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

已建立的豆类-根瘤菌共生关系导致了一种互惠关系，即植物宿主从细菌那里获得固定氮，以换取二元酸。这种植物-微生物的相互作用需要调节寄主和共生体的多种代谢和生理过程。富含根瘤特异性半胱氨酸(NCR)的多肽仅在根瘤菌接种的缺乏反向重复的豆科植物的不确定根瘤中表达。NCRs在调节根瘤菌分化为固氮类细菌方面具有已知的功能，越来越多的新证据表明，NCR多肽在调节植物-微生物相互作用方面具有额外的不同调节作用，包括在根瘤发育中。最近的工作发现了NCR在根瘤中的表达的昼夜节律控制；我们假设植物的昼夜节律时钟通过NCR的表达来调节植物与微生物在根瘤中的相互作用。本研究将利用模式系统紫花苜蓿及其共生菌Sinorhizobium Medicae来研究植物生物钟与NCRs结瘤的关系。为此，利用含有45个有节奏表达的NCR启动子的荧光素酶报告载体瞬时转化元胞根毛，将研究NCR的节律性表达。进一步的实验将解决：1)共生类菌是否通过将不同的根瘤菌接种到NCR转化的M.truncatula来影响NCR在根瘤内的节律性；2)在不同的昼夜周期和恒定的光照条件下筛选转化的M.truncatula的发光将使我们能够研究NCR的表达是否真正受昼夜节律的调节；3)NCRs在根瘤活动中的重要性可以通过使用定点突变来调控NCR的表达来研究。综上所述，这将有助于描述NCRs在调节结瘤的昼夜节律效应中的作用。

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.