Figure 4

The B. diazoefficiens bclA mutant is not affected in symbiosis with soybean. (a) Plant growth and nodule phenotype of G. max plants inoculated with B. diazoefficiens USDA110 wild type and the bclA mutant at 14 dpi. Scale bars are 3.5 cm (top panels) and 1 mm (bottom panels). (b) The nitrogen fixation activity of G. max plants inoculated with B. diazoefficiens USDA110 wild type and the bclA mutant, measured by the acetylene reduction assay per mg of fresh nodule weight (nmol ethylene produced per hour incubation and per mg nodule weight). 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. (c,d) Toluidine blue stained thin sections (two top rows) and confocal microscopy of bacteroid viability determination by live/dead staining (bottom row) of G. max nodules induced by USDA110 or USDA110ΔbclA. Scale bars are 100 µm for the top panels and 10 µm for the other panels. (e) Flow cytometry analysis of DNA content by DAPI fluorescence (top) and of cell size by forward scatter (bottom) in free-living B. diazoefficiens USDA110 (pink) or bacteroids isolated from G. max nodules infected with USDA110 wild type (blue) or USDA110ΔbclA (orange). The forward scatter and DAPI fluorescence profiles are completely overlapping for the 3 samples.