Nucleic Acids Research, 1994, Vol. 22, No. 8 1335-1341
© 1994
MOLECULAR BIOLOGY |
Subtraction hybridisation and shot-gun sequencing: a new approach to identify symbiotic loci
1Laboratoire de Biologie Moléculaire des Plantes Supérieures, Université de Genéve 1 chemin de I'Impératrice, 1292 Chambésy, Switzerland 2Molecular Genetics, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital Hills Road, Cambridge CB2 2QQ 3Department of Mycology and Plant Pathology, The Queen's University of Belfast Belfast BT9 5PX, UK
*To whom correspondence should be addressed
Received March 14, 1994. Accepted March 22, 1994.
Traditionally, new loci involved in the Rhlzobium-legume symbiosis have been identified by transposon mutagenesls and/or complementation. Wide dispersal of the symbiotic loci in Rhlzoblum species NGR234, as well as the large number of potential host-plants to be screened, greatly reduces the efficiency of these techniques. As an alternate strategy designed to identify new NGR234 genes involved in the early stages of the symbiosis, we combined data from competitive RNA hybridisation, subtractive DNA hybridisation and shot-gun sequencing. On the assumption that the expression of most nodulatlon genes is triggered by compounds released by the host-plant, we identified, in the ordered cosmld library of the large symbiotic plasmid pNGR234a, restriction fragments that carry transcripts induced by flavonolds. To target genes not present in the closely related strain R.fredll USDA257, we selected fragments that also carried sequences purified by subtractive DNA hybridisation. Shot-gun sequencing of this subset of fragments lead to the identification of sequences with strong homology to diverse prokaryotic genes/proteins. Amongst these, a symblotically active ORF from pNGR234a, is highly homologous to the leucine responsive regulatory protein of Escherichia coll (Lrp), is induced by flavonolds, and is not present in USDA257
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