Fig. 4: LCR associates with TPL.
From: miR394 and LCR cooperate with TPL to regulate AM initiation
a IP-MS using p35S::LCR-GFP and p35S::LCR4m-GFP as baits to identify LCR-interacting proteins in Arabidopsis thaliana. Important proteins are shown in the list. p35S::GFP served as the negative control. b The interaction between LCR and TPL was verified via Y2H assays. The protein domains of LCR and TPL used in Y2H are shown at the top. Yeast cells transformed with BD-LCR, BD-LCRF-box and BD-LCRΔN/AD-TPL were grown and turned blue on SD-L/-T/-H/-A + X-α-gal medium. Yeast transformed with BD-LCRΔC/AD-TPL or BD-LCR/AD-Blank did not grow on SD-L/-T/-H/-A + X-α-gal medium. pGADT7-T and pGBKT7-53 were used as positive controls, whereas empty vectors were co-transformed as negative controls. c BiFC assays revealed that LCR and LCRΔF-box interacted with TPL in vivo in N. benthamiana (tobacco) leaves. H2B-mCherry is a nuclear-localized marker. The combination of 35S::cYFP and 35S::TPL-nYFP was used as a negative control. Scale bars, 5 μm. d, e Stability test of TPL in the WT and lcr-1 backgrounds. Total protein was extracted from p35S::TPL-GFP (blue) and p35S::TPL-GFP lcr-1 (red) plants with or without MG132 treatment. TPL degradation was measured by immunoblotting with an anti-TPL antibody at the indicated time points. Actin was used as a loading control. Band intensities were quantified with ImageJ in (e). Data are mean ± s.d. (***p < 0.001, ns no significant difference, two-way ANOVA with Tukey’s honestly significant difference test. n = 3 biological replicates). The experiments in (b–e) were performed at least three times with similar results.
