REGULATION OF CALCIUM INFLUX AND REACTIVE OXYGEN SPECIES PRODUCTION DURING INFECTION OF LEGUMES BY RHIZOBIA
Nod factor (NF) can induce two separate calcium responses in legume root hairs. Nuclear-associated calcium spiking is central to the symbiosis signalling (Sym) pathway, which is necessary for the activation of genes required for nodule formation and bacterial infection. In addition NF activates a tip-focused calcium influx, which is less-well studied but is thought to be involved in bacterial infection. NF also activates ROS transient production at the tip of root hair cells. In this thesis I used fluorescent probes (Ca2+-sensitive Cameleons YC2.1 and YC3.6 and the ROS-sensitive CM-H2DCFDA dye) to characterise the NF-induced calcium influx and ROS transient responses in Medicago truncatula. Along with being spatially and temporally co-incident, the responses require similar concentrations of NF to be activated, are inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and are dependent on the NF receptor NFP but independent of the Sym pathway components DMI1 and DMI2. These results suggest the NF-induced calcium influx and ROS transient are part of a common signalling pathway during bacterial infection. ROP signalling is associated with ROS production and calcium influx during developmental root hair elongation. I assessed the role of ROPs during rhizobial infection in M. truncatula and found a ROP-activating protein, MtGAP1, was upregulated in root hairs during bacterial infection and is involved in normal root hair curling and infection thread development. Two pieces of evidence directly link ROP signalling with the NF-induced calcium influx: gap1 mutants were hypersensitive for induction of the calcium influx, and there was a reduction in the number of calcium influx responses in ROP9 RNAi knockdown lines. Drawing parallels between developmental root hair elongation and bacterial infection I propose a model for the regulation of ROP signalling by NF leading to root hair curling, the activation of the calcium influx and ROS transient, and infection thread formation.
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