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

Shino Suzuki, Toshihiro Aono,^,* Kyung-Bum Lee, Tadahiro Suzuki, Chi-Te Liu, Hiroki Miwa, Seiji Wakao, Taichiro Iki, and Hiroshi Oyaizu

Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received 5 July 2007/ Accepted 17 August 2007

The molecular and physiological mechanisms behind the maturation and maintenance of N₂-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 N₂-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 N₂ 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, C₄ 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.

Published ahead of print on 24 August 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

These authors contributed equally to the work.

Present address: National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan.