FIG 6 .

Phenotypes of PraR-bs-A and/or RebR-bs mutants. (A) Mutants with base substitutions in PraR-bs-A [P_reb(PraR-bs-A⁻) mutant], RebR-bs [P_reb(RebR-bs⁻) mutant], or both genes [P_reb(PraR-bs-A⁻ RebR-bs⁻) double mutant] on the reb promoter. The sequences of the PraR binding site A and RebR binding site regions are shown in the right-hand side. The sequences corresponding to consensus palindromes are indicated in capital letters. Restriction endonuclease recognition sites (XbaI and EcoRI) were generated using the base substitutions. Mutants were inoculated into stems of Sesbania plants grown at 30°C, and stem nodules were analyzed at 14 dpi. (B) Hand-cut images (upper) and ARAs (lower) of the stem nodules formed by the indicated strains. Data are presented as means ± standard deviations from five separate plants. Different letters indicate significant differences (P < 0.05, Tukey-Kramer). (C) OM observations of infected host cells in stem nodules and TEM observations of residing symbiotic bacterial cells. Arrowheads indicate R bodies. (D) Relative expression levels of the reb operon in bacteria from stem nodules and free-living cultures. Total bacterial RNA was isolated from bacteria that were grown in stem nodules and from free-living cultures grown in the defined medium at 38°C to an OD₆₀₀ of approximately 1.0. Expression levels of the reb operon were estimated using quantitative RT-PCR, normalized to 16S rRNA levels, and expressed relative to corresponding mRNA levels in free-living WT cultures and are presented as means ± standard deviations from three replicate cultures and plants. Statistical analyses were carried out for stem nodules and free-living cultures, respectively. Different letters indicate significant differences (P < 0.05, Tukey-Kramer).