Investigating the role of Medicago sativa lectin 1 (MsLec1) in the Sinorhizobium meliloti symbiotic association with plant roots

The role of lectins in the legume--Rhizobium symbiosis has yet to be fully elucidated. One hypothesis is that lectins can assist in the binding of compatible Rhizobium to plant roots, thus facilitating the early stages of this important symbiosis. Previous antisense mediated whole--plant RNA knockdown experiments in Medicago sativa Lectins 1 & 2 (MsLec1 & MsLec2), have demonstrated possible pleiotropic roles of the gene in various aspects of plant development. White Sweetclover (Melilotus alba Desr.), a plant with only one known copy of the highly homologous lectin gene, (MaLec1) is an attractive model in which to study this interaction. To isolate and study the root--specific role of this lectin, an Agrobacterium rhizogenes plant transformation system was successfully developed for this previously untransformable plant model system. Utilization of a powerful hairpin RNAi system illustrated that there are still pleiotropic effects seen by the knockdown of the MaLec1 gene. This indicates that either root to shoot translocation of the RNAi signal is altering the expression of the MaLec1 gene, or that an alternate root to shoot signal, brought about by root specific reduction in MaLec1 gene expression is responsible.;To perform biochemical studies on the function of MsLec1, nearly identical to MaLec1, multiple heterologous protein expression systems were employed to yield purified protein. The protein was found to be toxic when expressed in Drosophila melanogaster derived S2 cells, and insoluble in E. coli expression systems. Various commercially available N-terminal folding assistance polypeptide tags were examined to produce soluble MsLec1 protein to no effect. Alterations to the redox state of the bacteria, as well as expression of chaperonin proteins did permit the production of small quantities of the soluble protein. After significant effort, a SUMO N-terminal protein tagging system was developed, found to be far superior to the other systems tried, and used to purify soluble MsLec1 protein from E. coli..;Previous work with this lectin, and others, in transgenic legumes, indicates that the lectin may assist in the binding of the bacteria to the root surface. Transgenic Arabidopsis thaliana plants were generated to produce the MsLec1 protein. These plants, without the added complexity of having a genetic background geared towards symbiotically associating with Rhizobia, were used to determine if the MsLec1 protein was capable, by itself, of enhancing the binding of compatible Rhizobia to the roots. Surprisingly, expression of the MsLec1 protein was found to generally decrease the amount of root binding by the compatible Rhizobia. This indicates that in legumes, this protein may act in concert with others for the enhanced binding effects. In A. thaliana, without the background legume root proteins, it appears to function like a defensive protein. This indicates that, under certain circumstances, one protein can either function as a symbiotic enhancer and as plant defensive protein.