A CENTRAL DOMAIN OF RHIZOBIUM NODE PROTEIN MEDIATES HOST-SPECIFICITY BY DETERMINING THE HYDROPHOBICITY OF FATTY ACYL MOIETIES OF NODULATIONFACTORS

Previously, we have shown that the nodE gene is a major determinant of the difference in host range between Rhizobium leguminosarum biovars viciae and trifolii. A new genetic test system for stringent functiona l analysis of nodE genes was constructed. By testing chimeric nodE gen es constructed by the exchange of polymerase chain reaction (PCR)-gene rated restriction cassettes, we show that a central domain, containing only 44 non-conserved amino acid residues, determines the host specif icity of the NodE protein (401 amino acid residues). Mass spectrometri c analysis of the lipo-chitin oligosaccharides (LCOs) produced by the new test strain containing the biovar viciae nodE gene shows that mole cules containing a polyunsaturated C18:4 (trans-2 trans-4 trans-6, cis -11-octadecatetraenoic) fatty acyl moiety are produced, as is the case for wild-type R. legumino-sarum bv. viciae. The LCOs determined by th e biovar trifolii nodE gene, which was overproduced in our test strain , carry C18:2 and C18:3 fatty acyl chains containing two or three conj ugated trans double bonds, respectively. Therefore, the main differenc e between the nodE-determined LCOs of biovar viciae and trifolii in th is system is the presence or absence of one cis double bond, resulting in the very different hydrophobicity of the LCOs. Using a newly devel oped spot application assay, we show that the C18:2- and C18:3-contain ing LCOs are able to induce the formation of nodule primordia on roots of Trifolium pratense. On the basis of these and other recent results , we propose that the host range of nodulation of the R. leguminosarum biovars viciae and trifolii is determined by the degree of hydrophobi city of the polyunsaturated fatty acyl moieties of their LCOs, which i s mediated by the host-specific central domain of the NodE protein.