Figure 4.

CKI1-Mediated Signaling Is Connected to the Two-Component Signal Transduction Pathway.

(A) CKI1 induces cytokinin-independent ARR2 phosphorylation. Protoplasts from ahk2 ahk3 plants were cotransfected with ARR2-HA along with CKI1-HA or CKI1^H405Q-HA, incubated for 6 h, and treated with 100 nM t-zeatin (cytokinin) in the presence of 100 μM cycloheximide for 1 h. Wild-type protoplasts transfected with ARR2 served as a control. The mobility shift of ARR2 induced by phosphorylation was detected with an anti-HA antibody. Equal amounts of protein were loaded on each lane.

(B) A negative form of CKI1 protein represses the AHK4-mediated induction of ARR6. Protoplasts from wild-type plants were transfected with ARR6-LUC alone, wild-type AHK4, wild-type AHK4 plus mutant CKI1, wild-type CKI1, or wild-type CKI1 plus mutant AHK4. Error bars indicate se (n = 2). CKI1^H405Q and AHK4^H459Q are negative versions of CKI1 and AHK4, respectively. Rubisco large subunit (RbcL) stained by Coomassie blue was used as a protein loading control.

(C) CKI1-HA but none of the tested AHKs-HA proteins c-immunoprecipitate with myc-tagged CKI1. Mesophyll protoplasts from wild-type plants were transfected with CKI1-HA, CKI1^H405Q-HA, AHK3-HA, or AHK4-HA, with or without CKI1-myc, incubated for 6 h, and then immunoprecipitated with anti-myc antibodies. CKI1 proteins were detected with an anti-HA antibody.

(D) CKI1 forms homodimers in tobacco leaf cells. Confocal images of abaxial epidermal tobacco leaf cells expressing the indicated YFP-N and YFP-C fusion proteins demonstrate YFP fluorophore reconstitution due to protein–protein interaction of the tested proteins (top row). The bottom row shows the corresponding bright-field images of the transiently transformed cells. Bars = 50 μm.

(E) CKI1 forms dimers. Protoplasts expressing CKI1-HA were solubilized with Triton X-100. Total protein was treated with increasing amounts of the cross-linker BS³ and subjected to SDS-PAGE. Two bands corresponding to the predicted sizes of the CKI1 monomer and dimer were detected with the anti-HA antibody.

(F) and (G) Genetic manipulation of CKI1 activity affects two-component signaling in planta. Transgenic plants overexpressing CKI1 or CKI1^H405Q (F) or CKI1 T-DNA insertion lines (G) show changes in the expression of specific type-A ARRs. Quantitative RT-PCR was performed with total RNA extracted from 3-week-old seedlings (F) or inflorescence stems (G) using gene-specific primers for type-A ARRs (see Supplemental Table 1 online for primer sequences). Error bars indicate se (n = 8 [F] and 3 [G]). Asterisks indicate statistically significant differences from wild-type transgenic plants analyzed by Student's t test (*P < 0.05; ** P < 0.01).