title: Roles and Interplay of Flavonoids, Reactive Oxygen Species, and Cytokinin in the Early Infection of Medicago truncatula by Sinorhizobium meliloti
Creators: name: Rae, Angus
Publisher: name: The Australian National University
Year: 2024

description: Understanding the mechanisms by which legumes are infected by symbiotic, nitrogen-fixing bacteria (rhizobia) may prove crucial in the pursuit of bio-engineering nitrogen-fixation in current food crops, and as a result, reducing reliance on nitrogen fertilisers. In this thesis, I have examined the roles and interplay of key signalling factors required for the establishment of symbiotic infection of rhizobia in the roots of the model legume Medicago truncatula. Infection of Medicago roots is driven by the production of 'infection threads' by the plant. Infection threads are infection structures that are unique to nitrogen-fixing symbioses, and critical to our understanding of the establishment of the symbiosis in legumes. To facilitate the study of infection, I first developed a new method of imaging infection threads via fluorescent labelling and confocal microscopy, that has allowed high resolution imaging of their structure in 3D cell space. One of the earliest signals during the symbiosis is the production of secondary metabolites called flavonoids by the host root, which activate nodulation genes in rhizobia. The first aim of my thesis focussed on a new role for flavonoids in the formation of infection threads, which has so far not been characterised. Based on the observation that genetic elimination of flavonoids from the root prevents infection thread formation, I sought to identify the responsible flavonoid and to define its mechanism of action. Through isolation of purified root hairs of Medicago roots, I identified flavonoids in root hairs that are induced during the early stages of infection using mass spectrometry. These candidates were then tested for their ability to rescue infection thread formation in flavonoid-deficient roots, which led to the identification of two co-required flavonoids for infection. To characterise a possible mechanism of action, I investigated the role of reactive oxygen species (ROS), which are produced in root hairs during infection and are believed to be necessary for infection thread formation. I showed that a specific flavonoid, 4,4'-dihydroxy-2'-methoxychalcone, is required for ROS production in root hairs during infection, and in combination with another flavonoid identified in the same tissue by the mapping above, can rescue infection in roots in which all other flavonoids are silenced. These results suggest that flavonoids act directly or indirectly to regulate ROS formation in root hairs, and that this is necessary for infection thread formation. After infection threads are initiated, they need to grow towards the cortical cell layers inside the root. The mechanism controlling this process is not understood. I investigated an aberrant infection phenotype in the Medicago cytokinin perception mutant cre1-1, where the nodulation-specific cytokinin receptor CRE1 is mutated. Through this, I have developed a new model for cytokinin activity in infection, whereby cytokinin signalling via CRE1 is required to control cell cycle arrest that is necessary for the directed infection thread progression through the cortex. This links the process of infection thread progression with the process of nodule development in the cortex.