UNRAVELLING CALCIUM-MEDIATED SIGNALLING PATHWAYS IN ARBUSCULAR MYCORRHIZAL SYMBIOSIS

The research activity underlying this PhD thesis has focused on calcium-mediated intracellular signalling pathways activated in plant roots during the onset and functioning of the arbuscular mycorrhizal (AM) symbiosis. In particular, main emphasis has been given to the role of the complex cellular Ca2+ signalling circuits in the recognition of beneficial and detrimental fungi by plant roots and on the identification of novel putative molecular players involved in this process. The first aim of my PhD was to investigate how plants can discriminate symbiotic from pathogenic fungi via Ca2+-mediated intracellular cascades in root cells of the model legume Lotus japonicus. Combining different genetic backgrounds, pharmacological treatments and progressive dilutions of the stimuli, differentially targeted aequorin-based Ca2+ measurements were conducted in response to chitin-derived fungal signals. The occurrence of two distinct temporal Ca2+ phases, underlying the activation of both plant symbiosis and immunity, was found and confirmed at the single-cell level with the fluorescent Ca2+ probe cameleon. This first line of research led to a publication in Journal of Experimental Botany and is presented in Chapter 2 of this thesis. The results of this first line of research integrate with the emerging complex scenario of a plant symbiosis-immunity continuum in plant-fungus interactions, which does not support the simplistic hypothesis that chitin-derived molecules alone can drive the activation of one process or the other in the host plant. This scientific dilemma and possible solutions to it were discussed in a Viewpoint paper published in New Phytologist (Chapter 3), which I contributed to write and edit. The second major line of research aimed at investigating the role of Mildew Locus O genes in AM symbiosis. Three different insertional mutant backgrounds for a symbiotic L. japonicus MLO (called MLO4) were studied regarding AM colonization, confirming and extending previous findings. Given the novel identification of MLOs as Ca2+-permeable, the role of MLO4 in Ca2+-mediated plant-fungus communication circuits was analysed. Moreover, a novel and unexpected role for MLO4 in root development was uncovered. Overall, the results obtained during my PhD activity about the role of LjMLO4 in AM symbiosis and root development are reported in Chapter 4. Given the importance of the whole cell Ca2+ signalling network in regulating plant development and physiology, a further aim of my PhD was to investigate organellar contribution to Ca2+-mediated signalling pathways in beneficial plant-microbe interactions. As reported in Chapter 5, plastid- and endoplasmic reticulum-targeted aequorin chimeras were engineered and expressed in L. japonicus roots. The subcellular localization of the two probes and preliminary measurements of plastid stromal Ca2+ changes triggered by fungal signals were performed. This chapter also hosts the molecular cloning of a novel highly fluorescent Ca2+ reporter (mNeonGreen-GECO1) and preliminary Ca2+ imaging assays in L. japonicus roots, performed with the final aim of investigating the possible occurrence of systemic Ca2+ signalling during AM symbiosis. Finally, I took part in a project of applied interest carried out in collaboration with physicists, engineers and chemists based in Padova (University of Padova and CNR - Institute for Plasma Science and Technology), concerning the potential applications in agriculture of the so-called plasma-activated water (PAW), obtained via the controlled exposure of water to cold plasma. Following on the recently emerged use of PAW as an eco-friendly technology to reduce the use of pesticides and fertilizers in agriculture, this side project aimed to investigate the effects of plant treatment with PAW on the AM symbiosis. Our findings, reported in Chapter 6, demonstrate that plant irrigation with PAW may finely tune the AM symbiosis establishment and development.