Fig 1.
Endophytic colonisation of Lotus japonicus roots and nodules by R. mesosinicum KAW12.
A) Nodule section displaying a cortical infection thread (arrow) that contains both the M. loti wild-type and KAW12 bacteria, among fully infected nodule cells (arrow head) containing the M.loti symbiont. B) Nodule section showing that KAW12 (*) multiplication inside nodules is limited to small sectors compared to M. loti wild-type (arrow), which is the predominant coloniser. C) Root hair infection thread (arrow) containing both the M. loti wild-type symbiont and the KAW12 endophytic bacteria. D) Lotus plants inoculated with KAW12 endophyte display a nod-minus and nitrogen-starved phenotype (arrow) in comparison to E) Lotus plants that form nodules (arrow) and establish a nitrogen-fixing symbiosis after inoculation with M. loti wild-type. F) Root section illustrating the capacity of KAW12 (arrow) to colonise the intercellular space of Lotus roots. G) Section of an M. loti nodZ-induced nodule presenting KAW12 (*) and M. loti nodZ (arrow) infection. H) The infection and accommodation of compatible endophytes within Lotus nodules is regulated in at least three steps. Scale bars: A) and C) 20 μm, B) F) and G) 50 μm, D) and E) 1 cm. Mesorhizobium loti bacteria are visualized in green, and KAW12 in red. Mixed inoculum has been used in A) to C) and G), and single inocula with the aforementioned bacteria have been used for E) to F).
Table 1.
Colonisation of Lotus japonicus nodules by endophytic bacteria.
Fig 2.
R. mesosinicum KAW12 colonisation pattern in Lotus japonicus nodules induced by M. loti exoU.
A) to C) Nodules colonised by M. loti exoU-GFP (arrow) or KAW12-DsRed (arrowhead). The two nodules were visualized in bright field (A) with the GFP filter (B) or with the DsRed filter (C). D) Root hair infection threads (arrows) colonised by M. loti exoU (green) and KAW12 (red). E) Confocal laser scanning microscopy (CLSM) image of a nodule section illustrating internal nodule infection (dashed line) by KAW12 (red) and M. loti exoU (green). F) Thin section of a nodule primordium showing KAW12 infection (dashed line) in the inner zone. G) Detailed view of the same nodule as in (F) illustrating the inter- (*) and intra-cellular (arrow) KAW12-containing lagoons. H) Section of a mature nodule presenting multiple and enlarged lagoons colonised by KAW12 (*). (I) Transmission electron micrograph of an intercellular lagoon (arrow) containing bacteria surrounded by a white, undefined matrix (*). Scale bars = 500 μm (A to C), 20 μm (D, G), 50 μm (E), 100 μm (F, H), and 2 μm (I). The M. loti exoU is visualized in green and KAW12 in red (A to E).
Fig 3.
Transmission electron micrographs of Lotus japonicus nodules colonised by M. loti exoU or by R. mesosinicum KAW12.
The nodule sections were immunogold labelled (arrows) with an antibody against the GFP protein (A, B), or against the DsRED protein (C, D). GFP was detected (arrows) in the M. loti exoU-selected nodule (A), and DsRED (arrows) in the KAW12-selected nodule (D). There is some minor nonspecific labelling by the GFP antibody (arrows) in the nodule colonised by the DsRed-tagged KAW12 (B) and by the DsRED antibody (arrows) in the nodule colonised by the GFP-tagged M. loti exoU (C). Immunogold labelling of homogalacturonan by the JIM5 monoclonal antibody shows the presence of cell wall material (arrow) in the infection thread that contains M. loti exoU (E), and in the lagoons containing KAW12 (F, G). KAW12 is released inside the plant cell (*) (F). Immunogold labelling of glycoproteins (arrows) by the MAC236 monoclonal antibody reveals their location within the plant cells containing the KAW12-containing lagoons (H, I). Detailed images of the regions marked by rectangles in F) and H) are shown in G) and I), respectively. Scale bars = 1 μm (A to D, and F), 0.5 μm (E, G, I). b = bacteria.
Table 2.
Colonisation of M. loti exoU induced nodules by R. mesosinicum KAW12 on Lotus japonicus wild type and symbiotic mutants.