Mapping and Subcloning of the Trifolitoxin Production and Resistance Genes from Rhizobium leguminosarum bv. trifolii T24. Eric W. Triplett. Department of Agronomy and the Center for the Study of Nitrogen Fixation, University of Wisconsin-Madison, 1575 Linden Drive, Madison 53706 U.S.A. Myra J. Schink, and Kerri L. Noeldner. Department of Agronomy and the Center for the Study of Nitrogen Fixation, University of Wisconsin-Madison, 1575 Linden Drive, Madison 53706 U.S.A. MPMI 2:202-208. Accepted 5 April 1989. Copyright 1989 The American Phytopathological Society.

In mixed inoculum, Rhizobium leguminosarum bv. trifolii T24 limits nodulation of clover roots by other strains of R. l. bv. trifolii. The nodulation competitiveness expressed by T24 is caused by that strain’s ability to produce the antirhizobial peptide trifolitoxin. A recombinant plasmid, pTFX1, that confers trifolitoxin production in trifolitoxin-sensitive strains of Rhizobium has been identified in a genomic library of T24. In this study, transposon mutagenesis and restriction enzyme analysis were used to map and subclone the trifolitoxin genes in pTFX1. A 4.4-kb region of DNA, referred to as tfx, was found to be necessary for the expression of trifolitoxin production and resistance in Rhizobium. This region was subcloned into pRK415. The resulting recombinant plasmid, pTFX2, conferred the ability to produce trifolitoxin when conjugated into trifolitoxin-sensitive strains of Rhizobium. This demonstrates that pTFX2 possesses all of the genetic determinants necessary for trifolitoxin production and resistance. Chromatographic and bioassay evidence was used to confirm trifolitoxin production in transconjugants containing pTFX2. The genes for trifolitoxin resistance and production were separated by cloning a 10-kb fragment of pTFX1, containing part of tfx, into pDSK519. The resulting plasmid, pTFX4, conferred trifolitoxin resistance, but not production, in a trifolitoxin-sensitive strain of Rhizobium following conjugation.