Rhizobial Factors Required for Stem Nodule Maturation and Maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 Symbiosis

Abstract

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...

phrR -Like Gene praR of Azorhizobium caulinodans ORS571 Is Essential for Symbiosis with Sesbania rostrata and Is Involved in Expression of reb Genes

“…Furthermore, we constructed a whole-genome microarray of A. caulinodans and performed transcriptional profiling experiments with A. caulinodans in the free-living and symbiotic states (48). By the screening of Tn5 mutants, we identified 86 genes that were required for A. caulinodans to establish symbiosis (43). Among these genes, 17 were more highly expressed in bacteroids than in free-living bacteria (48).…”

“…Alongside the genome sequence analysis, we performed a concurrent large-scale screening of rhizobial genetic factors involved in nodule development using A. caulinodans mutants created by random Tn5 mutagenesis (43). Furthermore, we constructed a whole-genome microarray of A. caulinodans and performed transcriptional profiling experiments with A. caulinodans in the free-living and symbiotic states (48).…”

<i>Rhizobium</i> alters inducible defenses in broad bean, <i>Vicia faba</i>

“…Despite their ubiquity, nitrogen-fixing bacteria -host plant mutualisms are exceedingly specific interactions [26][27][28][29]. Vicia faba, for example, is one of the most discriminating of the legume hosts towards different strains of R. leguminosarum [5].…”

Effects of alteration in LPS structure in <i>Azorhizobium caulinodans</i> on nodule development

“…The trays were placed for three days at 37°C under dark conditions. After germination, S. rostrata were transferred into a commercial horticultural soil (Kureha Chemical, Japan) and grown at 35°C under a 24 h light regime at an intensity of 50,000 lux, as described previously (Suzuki et al, 2007). Bacterial cultures grown overnight were inoculated onto the stems 2 weeks after transplantation.…”

“…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.…”