Article Figures & Data
Figures
- Figure 1.
CIP73 contains a Scythe_N ubiquitin-like domain and belongs to the large ubiquitin superfamily. A, Schematic illustration of the CIP73 protein. The deduced amino acid sequence of CIP73 contains 691 amino acid residues with a calculated molecular mass of approximately 73 kD. Notable features include the Scythe_N (Scythe is also known as BAT3) ubiquitin-like domain at the N terminus (21–93) and a putative NLS (686–689) shown by the asterisk. The CCaMK-binding region identified in the original Y2H screening (414–691) is in the CIP73 C-terminal region. B, Multiple sequence alignment of the N-terminal ubiquitin-like domain of CIP73 and the homologous sequence from Medicago (TC97370), Arabidopsis (TC312062), rice (TC302632), human BAT3_N (NP_542433), Xenopus Scythe_N (NP_001080008), and L. japonicus ubiquitin (AW720576). The numbers on the left and right indicate the positions of amino acids. C, Homology tree of the N-terminal ubiquitin-like domain of CIP73 homologs, Scythe_N, and ubiquitin. The tree was constructed using the DNAMAN software (version 5.2.2; Lynnon Biosoft). Note that the N-terminal ubiquitin-like domain of CIP73 is more closely related to the animal Scythe_N domain than to LjUbiquitin.
- Figure 2.
CIP73 interacts with CCaMK in yeast. A, Y2H assays for interaction between CCaMK and CIP73. The kinase domain (1–300) was used in the initial isolation of CIP73 from the Y2H Library. SD-Leu-Trp medium was used for testing successful mating, and SD-Leu-Trp-His-Ade medium was used for testing the interaction. The strength of interaction was measured through the β-galactosidase activity. The combination of BD-53/AD-SV40 or BD-CCaMK/AD-CYCLOPS (Yano et al., 2008) was used as a positive control, and BD-Lam/AD-SV40 was used as a negative control (Clontech). CIP73 could only interact with CCaMK when the CaM-binding domain and three EF-hand motifs were removed from the kinase domain. The N-terminal 80 amino acid residues (80–160) of CCaMK are sufficient for interacting with CIP73 in yeast cells. B, The kinase domain of CCaMK (1–300) can interact with full-length CIP73 and CIP73-C (414–691) but cannot interact with CIP73-N (1–413). [See online article for color version of this figure.]
- Figure 3.
CIP73 interacts with CCaMK in vitro and in planta. A, In vitro protein pull-down assay for testing the interaction between CCaMK and CIP73. His-tagged CIP73 (414–691) was incubated with the immobilized GST-CCaMK fusion protein or GST alone in the presence of EGTA (lanes 2–8) or Ca2+ (lane 9). After washing, the proteins were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue R250 (top). A similar gel was used for immunoblot using anti-His tag antibody (bottom). B, Interaction of CCaMK and CIP73 in planta. N. benthamiana leaves were cotransformed with SCFPC::CCaMK and CIP73::SCFPN (a–c) or with SCFPC::CCaMK and CYCLOPS::VenusN (d–f). Left images show fluorescence signal of the cells (a and d); middle images show the cell architecture (b and e); right images show the overlays of fluorescence and bright-field images (c and f). Bars = 20 μm.
- Figure 4.
CIP73 expression and subcellular localization. A, CIP73 and CCaMK expression was investigated by semiquantitative RT-PCR before and after M. loti inoculation using polyubiquitin as an internal control. Uninoculated roots (1D–12D) are shown as controls for the same periods post inoculation with M. loti. B, Nuclear localization of GFP::CIP73 in onion epidermal cells. The control GFP gene (a–c) and GFP::CIP73 fusion gene (d–f), delivered by particle bombardment, were transiently expressed in onion epidermal cells and observed with a laser-scanning confocal microscope 24 h after bombardment. Note that the control GFP is distributed both in the cytoplasmic and nuclear compartments, whereas GFP::CIP73 only localizes in the nucleus. Top images show GFP fluorescence of the cells (a and d); middle images show the cell architecture (b and e); bottom images show the overlays of GFP and bright-field images (c and f). C, The C terminus of CIP73 (486–691) shows strong nuclear localization in epidermal and cortex cells of transformed hairy roots. Bars = 50 μm for B and 20 μm for C. [See online article for color version of this figure.]
- Figure 5.
Phosphorylation of CIP73 by CCaMK in vitro. A, Autophosphorylation and phosphorylation assay of CCaMK in the presence of Ca2+ and CaM. CCaMK can phosphorylate CIP73-N (1–413) but not CIP73-C (414–691). Casein served as a positive control. B, Phosphorylation of CIP73-N (1–413) in the presence (+) or absence (−) of Ca2+ EGTA and CaM. Bottom images show autoradiographs of kinase assays, and top images show Coomassie Brilliant Blue staining of the same gels. The autophosphorylation activity of CCaMK was increased in the presence of Ca2+, and substrate (CIP73) phosphorylation was accelerated by the addition of Ca2+/CaM. [See online article for color version of this figure.]
- Figure 6.
Nodulation phenotype of CIP73-specific RNAi-transformed hairy roots. A, Five representative images of plants transformed with the CIP73 RNAi-1 construct compared with plants that were transformed with an empty vector (control). The plants were inoculated with M. loti MAFF303099 and grown in the absence of nitrogen, and the photographs were taken at 4 weeks after inoculation. B and C, Nodulation phenotype of control vector transgenic roots (B) and RNAi-1 transgenic roots (C). The transgenic hairy roots were further confirmed by GUS staining before photographing. Arrowheads indicate nodule positions on the roots. D, Total nodule number of vector control and two RNAi construct transgenic roots determined 4 weeks after inoculation with M. loti MAFF303099. E and F, Estimation of CIP73 transcripts in transgenic hairy roots by quantitative real-time RT-PCR (E) and RT-PCR (F). Bars = 2 cm for A and 2 mm for B and C. [See online article for color version of this figure.]
- Figure 7.
Analysis of rhizobia infection of CIP73 RNAi hairy roots. A to D, Histochemical staining of lacZ-labeled M. loti strain to follow the infection events at 7 d post inoculation. Representative ITs observed in vector control roots (A) and CIP73 RNAi-1 roots (B–D) are shown (blue). Arrows indicate the cellular localization of IT ends. E, Frequencies of the infection events per root. The data are presented as 15 individual transgenic plants for each construct and randomly scored in four roots between 4 to 6 cm per plant. Different letters above the bars indicate significant differences (P < 0.05, t test) between pairwise comparisons. Bars = 50 μm for A and 25 μm for B to D. [See online article for color version of this figure.]
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