Figure 5

The B. diazoefficiens bclA, DD-CPase1, bclA/DD-CPase1 mutant phenotypes in symbiosis with A. afraspera. (a) Plant growth and nodule phenotype of A. afraspera plants inoculated with B. diazoefficiens USDA110 wild type and indicated mutants at 14 dpi. Scale bars are 2 cm (top panels) and 1 mm (bottom panels). (b) Confocal microscopy of bacteroid viability determination by live/dead and calcofluor white staining of A. afraspera nodules induced by USDA110 wild type and indicated mutants. Scale bars are 10 µm. (c) Flow cytometry analysis of DNA content by DAPI fluorescence and of cell size by forward scatter (FS) in free living B. diazoefficiens USDA110 (blue) or bacteroids isolated from A. afraspera nodules infected with USDA110 wild type and indicated mutants (pink). (d) Relative nitrogen fixation activity (% of maximum/h/plant) of A. afraspera plants inoculated with B. diazoefficiens USDA110 wild type and indicted mutants, measured by the acetylene reduction assay per plant. Box-plots represent in the rectangle the first quartile to the third quartile, divided by the median value, whiskers above and below the box show the minimum and maximum measured values. (e) Bleomycin sensitivity in B. diazoefficiens strain USDA110 and its bclA, DD-CPase1, bclA/DD-CPase1 mutant derivatives at 0,0375 µg bleomycin/ml. Growth was determined by optical density measurement at 600 nm with a plate reader after 72 h. Error bars in panels (d) and (e) are standard deviations.