Fig. 4: NIN binds promoters and transactivates the expression of CLE13 and CEP7.

a Alignment of NIN-binding site (NBS) motifs previously identified in LjNF-YA1, LjCLE-RS1, and LjCLE-RS2 promoters³² with homologous regions of MtNF-YA1, MtCLE13, and MtCEP7 promoters. Bold characters represent nucleotides conserved in more than four sequences out of the six identified. The sequence logo is derived from the six candidate NBSs, aligned using the MEME algorithm (http://meme-suite.org/index.html). b Schematic representation of MtCLE13 and MtCEP7 genes highlighting promoter regions used for ChIP-qPCR analyses. Red boxes represent the predicted NBSs identified on each promoter sequence. c NIN binds to the MtCLE13 and MtCEP7 promoters. Chromatin immunoprecipitation (ChIP)-qPCR analysis of NIN binding to MtCLE13 (left panel) and MtCEP7 (right panel) promoters, in wild-type (WT) or nin mutant roots 5 days post rhizobium inoculation (dpi) using either an anti-NIN antibody or IgG as a negative control. The fold enrichment of NIN binding was determined relative to IgG (control) IPs. One representative biological replicate out of two is shown. Data points from three technical replicates are plotted as open circles. d NIN transactivates MtCLE13 and MtCEP7 gene expression in M. truncatula mesophyll protoplasts. The promoter regions of MtCLE13 (left panel) and MtCEP7 (right panel) were fused to the luciferase (LUC) reporter gene and co-transformed in protoplasts with a p35S:NIN or an empty vector (ev). An AtUbi:GUS construct was used to measure the transformation efficiency. LUC/GUS ratios were normalized relative to values obtained in protoplasts transformed with an ev. To highlight fold changes, the dotted line corresponds to a ratio of 1. Data points from three biological replicates are plotted as open circles (n = 6). Mann–Whitney test was used for each promoter to assess significant differences between LUC/GUS ratios in the presence or absence of the NIN construct, as indicated by asterisks (***α < 0.001). e NIN activates MtCLE13 and MtCEP7 gene expression in M. truncatula roots. Expression analysis by qRT-PCR of MtNIN, MtCLE13, and MtCEP7 in wild-type (WT) uninoculated roots transformed with a pAtUbi:NIN construct or an ev. Plants were grown on a nitrogen-free Fåhraeus medium. To highlight fold changes, expression levels were normalized relative to roots transformed with the ev. Data points from six biological replicates are plotted as open circles (n ≥ 12). Mann–Whitney test was used for each gene to assess significant differences between pUbi:NIN-transformed roots and control roots, as indicated by asterisks (***α < 0.001). f NIN expression driven by the pNIN_5kb promoter is sufficient to restore the regulation of MtCLE13 and MtCEP7 gene expression in response to rhizobium. nin mutant roots were transformed with either an ev or the pNIN_5kb:NIN construct. MtCLE13 and MtCEP7 gene expression was analyzed by qRT-PCR in roots grown in vitro on a nitrogen-free Fåhraeus medium, 2 dpi with rhizobium. Expression levels were normalized relative to uninoculated roots for each genotype. To highlight fold changes, the dotted line corresponds to a ratio of 1. Data points from five biological replicates are plotted as open circles (n ≥ 9). Mann–Whitney test was used for each gene to assess significant differences between roots transformed with the pNIN_5kb:NIN construct and the control, as indicated by asterisks (***α < 0.001). In c–f, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; and crosses represent sample means.