Fig. 5

Targeted yeast-two-hybrid assays. a Targeted yeast hybrid assays were performed between AtAHL20, AtAHL6, AtAHL19, AtAHL22 and AtAHL29. 1 mM 3-amino-1,2,4-triazole (3-AT) was used to suppress auto-activation of bait proteins. b Yeast cells that were co-transformed with both the bait and prey constructs were plated on synthetic dropout II (SDII) media, which lacked tryptophan and leucine, as positive controls to demonstrate successful transformation. c Four individual colonies were picked and plated on synthetic dropout IV (SDIV) media lacking tryptophan, leucine, histidine, and uracil but supplemented with 1 mM 3-AT to suppress autoactivation. Clade-B member AtAHL6 and Clade-A member AtAHL20 physically interacted with themselves but not with each other. AtAHL20 and its closest family member AtAHL19 physically interacted with each other, as well as themselves. AtAHL20 also interacted with other Clade-A AHLs: AtAHL22 and AtAHL29. These results may suggest that AHLs function redundantly to regulate flowering time, possibly as part of homo-, hetero-dimers or multiple AHLs protein complex that includes AtAHL19 AtAHL20, AtAHL22, and AtAHL29. AtAHL6, a Clade-B member, was used as a control to show that not all AHLs interacted with each other. Light brown lines show demarcation between panels of Y2H assays that were performed separately, but have been pasted together to make this figure