Fig. 4: NIN is required for pre-infection priming and MtAnn1 expression.
From: Annexin- and calcium-regulated priming of legume root cells for endosymbiotic infection
pMtAnn1:GUS activity in roots of M. truncatula A17 (a, b) or nin (c, d) inoculated or not with S. meliloti at 4 dpi. GUS activity (blue) is visualised in endodermis (asterisks), root tips or lateral roots (arrowheads) and rhizobial infection sites (arrows). LacZ-expressing rhizobia are in magenta (Close-up in b). c Infection-induced pMtAnn1:GUS activity is abolished in nin. d NIN under pEXPA promoter induces pMtAnn1:GUS activity in root epidermis without rhizobia, also shown in Basic Fuchsin counterstained 10 µm sections. Data are from two independent experiments (A17, n = 41; nin, n = 31; nin + pEXPA:NIN, n = 37). e Transactivation assay of pMtAnn1:GUS with NIN or NINΔ (DNA-binding domain deletion version) in N. benthamiana leaves. Box plots show distribution of values (open circles) of individual plants (n = 25 per sample) from 4 independent experiments. First and third quartile (horizontal box edges), minimum and maximum (outer whiskers), median (centerline), mean (solid black circle) and outliers (crosses) are indicated. Different letters above boxes indicate statistically significant differences (p < 2e-16, by One-way ANOVA α = 5% followed by Tukey honest significant difference tests). Representative leaf disc images are shown. Expression of pENOD11:GFP-ER and pUBQ10:DsRed fusions in nin control (+pEXPA:GUS) (f, h) and complemented (+pEXPA:NIN) roots (g, i). Red DsRed fluorescence labels cytosol and nucleoplasm. Green GFP-ER fluorescence labels ER network and growing root hair tips (f, g), and rhizobia-induced cytoplasmic columns in complemented roots (arrowhead in g). Arrow indicates enclosed rhizobia (RHE). Cortical cells of control or epidermally-complemented nin do not show GFP-ER or DsRed-labelled cytoplasmic rearrangements (h, i), whereas these are clearly visible with DsRed in sunn (see Supplementary Fig. 11). Data are from 2 independent experiments (n = 7 for nin + pEXPA:GUS and n = 19 for nin + pEXPA:NIN). Asterisks mark xy positions of infected root hair base. Scale bars: a–d = 100 µm, f–i = 40 µm. See also Supplementary Figs. 10, 11. Source data, including split channels for merged fluorescence, are provided as a Source Data file.
