meliloti hfq mutants. Sets of 24 alfalfa plants grown hydroponically in test tubes were independently inoculated with bacterial suspensions of the

wild-type strains (1021 and 2011) and the knock-out hfq Screening Library chemical structure mutants (1021Δhfq and 2011-3.4). The number of nodules per plant induced by each strain and the percentage of nodulated plants were recorded at daily intervals post-inoculation (dpi). No significant differences were observed in the onset of this website nodulation (i.e. time of appearance of the first nodule) or the average number of nodules per plant at the end of the experiment (30 dpi) when the wild-type S. meliloti 1021 strain and the mutant 1021Δhfq were compared (Fig. 4a, left plot). The hfq mutant was also able to nodulate 100% inoculated plants, further supporting similar nodulation efficiency of both strains (Fig. 4a, right plot). However, a discrete delay in nodulation of the mutant when compared to the wild-type nodulation kinetics was revealed by both assays. Comparison of the symbiotic behaviour of the 2011-3.4 mutant with that of its parent strain 2011 led to identical conclusions (data not shown). Together these results suggest that the loss of Hfq does not affect the ability of S. meliloti to elicit nodule organogenesis on alfalfa roots but it probably influences on bacterial adaptations to the plant rhizosphere. Figure 4 Symbiotic phenotype of the S. meliloti hfq knock-out mutants. (a) Nodule formation

kinetics of the S. meliloti learn more 1021 wild-type strain and its mutant derivative 1021Δhfq determined as the number of nodules per plant (left plot) and % nodulated plants (right plot). Each point represents the mean ± standard error of determinations in two independent sets of 24 plants grown hydroponically in test tubes. Dpi, Ribose-5-phosphate isomerase days post inoculation. (b) Competition assays between the S. meliloti wild-type strain 2011 and its hfq insertion mutant derivative 2011-3.4. Nodule occupancy (expressed as

% of invaded nodules by each strain) was determined in plants grown in either Leonard assemblies or agar plates and co-inoculated with both strains at 1:1 ratio. (c) Symbiotic efficiency of the 1021 and 1021Δhfq strains. Left histogram, % nitrogen fixing nodules induced by each strain in plants grown either in test tubes (two sets of 24 plants) or agar plates (5 plates of 10 plants) 30 dpi. Right panels: growth of 1021- and 1021Δhfq-inoculated plants 30 dpi in Leonard jars and dry-weigh of the same plants expressed as the mean ± standard error from measurements in 24 individual plants. Ni, not inoculated. Competition assays were then performed on alfalfa plants grown in two different solid media; Leonard assemblies and agar plates (Fig. 4b). Taking advantage of the tagging of the 2011-3.4 mutant with the Km resistance marker of pK18mobsacB co-inoculation suspensions were prepared in this case by mixing S. meliloti 2011 and 2011-3.