Selective interaction of plant homeodomain proteins mediates high DNA-binding affinity

Abstract

Understanding molecular mechanisms that control cell fate in the shoot apical meristem is a fundamental question in plant development. Genetic and molecular studies demonstrate that maize KNOTTED1 (KN1) of the TALE (3-aa acid loop extension) class of homeodomain (HD) proteins is involved in shoot apical meristem function. We show that KN1 interacts with knotted interacting protein (KIP), a BEL1-like TALE HD protein. Interaction between KN1 and KIP is mediated by conserved domains in the N termini of both proteins. The KN1 DNA-binding sequence, TGACAG(G/C)T, was biochemically identified, and in vitro DNA-binding assays show that individually KN1 and the HD of KIP bind specifically to this motif with low affinity. The KN1-KIP complex, however, binds specifically to this DNA-binding motif with high affinity, indicating that the association of KN1 and KIP may function in transcriptional regulation.

Figure 1

Yeast two-hybrid and sequence alignments. (A) KIP interacted with the MEINOX domain (M) of KN1 and full-length KN1. (B) Interaction of the MEINOX domain with KIP depended on both conserved regions, M1 and M2, in the N terminus (N) of this protein. (C) The amino acid sequence of KIP showing M1 and M2 of the MID (blue) and HD (red). (D) Alignment of the HD sequences of KIP and BELL proteins of Arabidopsis. (E) Alignment of the MID, M1 and M2, in KIP and the Arabidopsis BELL proteins. The following GenBank accession numbers were used: BEL1, U39944; BLH2, AF173816; ATH1, X80126.

Figure 2

In vitro binding studies with KIP and KNOX proteins. (A) In vitro-synthesized [³⁵S]Met-KN1 and KIP were analyzed by SDS/PAGE. (B and C) Coimmunoprecipitation was used to analyze the interaction of KIP and KN1. The ligand blot assay used in our experiments has been described (49, 50). About 1 μg of each protein fusion as well as GST and BSA controls were separated by SDS/PAGE (D) and blotted to nitrocellulose. After the proteins were denatured and renatured, the blots were probed with 20 μl of [³⁵S]Met-KIP in renaturation buffer for 4 h at 4°C, gently shaking. Blots were washed in Tris buffer solution plus 0.05% Tween 20 (49), air-dried, developed for 12 h, and analyzed by autoradiography (E).

Figure 3

Selection and amplification of the KN1 DNA-binding motif. In some cases more than one DNA motif was found on SAAB clone. * designates identical SAAB clone.

Figure 4

EMSA analysis of purified T7-KN1 and GST-HDKIP. (A) WT-DNA and MT-DNA binding sites were used as probes. (B) T7-KN1 was incubated with WT-DNA probe (W) or MT-DNA probe (M). (C) The third helices in the HDs of MEIS, KN1, and KIP were aligned. (D) In EMSA, GST or GST-HDKIP was incubated with W or M probes. (B and D) DNA-binding specificity for T7-KN1 and GST-HDKIP were determined by competing with unlabeled WT-DNA (W) or MT-DNA (M), at 5-, 50-, and 500-fold molar excess over labeled probe.

Figure 5

EMSA analysis of the KN1–KIP complex. (A) DNA-binding assays were performed with 1 ng or 10 ng of KN1 or T7-KIP alone. KN1 and T7-KIP were mixed together in DNA binding assays at 1 ng or 10 ng of each protein per reaction. DNA-binding specificity was determined by competing with unlabeled WT-DNA (wt comp) or MT-DNA (mt comp), at 10- and 100-fold molar excess over labeled probe. (B) Antibodies to KN1 (α-KN1) or the T7 epitope (α-T7) were added to the DNA-binding reaction. As a control, preimmune (α-pre) sera were also added to the KN1-KIP DNA-binding reaction. Mixing 100 ng GST with 10 ng KN1 or 10 ng of KIP did not form a DNA-binding complex.