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

豆科植物与根瘤菌的共生关系建立在互惠互利的基础上，植物宿主从根瘤菌那里获得固定氮以交换二羧酸。这种植物-微生物的相互作用需要宿主和共生体的多种代谢和生理过程的调节。根瘤特异性富含半胱氨酸（NCR）肽只在根瘤菌接种的不确定根瘤中表达。已知NCR在调节根瘤菌分化为固氮类杆菌中具有功能，新的证据表明NCR肽在调节植物-微生物相互作用（包括根瘤发育）中具有额外的多种调节作用。最近的工作揭示了根瘤内NCR表达的昼夜节律控制;我们假设植物昼夜节律钟通过NCR的表达调节根瘤内的植物-微生物相互作用。本研究以蒺藜苜蓿及其共生菌Sinorhizobium medicae为模型系统，通过NCR研究植物生物钟与光合作用之间的关系。为了做到这一点，将在M.用荧光素酶报告载体转染蒺藜根毛，所述荧光素酶报告载体含有45个节律表达的NCR的启动子。进一步的实验将解决：1）共生类杆菌是否影响根瘤内的NCR节律性通过NCR-NCR转化的M。2）在不同的昼夜周期和恒定光照条件下筛选转化的M.truncatula的发光将允许我们研究NCR表达是否真正受昼夜节律调节，3）最后，可以通过使用定点诱变操纵NCR表达来研究NCR在根瘤活性中的重要性。总之，这将有助于阐明NCR在介导昼夜节律对排卵的影响中的作用。

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.