No abstract

The collection of symbiotic (sym) mutants of white sweetclover (Melilotus alba Desr.) provides a developmental sequence of mutants blocked early in infection or nodule organogenesis. Mutant phenotypes include non-nodulating mutants that exhibit root-hair deformations in response to Rhizobium meliloti, mutants that form ineffective nodules lacking infection threads, and mutants that form infection threads and ineffective nodules. Mutant alleles from both the sym-1 and the sym-3 loci exhibited a non-nodulating phenotype in response to R. meliloti, although one allele in the sym-1 locus formed ineffective nodules at a low frequency. Spot-inoculation experiments on a non-nodulating allele in the sym-3 locus indicated that this mutant lacked cortical cell divisions following inoculation with R. meliloti. The auxin transport inhibitor K ( 1 -naphthyl)phthalamic acid elicited development of pseudonodules at a high frequency on all of the sweetclover sym mutants, including the nonnodulating mutants, in which the early nodulin ENODZ was expressed. This suggests that K ( 1 -naphthyl)phthalamic acid activates cortical cell divisions by circumventing a secondary signal transduction event that is lacking in the non-nodulating sweetclover mutants. The sym-3 locus and possibly the sym-1 locus appear to be essential to early host plant responses essential to nodule organogenesis.

In an effort to obtain a developmental sequence of mutations in the Rhizobium-legume interaction within a single legume species, we have characterized the early events of nodule development in

The expression of genes for the nodulins, including leghemoglobin, is independent of the nitrogen-fixing ability of the nodule and appears to correlate with the differentiation of densely cytoplasmic host cells in the nodule and, to some extent, with bacterial release from infection threads.The development of symbiotic nitrogen-fixing root nodules resulting from the infection of leguminous plants by host-specific species of Rhizobium and Bradyrhizobium is dependent on the concerted expression of bacterial and host plant genes. The role of rhizobial genes in host specificity, induction of nodule development, and nitrogen fixation has been the subject of intensive study (21). However, the role of the eukaryotic host genes involved in symbiosis has only recently come under scrutiny. Classical genetic analysis of the host legume has demonstrated that plant genes play a part at every stage of nodule development(1 1).The plant proteins specifically formed as a result of the plantmicrobe interaction are termed "nodulins" (32).

Alfalfa plants form bacteria-free nodules in response to a number of agents, including Rhizobium meliloti exo mutants, Agrobacterium tumefaciens transconjugants carrying cloned R. meliloti nodulation genes, and compounds that function as auxin transport inhibitors, N-( l-naphthy1)phthalamic acid or 2,3,5-triiodobenzoic acid. These bacteriafree nodules contain transcripts for the nodulins Nms30 and MsENOD2; transcripts for late nodulins like leghemoglobin are not detected. In situ hybridization studies demonstrated that ENOD2 transcripts were localized in parenchyma cells at the base and along the periphery of nitrogen-fixing alfalfa root nodules. The ENOD2 gene was also expressed in a tissue-specific manner in nodules elicited by N-( l-naphthy1)phthalamic acid and 2,3,5-triiodobenzoic acid. In bacteria-free nodules induced by R. meliloti exo mutants and A. tumefaciens transconjugants carrying either one or both R. meliloti symbiotic plasmids, ENODS transcripts were also detected but were usually localized to parenchyma cells at the base instead of along the periphery of the nodule. On the basis of the pattern of ENODS gene expression, we conclude that the developmental pathway of bacteria-free nodules, whether bacterially or chemically induced, is the same as that of nitrogen-fixing nodules, and, furthermore, that the auxin transport inhibitors in their action mimic some factor(s) that trigger nodule development.