Fig. 6: CEP7 peptides regulate positively nodule number and rhizobial infections.

a Schematic representation of the predicted MtCEP7 prepropeptide. The blue rectangle represents the N-terminal signal peptide predicted using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP-3.0/). Red rectangles represent the predicted MtCEP7 Domain 1 (CEP7 D1) and Domain 2 (CEP7 D2) peptide sequences with hydroxyl modifications to proline as indicated in bold. b Representative images of the nodulation phenotype of wild-type (WT) and cra2 mutants treated with synthetic CEP7 D1 or CEP7 D2. Scale bars = 1 cm. c Quantification of the nodule number in WT and cra2 mutants, grown in vitro on a nitrogen-free Fåhraeus medium supplemented or not with 1 µM CEP7 D1 or CEP7 D2 peptides. Nodule number was scored at 14 days post rhizobium inoculation (dpi). Graphs are based on data points from three biological replicates (n ≥ 59). Mann–Whitney test was used for each genotype to assess significant differences between peptide-treated and control plants, as indicated by asterisks (***α < 0.001). d The nodulation phenotype of plants expressing an artificial microRNA targeting MtCEP7 transcripts (amiRCEP7). Representative images of nodulated roots transformed either with an empty vector (ev) or with the amiRCEP7 construct described in d. Scale bars = 1 cm. e Quantification of the nodule number of plants transformed either with an ev or with an amiRCEP7 construct. Plants were grown in vitro on a nitrogen-free Fåhraeus medium and nodules were scored at 14 dpi. Graphs are based on data points from 1 representative biological replicate out of 3 (n ≥ 14). Mann–Whitney test was used to assess significant differences, as indicated by asterisks (*α < 0.05). f Quantification of infection thread (IT) number in six dpi roots transformed either with an ev or with the amiRCEP7 construct and inoculated with a rhizobium strain constitutively expressing a LacZ reporter. ITs that reached the epidermis (epidermal ITs) were distinguished from ITs that reached the cortex (cortical ITs). Graphs are based on data points from two biological replicates (n ≥ 15). Mann–Whitney test was used to assess significant differences between amiRCEP7 roots and control roots, as indicated by asterisks (*α < 0.05; ***α < 0.001). g Representative images of the nodulation phenotype of plants transformed either with a pCLE13:GUS or a pCLE13:CEP7 construct. Scale bars = 1 cm. h Quantification of the nodule number in plants transformed either with the pCLE13:CEP7 construct or the pCLE13:GUS construct as a negative control. Plants were grown in pots containing a perlite/sand (3/1) mixture and watered with a “i” low nitrogen medium, and nodules were scored at 21 dpi. Graphs are based on data points from two biological replicates (n ≥ 28). Mann–Whitney test was used to assess significant differences, as indicated by asterisks (**α < 0.01). i Quantification of IT number in six dpi roots transformed either with the pCLE13:CEP7 construct or the pCLE13:GUS construct as a negative control and inoculated with a rhizobium strain constitutively expressing a LacZ reporter. ITs that reached the epidermis (epidermal ITs) were distinguished from ITs that reached the cortex (cortical ITs). Graphs are based on data points from two biological replicates (n ≥ 15). Mann–Whitney test was used to assess significant differences between the pCLE13:CEP7 roots and control roots, as indicated by asterisks (**α < 0.01; ***α < 0.001). In c, e, f, h, i, 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.