Fei Zhang, Pascale Rossignol, Tengbo Huang, Yewei Wang, Alan May, Christopher Dupont, Vladimir Orbovic, Vivian F. Irish
(A) SEM of a stage 8 leaf base from the ti1 ti2 double mutant showing a thorn transformed to a branch primordium and outgrowth of the bract. The scale bar represents 100 μm.
(B) Quantitative real-time PCR to detect CsWUS expression using RNA from vegetative apices of WT and ti1, ti2, and ti1 ti2 mutants. Expression of CsWUS was increased in the ti1, ti2, and ti1 ti2 mutants compared with WT. SEs were calculated from the means of three biological replicates.
(C) Diagram of the reporter and effectors used in dual-luciferase assays. Mini35S, 60-bp 35S minimum promoter element; REN, Renilla luciferase; LUC, firefly luciferase.
(D) Relative activity of different effectors on the CsWUS promoter in Carrizo citrange leaf protoplasts. ∗p < 0.05, ∗∗p < 0.01 (t test). SEs were calculated from the means of three biological replicates.
(E) Diagram of the CsWUS promoter reporters with or without mutations. Mutations in two putative TI1 binding sites (P1 and P2) are indicated as red crosses and the mutated sequences are indicated in lowercase.
(F) Relative activity of TI1 on the CsWUS promoter with or without mutations as indicated in (E). Light gray bars indicate interactions with the control BD effector; dark gray bars indicate interactions with the TI1 effector. ∗p < 0.05 (t test). SEs were calculated from the means of three biological replicates.
(G) Chromatin immunoprecipitation (ChIP) of TI1-eGFP protein with CsWUS chromatin regions, with amplicon locations (P1, P2, 3′) diagrammed. Dark-colored bars represent ChIP using an anti-HA (hemagglutinin) antibody (control), whereas light-colored bars represent ChIP using a GFP antibody. The ChIP experiments were repeated three times using independent biological replicates with similar results, and one representative dataset is shown. ∗∗∗p < 0.001 (t test).
(H) Electrophoretic mobility-shift assay (EMSA) using recombinant MBP-TI1 fusion protein and MBP protein. Biotin-labeled oligo probes corresponded to the P1 or P2 CsWUS promoter elements. 20X indicates 20 times unlabeled competitor probe, whereas m20X indicates 20 times unlabeled competitor probe with the same mutations as in (E).
(I and J) Transgenic plants containing a p35S::CsWUS-GR construct were treated with 0.1% ethanol buffer (I; mock) or 20 μM dexamethasone (J; DEX).
(K and L) Transgenic plants with a pTI1::CsWUS-GR construct were treated with 0.1% ethanol buffer (K; mock) or 20 μM DEX (L).