Figure 1.

Nodulation phenotypes of MtRRB3 RNAi roots and of the rrb3 mutant. A, Schematic representation of the MtRRB3 coding sequence. The mutation site leading to a stop codon in the rrb3 mutant is indicated by a black arrow, and sequences targeted by the two RNAi constructs (RNAi-1 and RNAi-2) are marked by horizontal gray lines. REC, Phosphate receiver domain; MYB, MYB-like DNA binding domain. B, RT-qPCR analysis of MtRRB1, MtRRB2, and MtRRB3 expression in roots expressing MtRBB3 RNAi constructs 1 and 2 or GUS (as control). The GUS RNAi control was set to 1 (dashed line) for each gene to highlight fold changes. Values are the mean of two independent experiments, and error bars represent standard deviations. Student’s t test was performed to assess significant differences (*P < 0.05) relative to the corresponding nontreated control, indicated by asterisks . C, Quantification of nodules formed in vitro on roots expressing MtRRB3 RNAi constructs and GUS (as control) 14 d postinoculation (dpi). Error bars represent the confidence interval (α = 0.05; n > 30 independent transgenic roots per construct). A Kruskal-Wallis test was performed to assess significant differences (α < 0.05), indicated by lowercase letters. D, Quantification of nodules formed 6 and 14 dpi on wild-type (WT) and rrb3 mutant roots. Values are the mean of two independent experiments, and error bars represent the confidence interval (α = 0.05; n > 30 independent plants/genotype for each experiment). A Mann-Whitney test was performed to assess significant differences (α < 0.05), indicated by asterisks. E, Quantification of infection events in wild-type and rrb3 mutant roots 6 dpi using a LacZ rhizobium strain. Values are the mean of two independent experiments, and error bars represent the confidence interval (α = 0.05; n > 5 independent roots/genotype for each experiment). A Mann-Whitney test was performed to assess significant differences (α < 0.05), indicated by asterisks. F, RT-qPCR analysis of MtENOD11 expression in roots treated with NFs (10⁻⁸ m NF for 3 h) in wild-type and rrb3 mutant roots. The MtENOD11 expression values in respective untreated roots were set to 1 to highlight fold changes, as indicated by the dashed line. Values are the mean of two independent experiments, and error bars represent standard deviations. Student’s t test was performed to assess significant differences (*P < 0.05) relative to the corresponding nontreated control, indicated by asterisks.