Knockout Of An Azorhizobial dTdplrhamnose Synthase Affects Lipo And Exopolysaccharide Synthesis And Symbiosis With Sesbania Rostrata

W. D'Haeze, D. Vereecke, M. Gao, R. De Rycke, M. Holsters

Dept of Plant Genetics, V.I.B., K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

The micro-symbiont, Azorhizobium caulinodans ORS571, induces nitrogen-fixing nodules on both stem and roots of the tropical leguminous host plant, Sesbania rostrata. Besides azorhizobial lipochito-oligosaccharidic Nod factors (D'Haeze et al. 1998), surface polysaccharides (SPSs), comprising lipo- (LPSs), capsular (CPSs), and exopolysaccharides (EPSs), play a pivotal role during successful nodule invasion and development. To better understand the role of SPSs, a non-polar mutation was made in the oac2 gene (Gao et al. 2001). oac2 is part of a locus containing oacO, oacl, oac2, and oac3, supposedly involved in the synthesis of dTDP-L-rhamnose, and encoding a dTDP-L-rhamnose synthase, which catalyzes the last step of this pathway. Compared to wild-type LPSs, those synthesized by the oac2 mutant, ORS571-oac2, partitioned in the water phase upon hot phenol extraction, showed a lower degree of polymerization based on faster migration in detergent gel electrophoresis, and had a reduced rhamnose content. Using a hydrocarbon adherence method, ORS571-oac2 was shown to be slightly more hydrophobic than the parental strain.

Inoculation of S. rostrata stems or roots with ORS571-oac2 produced normal nodule initiation and early stages, but further nodule development was arrested, leading to defective organs with no nitrogen fixation. In situ localization of ORS571-oac2 indicated no bacterial internalization and bacteroid formation in the nodule-like structures. Sections showed abnormal tissue organization, without clear demarcation between central and peripheral tissues, or any sign of bacteria internalized into plant cells. Instead, many broad infection threads were present. In contrast to wild-type nodules, a severe blue auto-fluorescence was observed in a region resembling the infection center. Electron microscopy showed that intracellular infection threads, present in nodulelike structures, were 3 to 5 times wider than those in wild-type nodules. They were surrounded by a thick layer of cell wall material. Internalization events were not seen. Auto-fluorescence and the thick infection thread walls suggest the induction of a plant defense response, either to altered ORS571-oac2 SPSs, or because the oac2 mutant is unable to suppress a plant defense response induced by other unknown factors.

In some stem nodules, induced by co-inoculation in a 1:1 ratio with ORS571-oac2 and ORS571-V44 (an azorhizobial nodA mutant that is unable to produce Nod factors), ORS571-oac2 could enter plant cells. Normal central tissues were formed consisting of many plant cells occupied by the oac2 mutant. This observation suggests that normal SPSs, produced by ORS571-V44, are capable to complement in trans defects induced by ORS571-oac2.

To better understand the role of specific SPSs, electron microscopy showed wild-type bacteria surrounded by a thick layer of low electron-dense material, suggesting a massive amount of most likely EPSs. A similar, but thinner, layer also was present around ORS571-oac2.

Quantification of EPSs produced by ORS571 and ORS571-oac2 cultivated on nitrogen fixation medium together with the determination of global bacterial hydrophobicity suggest that the latter layer surrounding the oac2 mutant may resemble CPSs. Accordingly, CPSs may be involved in the transition from infection pockets to infection threads, whereas LPSs and/or EPSs may be required for the internalization into plant cells.

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