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Plant and Cell Physiology Advance Access originally published online on January 9, 2008
Plant and Cell Physiology 2008 49(3):301-313; doi:10.1093/pcp/pcn004
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Requirement for Mesorhizobium loti Ornithine Transcarbamoylase for Successful Symbiosis with Lotus japonicus as Revealed by an Unexpected Long-Range Genome Deletion

Elina Mishima1,2, Atsuko Hosokawa1, Haruko Imaizumi-Anraku3, Katsuharu Saito4,5, Masayoshi Kawaguchi4 and Kazuhiko Saeki1,2,*

1Department of Biology, Graduate School of Science, Osaka University, Toyonaka, 560-0043 Japan
2Department of Biological Sciences, Faculty of Science, Nara Women's University, Nara, 850-6503 Japan
3National Institute of Agrobiological Sciences, Tsukuba, 305-8602 Japan
4Department of Biological Sciences, Graduate School of Sciences, University of Tokyo, Tokyo, 113-0033 Japan

*Corresponding author: E-mail, ksaeki{at}cc.nara-wu.ac.jp; Fax, +81-742-20-3028.


  Abstract

With the original aim of surveying the role of exopolysaccharide (EPS) in Lotus–Mesorhizobium symbiosis, we carried out Tn5 mutagenesis of Mesorhizobium loti and obtained 32 mutants with defects in EPS biosynthesis. One of the mutants, HIA22, formed pseudonodules and failed to fix nitrogen with Lotus japonicus. However, complementation analysis unexpectedly revealed that the potential gene with the locus tag, mll2073, interrupted by Tn5 was responsible for neither normal EPS synthesis nor symbiosis. Further analysis uncovered that HIA22 had a genome deletion of approximately 20 kbp, resulting in the loss of two separate genes responsible for EPS biosynthesis and symbiosis. One gene with the locus tag, mll5669, was needed to synthesize normal EPS that fluoresced on medium containing Calcofluor and encoded a homolog of O-antigen acetyl transferase in Salmonella typhimurium. A specific mutant of mll5669, EMB-B58, successfully fixed nitrogen when infected onto L. japonicus. Another gene, mlr5647, was needed to establish fully functional nodules and encoded ornithine carbamoyl transferase [ArgF (EC 2.1.3.3 [EC] )], which participates in arginine biosynthesis. A specific mutant of mlr5647, EMB-Y2, showed arginine auxotrophy and formed infection threads, but the nodules formed by this strain had few infected cells filled with bacteroids. These mutant phenotypes were complemented by supplementation of arginine or citrulline to bacterial or plant medium. EMB-Y2 represented a novel class of rhizobial arginine auxotrophs with symbiotic deficiency, and its phenotypes indicated that sufficient supply of citrulline or its derivative is essential for successful infection or for a stage in the infection process in Lotus–Mesorhizobium symbiosis.

Keywords: Arginine auxotroph - Exopolysaccharide - Lotus japonicus - Mesorhizobium loti - Nitrogen fixation - Symbiosis

Abbreviations: EPS, exopolysaccharide; GFP, green fluorescent protein; LPS, lipopolysaccharide; ORF, open reading frame; OTCase, ornithine transcarbamoylase; RDM, rhizobium defined medium; TY, tryptone–yeast extract–CaCl2; YEM, yeast extract–mannitol medium.

5Present address: Faculty of Agriculture, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano, 399-4598 Japan.

(Received October 13, 2007; Accepted January 6, 2008)
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