Analysis of gene expression, regulation and function of three symbiotic ABC subfamily-B transporters in Medicago truncatula
The legume M. truncatula overcomes low soil nutrient conditions by forming symbiotic associations with nitrogen-fixing soil bacteria called rhizobia (Root Nodule Symbiosis) and fungi which assist in the acquisition of phosphate, collectively called arbuscular mycorrhizae (Arbuscular Mycorrhizal Symbiosis). Establishment of functional beneficial symbioses requires successful microbial infection. An initial exchange of signalling molecules between the host and the microbe sets off extensive transcriptional reprogramming of the host developmental programme to accommodate the incoming microbe. Microbial lipochitooligosaccharide molecules trigger a signalling pathway comprising a core set of around seven genes common to both symbioses, central to which is the calcium calmodulin kinase, CCaMK. Here I describe the identification and characterization of three novel M. truncatula ATPBinding cassette containing sub-family B transporters, transcriptionally induced upon infection by both rhizobia and mycorrhizae; therefore named AMN for ABC transporters in Mycorrhization and Nodulation. Promoter-GUS expression reveals that these genes are exclusive to infection structure containing root hair cells and arbuscule containing root cortical cells. I use different SYM pathway mutants to show that the induction of these transporters is dependent on CCaMK and other members of the symbiotic pathway. Conservation of these transporters across mycorrhizing angiosperms suggested an important evolutionary function therefore I identified and characterized single and double mutants. In the absence of any aberrant symbiotic phenotype a triple mutant was also generated but remains to be characterized. Since ABC sub-family B transporters in Arabidopsis are known to efflux auxin I also undertook a multipronged approach to identify a role for auxin in rhizobial infection. Using pharmacological and physiological assays I describe results that indicate a positive role for auxin in infection. Lastly I describe the nodulation phenotype of mtlax2 and mtiaa8; a homologue of the Arabidopsis AtAUX1 auxin influx carrier and an infection induced AUX/IAA repressor respectively. A lower nodule number in both mutants provide the first genetic evidence for auxin’s role in nodule development.
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