Fig 2: MKKK18 interacts with the ABI1 protein phosphatase. (A) Yeast two-hybrid analysis of the interaction between MKKK18 and ABI1/2 PP2Cs. Diploid yeast colonies were grown on double (DDO-SD medium without Leu and Trp) or quadruple selective medium (QDO-SD medium without Leu, Trp, His or Ade) with or without supplemented X-a-Gal and aureobasidin. The bait (MKKK18) did not autoactivate the reporter genes in yeast. (B) Detection of ABI1/2 and MKKK18 expression in diploid yeast strains. Expression of BD and AD fusion proteins in yeast was determined by immunoblotting using specific anti-ABI1, anti-ABI2 and anti-MKKK18 antibodies. (C and D) Pull-down assays to verify the interaction of ABI1/2 with MKKK18. Input lines represent 100% of the ABI1/2. Recombinant GST–MKKK18 or His-MKKK18 was pre-coupled to glutathione–Sepharose, and incubated with StrepTag-ABI1 or StrepTag-ABI2, respectively. Pulled-down StrepTagged ABI1 protein was detected (IB) with the epitope tag antibody. The presence of recombinant protein was confirmed using anti-MKKK18 antibody. (E and F) ABI1–MKKK18 interaction occurs within the nucleus. BiFC analysis in Arabidopsis protoplasts expressing full-length ABI1/2 and MKKK18 fused to cECFP or nVenus, respectively. RFP (E) was used as a transformation control. CFP–CBP20 (cyan fluorescent protein–Cap Binding Protein 20) (F) was used as a marker of nuclear localization.

Fig 3: Regulation of MKKK18 activity and stability by ABI1 PP2C. Under standard conditions, active ABI1 protein phosphatase inhibits MKKK18 kinase activity and protein stability. ABA receptors and dephosphorylated MKKK18 are directed for degradation via the proteasome pathway. In the presence of ABA, ABI1 PP2C activity is restrained by binding of ABI1 to the ABA–PYL complex (ABA signaling is turned on). Active MKKK18 activates the downstream cascade. ABA inhibits the degradation of ABA receptors by limiting their polyubiquitination.

Fig 4: ABI1 inhibits MKKK18 activity. (A) Phosphatase activity of recombinant ABI1 and ABI2 proteins. The enzyme reactions were performed in a 50 µl final volume containing 3–5 µg pf GST–ABI1 or GST–ABI2. The results presented are the means from three independent biological replicates. (B) ABI1 inactivates MKKK18. The MKKK18–GFP immunocomplex was incubated with 3 µg of GST–ABI1 and GST–ABI2 (as a negative control), after which the kinase activity was determined with MBP as a substrate. Equal loading was confirmed by Coomassie Brilliant Blue (CBB) staining of MBP. The 32P-labeled MBP bands were quantified and then normalized against the intensity of the corresponding control band using ImageJ software. Data are means ± SD of the relative band intensities from three independent experiments. An asterisk (*) indicates statistically significant changes determined using Student’s t-test. (C) Recombinant MKKK18 has no autophosphorylation activity in vitro. GST–MKKK18 was incubated with MBP or/and MKK3, without or in the presence of [32P]ATP. Bands of GST–MKKK18, MKK3–GST and GST–SnRK2.6 are indicated by a filled circle, a triangle and an arrow, respectively. Protein loading was confirmed by CBB staining. The blots shown are representative of three independent trials.