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 pr imitive soybean cultivars such as Peking but fails to nodulate agronom ically 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 subje ct to competitive nodulation blocking. We have isolated a cosmid conta ining DNA that corresponds to the site of transposon insertion in 257D H4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open readin g frames. Five of these, designated noIBTU, ORF4, and noIV, are closel y spaced and of the same polarity. noIW and noIX are of the opposite p olarity. 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 a nd NoIW proteins have putative membrane-spanning regions. The N-termin us of the hypothetical NoIW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has s ignificant homology to known nodulation gene products. Site-directed m utagenesis with mudII1734 confirms that inactivation of noIB, noIT, no IU, noIV, noIW, or noIX extends host range for nodulation to McCall so ybean. This phenotype could not be genetically dissected from sensitiv ity to competitive nodulation blocking. Expression of noIBTU and noIX is induced as much as 30-fold by flavonoid signal molecules, even thou gh these genes lack nod-box promoters. Histochemical staining of McCal l roots inoculated with noIB-, noIU-, or noIX-lacZ fusions verifies th at these genes are expressed continuously from preinfection to the sta ge of the functional nodule. Although a noIU-ORF4-noIV clone hybridize s to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and b road-host-range Rhizobium sp. NGR234, hybridizing sequences are not de tectable in other rhizobia.