In contrast, the number of Rt2472 and Rt2441 cells attached to roots during 0.5 h was drastically lower (3.6% and 4.7% of the wild type, respectively). After 48 h, the rosR mutant cells were still considerably less numerous than Rt24.2 (14.6% for Rt2472 and 16.5% for Rt2441). These assays INCB024360 in vitro confirmed that rosR mutation affects the first step of the infection process, i.e., bacterial adhesion

to root hairs (Figure 10I). To study the further stages of clover infection, seedlings were inoculated with Rt24.2 and Rt2472 tagged with gfp and observed under a light microscope during a 10-day experiment. The following were quantified: (i) tightly curled root hairs containing trapped rhizobia, (ii) initiated (immature or aborted) infection threads, and (iii) infection threads which successfully entered the root cortex of clover. As was shown in Figure 10J, wild type bacteria effectively colonized curled root hairs, and the first initiated infection threads were selleck compound observed after 4 dpi. Extended infection threads were formed from almost all colonized root hairs, giving, on average, 5.6 successful

infections per plant after 10 days. The rosR mutant exhibited notable differences in infection thread formation. Rt2472 cells colonized root hairs very rarely and with a delay in comparison to the wild type. As a consequence, the initiation of infection threads was observed only occasionally and a great majority of the infection threads was not properly extended and did not reach root cortical cells (Figure 10J). Discussion In this paper, we present data showing that RosR of R. leguminosarum bv. trifolii 24.2, besides its role in transcriptional regulation of EPS synthesis, is required for successful interaction with clover plants, stress tolerance, motility, and biofilm formation. Both the rosR mutants (Rt2440 and Rt2472) described earlier [23, 30] and the newly Branched chain aminotransferase isolated Rt2441, bearing a genomic wild type rosR with the regulatory region in addition to the mutated rosR copy, displayed pleiotropic phenotypes. Pleiotropy of the rosR mutants was fully restored in complementation tests using a low-copy

plasmid carrying rosR. Interestingly, the Rt2441 mutant showed a negative dominant effect on EPS production, which confirmed the regulatory role of RosR in EPS synthesis. This phenomenon could be explained, to some extent, by negative autoregulation of rosR expression [23], which may be strengthened by the presence of more RosR-boxes binding RosR (Figure 2). As a result, the diminished amount of functional RosR might be insufficient for positive regulation of EPS production. The negative dominance could be overcome by introducing additional copies of rosR in the complementation experiments (Table 1, Figure 2). A similar dominant-negative effect of rosAR mutation in A. radiobacter had been described by Brightwell et al. [43].