LIR1 expressed in <i>Arabidopsis thaliana</i> is targeted to the plasma membrane and decreases the iron content of leaves.

<p>(a) Confocal images of root sections of <i>Arabidopsis thaliana</i> expressing LIR1 fused to GFP at the C-terminus. Red, cell wall/plasma membrane staining with propidium iodide (PI). Green, LIR1-GFP. Scale bars: 50 μm (upper panel) and 10 μm (lower panel). (b) ICP-MS quantification of the iron total content from leaves of wild type (WT), WT expressing LIR1, and <i>irt1-/- Arabidopsis thaliana</i>. A-C, independent <i>Arabidopsis</i> transgenic lines containing the 35S promoter-driven <i>LIR1</i> cDNA (35S::LIR1). D-F, independent transgenic lines of 35S::LIR1+GFP (35S promoter-driven LIR1 cDNA fused to GFP). The data represent the mean ± SD of 3 samples for each strain, normalized for tissue weight. (c) Phenotype of <i>irt1-/-</i> and <i>irt1-/-</i> expressing LIR1 (<i>irt1-/-</i> + LIR1) <i>A</i>. <i>thaliana</i> plants grown in soil irrigated with either water (Control) or 0.5 g/L Sequestrene (+ Iron) for 20, 23, 26 and 30 days post germination (dpg). (d) ICP-MS quantification of the iron total content from leaves of <i>irt1-/-</i> and <i>irt1-/-</i> expressing LIR1 (<i>irt1-/-</i> + LIR1) <i>A</i>. <i>thaliana</i> irrigated with either water (control) or 0.5 g/L Sequestrene (+ Iron). Plant tissue was collected 30 days post germination (dpg). The data represent the mean ± SD of 3 leave samples for each strain, normalized for tissue weight. * p = 0.0147 (<i>irt1-/-</i> + Iron vs. <i>irt1-/-</i> + LIR1 + Iron).</p

LIR1 deficiency markedly reduces <i>L</i>. <i>amazonensis</i> virulence.

<p>(a) Microscopic quantification of intracellular parasites in BMM infected with purified metacyclic forms of wild type (WT), <i>LIR1</i> double knockout (KO), <i>LIR1</i> single knockout (SKO) and add-back (AB) <i>L</i>. <i>amazonensis</i> (multiplicity of infection = 3) for 3, 24, 48, 72 and 96 h. The values represent the mean ± SD of triplicate determinations in one experiment that is representative of 3 independent experiments. * p = 0.031 (KO vs. AB 24 h), ** p = 0.0063 (KO vs. AB 72 h); p = 0.0019 (KO vs. AB 96 h); *** p = 0.0007 (KO vs. AB 48 h). (b) AlamarBlue fluorescence viability assay of 10⁶, 2x10⁶ and 4x10⁶ purified metacyclics purified from WT and KO promastigote cultures. The values represent the mean ± SD of 3 independent experiments. (c) Immunofluorescence deconvolution images of BMM infected for 3 h with WT and KO metacyclic promastigotes. Blue, DAPI DNA staining; green, lysosomes and PV membranes stained with anti-Lamp-1 antibodies. Scale bars = 11 μm. (d) Cutaneous lesion development in C57BL/6 mice inoculated with 10⁶ purified metacyclic forms of wild type (WT), <i>LIR1</i> double knockout (KO), <i>LIR1</i> single knockout (SKO) and add-back (AB) <i>L</i>. <i>amazonensis</i>. The data represent the mean ± SEM of measurements from 5 different mice in each group. (e) Parasite load in footpad tissues determined at 10.5 weeks after infection (n = 5).</p