Investigating the link between the plant circadian clock and regulation of plant-microbe interactions in the Medicago truncatula - Sinorhizobium melil

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An established legume-rhizobia symbiosis results in a mutualistic relationship whereby the plant host receives fixed-nitrogen from the bacteria in exchange for dicarboxylic acids. This plant-microbe interaction requires the regulation of multiple metabolic and physiological processes in both the host and symbiont. Nodule-specific cysteine rich (NCR) peptides are exclusively expressed within rhizobia-inoculated indeterminate nodules of inverted-repeat lacking clade legumes. NCRs have known functions in regulating rhizobia differentiation into nitrogen-fixing bacteroids, new mounting evidence suggest NCR peptides have additional diverse regulatory roles in regulating plant-microbe interactions, including in nodule development. Recent work has uncovered circadian control of NCR expression within nodules; we hypothesise that the plant circadian clock regulated plant-microbe interactions within nodules through the expression of NCRs. This research project will investigate the relationship between the plant circadian clock and nodulation via NCRs using the model system Medicago truncatula and its symbiont Sinorhizobium medicae. To do this, NCR rhythmic expression will be investigated following transient transformation of M. truncatula root hairs with luciferase reporter vectors containing the promoters of 45 rhythmically expressed NCRs. Further experiments will address: 1) whether symbiotic bacteroids affect NCR rhythmicity within nodules by inoculating NCR-transformed M. truncatula with different rhizobial strains, 2) screening luminescence of transformed M.truncatula in different day/night cycles and constant light conditions will allow us to investigate whether NCR expression is truly circadian-regulated, 3) and lastly, the importance of NCRs in nodule activity can be investigated through manipulating NCR expression using site-direct mutagenesis. Together this will help to characterise the role of NCRs in mediating circadian effects on nodulation.