Cdna library construction kit

The standardized yeast-two hybrid protocol^{36 (link)} was used to perform the screen in which CmWIP1 protein served as bait and cDNA library as a prey in order to identify interacting protein partners of CmWIP1. Total RNA was extracted from male, female and hermaphrodite flowers just before and after the arrest of the inappropriate organ^{37 (link)} using the Trizol reagent (Invitrogen). The cDNA library was synthesized from 6 µg of total RNA using the cDNA Library Construction Kit (Invitrogen). The CmWIP1 bait gene was fused to the GAL4 DNA binding domain into pDEST^TM32 vector. The cDNA library was fused to the GAL4 transcriptional activation domain cloned into the pDEST^TM22 vector. For the two-hybrid assays, yeast cells MaV203 (Invitrogen) were cotransformed with the two constructs according to the protocol of Dohmen et al.^{38 (link)}. The ability to drive the HIS3 reporter gene was assessed by growing transformants on selective medium lacking tryptophan, leucine, and histidine and supplemented with 60 mM 3-amino-19,29,49-triazole (3-AT). Handling of yeast cultures and plate growth assays were performed as described in the Yeast Protocols Handbook (Clontech).