Plant factors induce expression of nodulation and host-range genes in Rhizobium trifolii

In Rhizobium trifolii ANU843, host specific nodulation capability is encoded within a 14kb HindIII fragment of the symbiosis plasmid. To gain a better understanding of the regulation of the nodulation (Nod) genes, we have isolated lac operon transcriptional fusions to several genes within this fragment, using the mini-Mu-lac bacteriophage transposon Mu dI1734. Using a broad-host-range vector, fragments containing Mu dI1734 insertions were introduced into R. trifolii ANU845, a derivative of ANU843 which lacks the symbiosis plasmid. Four distinct regions were identified within the Nod fragment, insertions in which resulted in nodulation phenotypes similar to those found previously for Tn 5. Region I mutants were Nod^- and defective in root hair curling (Hac^-) and corresponded to the nodABC and D genes identified by sequence analysis. Region II mutants showed an exaggerated root hair curling (Hac⁺⁺) response on clover plants and a greatly reduced nodulation ability. Region III mutants were affected in host-range properties, as they gained the ability to nodulate Pisum sativum (peas), but showed only poor nodulation ability on the normal host plant, Trifolium repens (white clover). Region IV mutants showed a delay in the nodulation of Trifolium repens, but only when plants were grown under high light-regimes. When ANU845 strains carrying the above Mu dI1734 insertions were grown in standard laboratory media, only insertions in nodD expressed β-galactosidase at high levels. However, when cells were placed in medium in which Trifolium repens was growing, insertions in nodA, nodB, region II, region III, and region IV were all induced from 5-10 times above basal levels. This allowed us to determine the directions of transcription in these regions.