After the first 90 min, single Rt24.2 cells were visible on the surface of root hairs (Figure 10A). After 24 h, attachment of numerous PI3K inhibitor Rt24.2 cells to root hairs was seen. Bacteria were located mainly on root hair tops, forming caps and rhizobial clouds (Figure 10B). In the zone of growing root hairs, the majority of the root hairs were coated with Rt24.2 cells (Figure 10C). After 6 days post infection (dpi), infection

threads inside some of the root hairs were initiated or already extended and reached root epidermal cells (Figure 10D). In contrast, Rt2472 cells were seen on the root surface but were attached to the root hairs only sporadically demonstrating a much weaker attachment ability (Figure 10E). The caps formed LOXO-101 by rosR cells on the top of root hairs were detected very Combretastatin A4 mouse rarely (Figure 10F). In addition, several root hairs had an atypical, expanded shape resembling ginger roots (Figure 10G) in contrast to the typical curled root hairs in clover inoculated with the wild type. In the case of rosR mutant-inoculated plants, infection threads inside root hairs were observed sporadically, and their elongation was frequently interrupted (Figure 10H). Figure 10 Root attachment and infection of clover roots by the rosR mutant and the wild type. Fluorescence microscopy analyses of clover root colonization and invasion by GFP-expressing cells of R. leguminosarum bv. trifolii wild type (A-D) and the rosR mutant (Rt2472)

(E-H). The Rt24.2 cells attached very fast and effectively to root hairs (A-B), and formed caps on the top of root hairs (C). (D) Curled root hairs with an extended infection thread filled with the wild type cells. The infection thread started from the Shepherd’s Methisazone crook of the curled root hair and reached the base of root hair. The ability of root attachment and root cap formation of the rosR mutant was substantially decreased (E-F). Only individual cells of the Rt2472 rosR mutant attached to root hairs (E) and root caps were formed sporadically (F). Several root hairs showed abnormal deformation (G). The root hair colonized by the rosR mutant, which had developed an aborted

infection thread (H). (I) Attachment to clover roots 0.5 h and 48 h post inoculation with the wild type, and the Rt2472 and Rt2441 rosR mutants, and their derivatives complemented with pRC24. For each strain, ten roots were examined. Data shown are the means of two replicates ± SD. (J) Kinetics of curled root hair (CRH) formation, infection thread (IT) initiation and extension on clover plants inoculated with the wild type and the rosR mutant (Rt2472). For each strain, 25 plants were used. Data shown are the means of two experiments. To quantitatively determine the attachment ability to the surface of clover roots, Rt24.2 wild type, Rt2472 and Rt2441 rosR mutants, and their derivatives bearing plasmid pRC24 were incubated with clover roots for 0.5 h and 48 h.