Multiple roles of flavonoids in plant-rhizobia symbioses and plant-pathogen interactions

Abstract

Flavonoids are secreted by plant roots to activate and attract nitrogen-fixing bacteria known as rhizobia. Rhizobia perceive flavonoids and reciprocate with secretion of nod-factors required to initiate nodule organogenesis. In this thesis I have explored the roles flavonoids play in roots during early nodulation of Medicago truncatula. To achieve this goal, I silenced or overexpressed genes encoding key enzymes in the flavonoid biosynthesis pathway in hairy-root cultures. I used RNA-interference as a mechanism to silence multiple copies of chalcone synthase, flavonol synthase, isoflavone synthase, dihydroflavonol-4-reducase and flavone synthase II. I also used a 35S promoter to overexpress the genes chalcone synthase, flavonol synthase, isoflavone synthase and dihydroflavonol-4-reducase, although only isoflavone synthase overexpression showed quantifiable increase in targeted metabolites. Flavonoids have been described to affect the spatio-temporal balance of the plant hormone auxin through the inhibition of polar auxin transport and its breakdown in response to rhizobia. Using constitutive nod-factor expressing rhizobia to inoculate flavonoid-silenced (CHSi) roots, I have demonstrated that flavonoids regulate nodule organogenesis in part through the control of auxin synthesis in the root, by regulating gene expression of YUCCA1, a central enzyme in auxin biosynthesis. In contrast, flavonoids did not alter the expression of genes encoding PIN and LAX auxin efflux and influx transporters. The inhibition of polar auxin transport was regulated most strongly by root flavonols and isoflavonoids, however only flavonols were found to be important for nodule formation. A synthetic auxin transport inhibitor, failed to complement CHSi roots, suggested that flavonoids play additional roles in nodule organogenesis apart from controlling auxin transport. To explore further functions for flavonoids in nodulation, I studied the transcriptomic changes in CHSi roots 6 and 24 hours post inoculation with constitutive nod-factor expressing rhizobia. Inoculated CHSi roots over-accumulated many defense-related genes compared to control roots, but failed to express structural enzymes that could be involved in infection thread formation. These roots also differentially expressed genes encoding proteins involved in the synthesis and response of hormones gibberelins, ethylene, cytokinin and auxin. GFP-labelled, constitutively nod-factor expressing rhizobia were shown to form infection-threads in control but not CHSi roots, suggesting that flavonoids are involved in infection of rhizobia. CHSi roots also did not trigger reactive oxygen species accumulation, suggested to be required for cross-linking of glycoproteins in the matrix of infection threads. Flavonoids have previously also been demonstrated to be important as plant-defense compounds. Using the transgenic hairy roots with altered flavonoid metabolite profiles, I suggest a link between auxin-mediated plant-defense and the presence of flavonoids in the roots challenged with the pathogens Rhizoctonia solani (AG8) and Aphanomyces euteiches. One mechanism by which roots show tolerance is through secondary root formation to outgrow and evade pathogens. Roots showing reduced expression of flavonol synthase showed reduced ability to form secondary roots and increased susceptibility to the fungus and oomycete. Conversely, isoflavonoids are precursors to many protective phytoalexins. Overexpression of isoflavone synthase led to an increase in tolerance to the pathogens. This could have important implications in increasing productivity of agricultural crops exposed to environmental stresses. Show more