Fig. 5: The NIN-binding site cis-element predicted in the proximal region of the CEP7 promoter is required for its induction by rhizobia.

a–c NIN expression driven by the pUbi promoter is sufficient to induce the expression of a MtCEP7:GUS fusion in the absence of rhizobium (b), compared to an empty vector (ev) control (a). Representative images of the GUS expression pattern associated only with the root stele (“stele”) or with both the root epidermis and stele (“epidermis and stele”). Wild-type plants were grown in vitro on a nitrogen-free Fåhraeus medium. In c, quantification of the proportion of roots (n ≥ 25) showing a GUS expression pattern associated only with the root stele or with both the root epidermis and stele, as described in a, b. d–h The NIN-binding site (NBS) in the proximal region of the MtCEP7 promoter is required for the induction of a MtCEP7:GUS fusion in response to rhizobium. In d, schematic representation of the MtCEP7 promoter, highlighting the predicted NBS cis-element identified as a red box and the corresponding deletion construct (pCEP7ΔNBS:GUS). In e, quantification of the proportion of roots (n ≥ 34) showing a GUS expression pattern associated only with the root stele (“stele”) or with both the root epidermis and stele (“epidermis and stele”), as described in f–i. In f, g, representative images of the GUS expression pattern associated only with the root stele or with both the root epidermis and stele of pCEP7ΔNBS:GUS 1 day post rhizobium inoculation (dpi) in longitudinal (f) or transversal (g) sections. In h, i, representative images for the same GUS expression patterns for MtCEP7:GUS roots 1 dpi in longitudinal (h) or transversal (i) sections. In a, b, f–i, a minimum of eight independent roots were analyzed for each time point from three independent experiments. Images show the GUS staining as a blue signal in bright field microscopy. Scale bars = 100 µm. In c, e, Fisher exact test was performed to assess significant differences in the proportion of roots (n ≥ 25) showing the different GUS expression patterns between the NIN overexpressing (c), or NBS-deleted MtCEP7:GUS (e) roots, and the control, as indicated by asterisks (***α < 0.001).