The behavior of Ralstonia pseudosolanacearum strain OE1-1 and morphological changes of cells in tomato roots

The soil-borne Gram-negative β-proteobacterium Ralstonia solanacearum species complex (RSSC) infects tomato roots through the wounds where secondary roots emerge, infecting xylem vessels. Because it is difficult to observe the behavior of RSSC by a fluorescence-based microscopic approach at high magnification, we have little information on its behavior at the root apexes in tomato roots. To analyze the infection route of a strain of phylotype I of RSSC, R. pseudosolanacearum strain OE1-1, which invades tomato roots through the root apexes, we first developed an in vitro pathosystem using 4 day-old-tomato seedlings without secondary roots co-incubated with the strain OE1-1. The microscopic observation of toluidine blue-stained longitudinal semi-thin resin sections of tomato roots allowed to detect attachment of the strain OE1-1 to surfaces of the meristematic and elongation zones in tomato roots. We then observed colonization of OE1-1 in intercellular spaces between epidermis and cortex in the elongation zone, and a detached epidermis in the elongation zone. Furthermore, we observed cortical and endodermal cells without a nucleus and with the cell membrane pulling away from the cell wall. The strain OE1-1 next invaded cell wall-degenerated cortical cells and formed mushroom-shaped biofilms to progress through intercellular spaces of the cortex and endodermis, infecting pericycle cells and xylem vessels. The deletion of egl encoding β-1,4-endoglucanase, which is one of quorum sensing (QS)-inducible plant cell wall-degrading enzymes (PCDWEs) secreted via the type II secretion system (T2SS) led to a reduced infectivity in cortical cells. Furthermore, the QS-deficient and T2SS-deficient mutants lost their infectivity in cortical cells and the following infection in xylem vessels. Taking together, infection of OE1-1, which attaches to surfaces of the meristematic and elongation zones, in cortical cells of the elongation zone in tomato roots, dependently on QS-inducible PCDWEs secreted via the T2SS, leads to its subsequent infection in xylem vessels.