© CSIRO 2003 10.1071/FP03093 1445-4408/03/121219 Functional Plant Biology , 2003, 30, 1219–1232 www.publish.csiro.au/journals/fpb CSIRO PUBLISHING Alfalfa nodulation by Sinorhizobium fredii does not require sulfated Nod-factors Sadaf Noreen A , Helmi R. M. Schlaman B , Ramón A. Bellogín C , Ana M. Buendía-Clavería C , María Rosario Espuny C , Marga Harteveld B , Carlos Medina C , F. Javier Ollero C , Maurien M. A. Olsthoorn D,F , M. Eugenia Soria-Diaz C , Herman P. Spaink B , Francisco Temprano C , Jane Thomas-Oates A,D,G , José M. Vinardell C , Su Sheng Yang E , Haiyu Zhang E and José E. Ruiz-Sainz C,H A Michael Barber Centre for Mass Spectrometry, Department of Chemistry, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester, M60 1QD, UK. B Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. C Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Apdo. 1095, 41080 Sevilla, Spain. D Department of Mass Spectrometry, Bijvoet Centre for Biomolecular Research, Faculty of Chemistry, Utrecht University, Utrecht, The Netherlands. E Department of Microbiology, China Agricultural University, 2 Yuan Ming Yuan West Road, 100094 Beijing, People's Republic of China. F Current address: DSM Food Specialties, Research and Development, Department of Analysis, Delft, The Netherlands. G Current address: Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK. H Corresponding author; email: rsainz@us.es Abstract. Rhizobium strain 042B(s) is able to nodulate both soybean and alfalfa cultivars. We have demonstrated, by mass spectrometry, that the nodulation (Nod) factors produced by this strain are characteristic of those produced by Sinorhizobium fredii, which typically nodulates soybean; they have 3–5 N-acetylglucosamine (GlcNAc) residues, a mono-unsaturated or saturated C16, C18 or C20 fatty-acyl chain, and a (methyl)fucosyl residue on C 6 of the reducing-terminal GlcNAc. In order to study Rhizobium strain 042B(s) and its nodulation behaviour further, we introduced an insertion mutation in the noeL gene, which is responsible for the presence of the (methyl)fucose residue on the reducing terminal GlcNAc of the Nod-factors, yielding mutant strain SVQ523. A plasmid (pHM500) carrying nodH, nodP and nodQ, the genes involved in sulfation of Nod-factors on C 6 of the reducing-terminal GlcNAc, was introduced into SVQ523, generating SVQ523.pHM500. As expected, strain SVQ523 produces unfucosylated Nod-factors, while SVQ523.pHM500 produces both unfucosylated and unfucosylated sulfated Nod- factors. Plant tests showed that soybean nodulation was reduced if the inoculant (SVQ523.pHM500) produced sulfated Nod-factors. In the Asiatic alfalfa cultivar Baoding, SVQ523 (absence of a substitution at C 6 ) failed to nodulate, but both 042B(s) (fucosyl at C 6 ) and SVQ523.pHM500 (sulfate at C 6 ) formed nodules. In contrast, SVQ523 showed enhanced nodulation capacity with the western alfalfa cultivars ORCA and ARC. These results indicate that Nod-factor sulfation is not a requisite for S. fredii to nodulate alfalfa. Keywords: alfalfa, LCO, Nod-factor, nodulation, Sinorhizobium, soybean. Introduction Gram-negative bacteria of the genera Rhizobium, Sino- rhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium and Allorhizobium, collectively called rhizobia, are able to enter into a species-specific relationship with leguminous plants, resulting in the formation of nitrogen fixing nodules on the host plant root. Within these root nodules, a differentiated form of bacterium, termed a bacteroid, is able Abbreviations used: APS, adenosine 5′ phosphosulfate kinase; AVG, aminoethoxyvinylglycine; ES-CID, electrospray collision-induced dissociation; ES-Q-ToF, electrospray quadrupole time-of-flight; FAB, fast atom bombardment; GlcNAc, N-acetylglucosamine; LCO, lipochitin oligosaccharide; LPS, lipopolysaccharide; MALDI-ToF, matrix-assisted laser desorption/ionisation time-of-flight; MS, mass spectrometry; TLC, thin layer chromatography.