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. 2009 Sep 29;106(41):17588–17593. doi: 10.1073/pnas.0907095106

Fig. 2.

Fig. 2.

Group A PP2Cs interact with subclass III SnRK2s. (A) Yeast two-hybrid analysis of SnRK2s and PP2Cs. GAL4BD:SnRK2s and GAL4AD:PP2Cs were introduced to yeast cells as indicated. Black slopes indicate screening stringency of SD media supplemented with: left, -LW; center, -LWH + 10 mM 3-AT; right, -LWHA + 30 mM 3-AT. (B) Yeast two-hybrid analysis of PP2C mutations. ABI1, abi1–1, abi1–1R6, ABI2, and abi2–1 were tested against subclass III SnRK2s. The black slope demonstrates stringency of screening media: SD -LW, SD -LWH + 10 mM 3-AT, SD -LWHA + 30, 60, or 100 mM 3-AT from left to right. (C) Subcellular localization of GFP-tagged SnRK2 or PP2C proteins that were transiently expressed in Arabidopsis mesophyll protoplasts. (D) Co-immunoprecipitation of GFP-tagged SnRK2s and HA-tagged PP2Cs was performed using Arabidopsis protoplasts. Immunoprecipitates against anti-GFP antibody (IP) or crude extracts (input) were analyzed via Western blotting (WB) using anti-GFP or -HA antibodies. (E) BiFC analyses were performed for the pairs of SRK2E-YFPN/ABI1-YFPC or SRK2I-YFPN/ABI1-YFPC in epidermal cells of fava bean leaves. The photos were YFP, DIC, autofluorescence (auto) and merged images. The results were confirmed through several replications.