| Medicago truncatula is a legume species with a small genome that has been adopted as a model organism for the study of nitrogen fixation and other aspects of plant biology. The aim of this work is to identify genes involved in the early events in the symbiotic pathway. This has been achieved using two different approaches, namely, characterizing plant mutants, and gene discovery using DNA microarray technology. Plant mutants, obtained by EMS mutagenesis, that are defective in early interactions with microbial symbiont Sinorhizobium meliloti were used for this study. Features of mutants under study include (1) the inability to make nitrogen-fixing nodules [nod−], (2) a very early arrest in the infection process [inf−], and (3) abnormal root hair deformation in response to rhizobia. Two single, recessive mutants, representing two distinct loci, have been selected for detailed analysis. Both mutants manifest defects in symbiotic responses within ten hours of inoculation. These two inf − mutants show loss of root hair growth polarity in response to Rhizobium, and fail to reinitiate growth leading to root hair curling. This phenotype is mimicked by the actin microfilament drug cytochalasin B. The mutants also show defects in reorganization of the microfilament cytoskeleton after inoculation, and in cortical cell activation Comparative molecular mapping of one of the mutants, an allele of dmi2, indicates that it could be an ortholog of a non-nodulating alfalfa mutant, nn1. This discovery will accelerate the cloning of this locus that is required for nodulation. The analysis of gene expression in the mutants has also shown that they are arrested very early in the symbiotic interaction. A second approach, using DNA microarrays, has been used to study temporal changes in gene expression during early stages of nodulation. A preliminary study has identified differentially expressed genes before and after inoculation. Further studies are underway to characterize these genes. This bilateral approach should help in dissecting the early signaling pathway of legume-Rhizobium interaction. |
