Investigation of the rhizobial and mycorrhizal symbioses that occur with plants by the analysis of mutants and ENOD40

The two best characterized symbioses that occur between plants and microbes are those with rhizobia and mycorrhizal fungi. The nitrogen-fixing symbiosis that occurs between rhizobia and the single family Fabaceae (with one interesting exception) involves a continuous exchange of signal molecules that culminates in the formation of nodules on plant roots. In contrast, over 80% of plant families are capable of establishing an association with mycorrhizal fungi. This symbiosis improves the plant's absorption of minerals from the soil, particularly phosphorous.; There are a number of significant similarities between arbuscular mycorrhizal (AM) and rhizobial symbioses, including the influence of exogenous flavonoids on the growth and colonization of these microbes. Several mutants defective in various aspects of flavonoid biosynthesis were characterized in Mehlotus alba, white sweetclover. These mutants showed alterations in growth, pigmentation and flavonoid content, however none of them profoundly affected the symbioses with rhizobia or mycorrhizal fungi, demonstrating the complexity of flavonoid biosynthesis in legumes. However, we found that nodulation mutants of M. alba were defective in their ability to form an AM-symbiosis, showing that there is conservation in the machinery governing both associations. However, the microbe signals involved do not appear to be as closely linked, as no complementation was seen by rhizobial mutants with mycorrhizal fungi.; ENOD40 is one of the early nodulin (ENOD) genes expressed in the Rhizobium-legume symbiosis, and has subsequently been implicated in the mycorrhizal association. We found that this gene is induced in alfalfa by nitrate. Furthermore, analysis of MsENOD40 promoter-GUS constructs in the non-legume Arabidopsis show that the regulatory controls are conserved in a species that does not engage in either symbiosis.; Finally, ENOD40 was found to be present in non-nodulating legume species representing the more basal members of the family. Transcript accumulation in stems and roots is similar to that reported in nodulating species, showing that the expression of this gene in the root preceded nodulation and may have a general function in root processes. Analysis of ENOD40 gene sequences representing all three subfamilies within the legumes, revealed that although there is considerable variation among species, core conserved regions and a potential small peptide remain intact. Furthermore, phylogenetic analysis of predicted secondary structure of ENOD40 supports the hypothesis that the ENOD40 is functioning, at least in part, as an RNA.