Extended Data Fig. 9: TOR stabilizes PLT2.
From: TOP1α fine-tunes TOR-PLT2 to maintain root tip homeostasis in response to sugars
a, CFP florescence showing PLT2 transcript levels in pPLT2::CFP and pPLT2::CFP top1α under the indicated treatments. Scale bars: 50 µm. b, e, Y2H assays to examine TOR-PLT2 and TOR-PLT1 interactions (b) and to identify particular domains of PLT2 for the TOR-PLT2 interaction (e). The indicated construct combinations were co-transformed into yeast cells and screened on plates containing SD/–Leu,–Trp or SD/–Ade,–His,–Leu,–Trp medium. c, Amino acid sequence alignment of PLT1 and PLT2. The AP2 domains are marked by red lines. d, Diagram of the constructs used in the yeast two-hybrid (Y2H) assay. F: full length; N: N-terminus; C: C-terminus; KD: kinase domain; AP2: AP2 domain. f, g, Examination of PLT2 stability in a cell-free system. MBP-PLT2 was purified and added to cell extracts from WT plants treated with DMSO or MG132 (f) or from WT and tor-es plants (g). Anti-MBP and anti-P-S/T antibodies were used to detect MBP-PLT2 and phosphorylated MBP-PLT2, respectively. h, MBP-PLT2 signal intensity of (g) quantified by ImageJ. Values represent the mean ± s.d. (n = 3). Statistical significance was determined by Student’s t-test (two-tailed). i, Examination of MBP-PLT2S12A stability in a cell-free system. MBP-PLT2 and MBP-PLT2S12A were purified and added to cell extracts from WT plants. Anti-MBP and anti-P-S/T antibodies were used to detect MBP-PLT2 and phosphorylated MBP-PLT2, respectively. j, l, CC number (j) and meristematic zone (l) of the indicted plants. Seedling was grown on -Suc under dark condition for 5 days. The CC number (j) and PLT2-GFP level (Fig. 3j) were examined. Data were analyzed from 15 seedlings for each genotype from 3 experiments. Statistical significance was determined by Student’s t test (two-tailed). k, The root phenotypes of the indicated plants. The seedlings were grown on 1/2MS plate under light condition for 5 days after germination. The PLT2-GFP level and RAM size were examined. The white arrows indicate the first elongated cortex cell and the yellow bars indicate the RAM size. Scale bars: 50 µm. For a and k, (m/n) indicates m in n of biological repeats showing the displayed features. For f, g and i, experiments were repeated three times with similar results. For gel source data, see Unmodified_Gels_Fig2.
