Fig. 7

Yeast two-hybrid interaction of assay of OsMPKs and OsAux/LAX1. The transformed yeast constructs were grown in selection media [drop out (DO) and double drop out (DDO)]. Drop out media lacks -Leu/-Trp and double drop out media lacks –Ade/- His/-Leu and –Trp amino acids. In the study, the OsAux/LAX1 gene was incorporated into BD vector and OsMPK genes were incorporated into AD vector and vice versa. A swapping assay was conducted by incorporating OsAux/LAX1 into AD vector and OsMPKs in BD vector. Transformed colonies were first grown in DO media. The colonies raised in DO were then plated in DDO media. The colony those grown in DDO media were considered to be interacting with each other. OsAux/LAX1 in AD vector and OsMPKs in BD vector and vice versa grew in DDO media, confirming that OsAux/LAX1 interacts with MPKs. When OsAux/LAX1 gene in AD and BD vector was transformed with empty vector (BD and AD, respectively), colonies were observed in DO media but not in DDO media. Similarly, when OsMPKs in AD and BD vector were transformed with empty BD and empty AD vector, no colonies were observed in DDO media. This confirms that neither OsAux/LAX1 nor OsMPKs were able to grow in DDO media due to lack of their interacting gene. Absence of colonies in DDO media in empty vector confirms that, there is no auto-activation of yeast-two hybrid assay and the interactions are positive