Yeast two-hybrid assay for AaPYL9-interacting proteins.

<p>Two-hybrid interaction test of AaPYL9 (native protein and two different point mutations) with AtABI1 (AT4G26080). Left columns show bait and prey plasmids. The photograph on the right shows the results of yeast two-hybrid assays on control medium that lacked leu, trp (SD/-T-L) and selection plate that lacked trp, leu, his (SD/-T-L-H), lacked trp leu, his, ade (SD/-T-L-H-A), and lacked trp leu, his, ade supplemented with 10 µM ABA(SD/-T-L-H-A+10 µM ABA). A: PYL9s from <i>Arabidopsis</i> and <i>Artemisia</i> were constructed as binding-domain (BD) fusion proteins, and tested their interactions with activation domain (AD)-fused AtABI1 with the Y2H assays by supplemented with or without ABA. Yeast strain expresses AD-AtABI1 and BD-AtPYL9 could grew well on SD/-T-L-H-A medium and SD/-T-L-H-A+10 µM ABA medium. However, Yeast cells harbouring AD-AtABI1 and BD-AaPYL9 only grew well on SD/-T-L-H-A+10 µM ABA medium. The empty vector used as negative control. B: Effect of point mutations on the interaction between AaPYL9 and AtABI1 in the yeast two-hybrid assay. (Left) Diagram of point mutation sites, H116A indicates a His to Ala mutation at the 116 latch residue sites. P89S indicates a Pro to Ser mutation at the 89 gate residue sites. (Right) The mutations of AaPYL9 were fused with the binding-domain and introduced into yeast with the AD-AtABI1 construct. The growth on selective medium was shown. Yeast expresses BD-AaPYL9H116A and AD-AtABI1 could not grew on all kinds of mediums except SD/-T-L. Yeast cells harbouring BD-AaPYL9P89S and AD-AtABI1 have similar results.