A New Rhizobium meliloti Symbiotic Mutant Isolated After Introducing Frankia DNA Sequence into a nodA::Tn5 Strain. A. Reddy. Department of Biology, University of California, Los Angeles, 90024, U.S.A. B. Bochenek, and A. M. Hirsch. Department of Biology, University of California, Los Angeles, 90024, U.S.A. MPMI 5:62-71. Accepted 7 October 1991. Copyright 1992 The American Phytopathological Society.

Our goal was to isolate Frankia genes by complementation of Rhizobium meliloti mutants. By mating a library of Frankia genes in pLAFR3 with a R. meliloti nodA::Tn5 recipient, we isolated a clone that appeared to confer nodulation functions, although the apparently complemented transconjugant (Rm5610 NS6) remained Fix‾ on alfalfa. Further experiments have shown that the Nod⁺ Fix‾ phenotype was due to genomic mutations in R. meliloti and was independent of the Frankia DNA. Rm5610 NS6 had apparently lost Tn5 and simultaneously acquired neomycin resistance. We subsequently isolated a spontaneous neomycin-resistant derivative of R. meliloti 1021 that appeared to be identical to NS6. The nodules elicited by the neomycin-resistant mutants contained infection threads that penetrated the nodule; rhizobia were released from the threads, but they did not differentiate into elongate bacteroids. We examined nodulin gene expression in the ineffective mutant-induced nodules by northern blot and by in situ hybridization analyses and found that transcripts for the nodulins MsENOD2, MsENOD12-1, and leg-hemoglobin were detectable in the same cellular location as in wild type-induced nodules. These results confirm that the expression of these nodulin genes is regulated by signals exclusive of bacteroid differentiation and nitrogen fixation.