The molecular and physiological mechanisms behind the maturation and maintenance of N 2 -fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N 2 -fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N 2 fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C 4 metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.Symbiosis between rhizobia and legumes results in the formation of nitrogen-fixing nodules. The symbiotic interaction begins with the induction of bacterial nod genes by flavonoids secreted from the plant roots (11). The nod genes encode proteins that synthesize nodulation factor (Nod factor), which initiates many of the developmental changes seen in the host plant early in the nodulation process (11,26,55). After the initial exchange and bacterial attachment at the surface, cortical cells begin dividing to form the nodule primordia. Bacteria penetrate the developing nodule primordia via host-derived infection threads (11,26,55). Upon release from the infection threads, bacteria invade the plant cell cytoplasm, where they differentiate into bacteroids and provide ammonium to the host plant by reducing atmospheric dinitrogen in exchange for carbon and amino acid compounds (18,54,58).It is deduced that multiple stages exist in the establishment of complete nitrogen-fixing symbiosis and that signal exchange between rhizobia and legumes might occur at each stage. The finding that the bacterial Nod factor switches on the nodulation program in the plant and the characterization of the plant genes of the Nod factor receptor or perception complexes have revealed a remarkable early event in the process of nodule development (23,46,47,60,75). However, the molecular and physiological mechanisms behind the maturation and maintenance of nodules still remain unclear. The transcriptional analysis of this process has been well-described in several legumerhizobium symbiosis systems, and the results have revealed that drastic transcriptional changes occur during nodule development (2,5,7,76). However, to our knowledge, large...
2005). LPS is composed of three parts: lipid A, containing sugar and fatty acid, which forms the outer leaflet of the outer membrane and anchors LPS to the cell envelope; a core oligosaccharide region, a non-repeating oligosaccharide, which links lipid A and O-antigen; and the Oantigen, consisting of repeating oligosaccharide units. The LPS containing or lacking O-antigen is usually called smooth LPS and rough LPS, respectively. In the majority of gram-negative bacteria, the core oligosaccharide can be subdivided into an outer core and an inner core. The outer core region provides an attachment site for O-antigen. The inner core typically contains residues of 3-deoxy-D -manno-2-octulosonic acid (Kdo) and L-glycero-Dmanno-heptose. Kdo connects the inner core to the lipid A (Heinrichs et al., 1998;Raetz and Whitfield, 2002).Azorhizobium caulinodans ORS571 is a microsymbiont of a tropical legume, Sesbania rostrata (Dreyfus and Dommergues, 1981;Dreyfus et al., 1983Dreyfus et al., , 1988. N 2 -fixing nodules are formed by A. caulinodans on the stems as well as on the roots of S. rostrata. Previously, the whole genome sequence of A. caulinodans was determined (Lee et al., 2008), and we performed a concurrent large-scale screening of rhizobial genetic factors involved in nodule development using A. caulinodans mutants created by random Tn5 mutagenesis (Suzuki et al., 2007). As a result of this screening, we isolated three mutants having a Tn5-insertion in the putative LPS biosynthesis genes (Suzuki et al., 2007). These three mutants, Ao13-C11, Ao77-C09 and Ao80-F04, were disrupted in the rfaF, rfaD, and rfaE genes, respectively. These genes are involved in the synthesis of the LPS inner core region. The rfaD and rfaE genes encode ADP-L-glycero-D-manno-heptose-6-epimerase and ADP-L-glycero-D-manno-heptose synthase, respectively. These enzymes are involved in the synthesis of ADP-L-glycero-D-manno-heptose, which is a component of the LPS inner core (Kneidinger et al., 2002; The lipopolysaccharide (LPS) of Azorhizobium caulinodans ORS571, which forms N 2 -fixing nodules on the stems and roots of Sesbania rostrata, is known to be a positive signal required for the progression of nodule formation. In this study, four A. caulinodans mutants producing a variety of defective LPSs were compared. The LPSs of the mutants having Tn5 insertion in the rfaF, rfaD, and rfaE genes were more truncated than the modified LPSs of the oac2 mutants. However, the nodule formation by the rfaF, rfaD, and rfaE mutants was more advanced than that of the oac2 mutant, suggesting that invasion ability depends on the LPS structure. Our hypothesis is that not only the wild-type LPSs but also the altered LPSs of the oac2 mutant may be recognized as signal molecules by plants. The altered LPSs may act as negative signals that halt the symbiotic process, whereas the wild-type LPSs may prevent the halt of the symbiotic process. The more truncated LPSs of the rfaF, rfaD, and rfaE mutants perhaps no longer function as negative signals inducing discontinu...
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