MOLECULAR-CLONING AND CHARACTERIZATION OF A SYM PLASMID LOCUS THAT REGULATES CULTIVAR-SPECIFIC NODULATION OF SOYBEAN BY RHIZOBIUM-FREDII USDA257

Rhizobium fredii strain USDA257 produces nitrogen-fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposon-mutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain USDA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated noIBTU, ORF4, and noIV, are closely spaced and of the same polarity. noIW and noIX are of the opposite polarity. The initiation codon for noIW lies 155 bp upstream from that of noIB, and it is separated from noIX by 281 bp. The predicted NoIT and NoIW proteins have putative membrane-spanning regions. The N-terminus of the hypothetical NoIW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site-directed mutagenesis with mudII1734 confirms that inactivation of noIB, noIT, noIU, noIV, noIW, or noIX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of noIBTU and noIX is induced as much as 30-fold by flavonoid signal molecules, even though these genes lack nod-box promoters. Histochemical staining of McCall roots inoculated with noIB-, noIU-, or noIX-lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a noIU-ORF4-noIV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad-host-range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.