGBKT7 (EV) to identify bait-interaction in the resulting diploid yeast cells. The transformants were plated using different dilutions on synthetically defined (SD) medium lacking tryptophan (Trp), and leucine (SD/-Trp/-Leu) (C), and on SD/-Trp/-Leu medium lacking histidine and adenine (SD/-Trp/-Leu/-His/-Ade) (D). The plates were incubated for 3 to 5 d at 30 °C and observed for growth. Growth of yeast culture on SD/-Trp/-Leu/-His/-Ade indicates interaction of CrROP3 and CrROP5 with CrPGGT-I (F). Three independent colonies were tested per combination and a representative colony is shown. E, F) Schematic representation of ΔCrROP3 and ΔCrROP5 showing lack of CSIL motif. “LKA” and “QKA” represent three amino acids preceding the CSIL motif in CrROP3 and CrROP5, respectively. G, H) Mating-based Y2H screening of interaction. CrROP3 or ΔCrROP3 and CrROP5 or ΔCrROP5 were separately transformed into AH109 (MATa) yeast strain, which were then co-cultivated with the compatible mating type Y187 (MATα) yeast strain having CrPGGT-I to identify bait-interaction in the resulting diploid yeast cells. The transformants were plated using different dilutions on SD/-Trp/-Leu (G), and on SD/-Trp/-Leu/-His/-Ade (H).