Establishment and evaluation of differentially targeted, genetically encoded, calcium indicators for the study of arbuscular mycorrhizal symbiosis ​

Plants are surrounded by many soil microorganisms affecting their growth and physiology. Many of them have no direct effects on the plant, others are pathogenic and only few are beneficial and can establish symbiotic relationships. Among the various plant-microbe symbioses, the most widespread and ancient is the arbuscular mycorrhizal (AM) symbiosis with Glomeromycotina fungi. The aim of this thesis work is the study of plant Ca2+ signalling events during the early phases of AM symbiosis establishment, focusing in particular on the plant discrimination process among symbiotic and pathogenic fungal signals. Lotus japonicus plants were transformed with YFP-aequorin probes specifically targeted to either the cytosol or the nucleus. Complex and multiphasic cytosolic and nuclear Ca2+ signatures were recorded by challenging transformed roots with fungal chitin-derived molecules. The involvement of the recorded Ca2+ signatures in symbiotic/defence signalling pathways was deepen using L. japonicus mutants, defective in different plasma membrane receptors (SYMRK, CERK6). In addition, aequorin-based chimeras targeted to plastids and the endoplasmic reticulum, respectively, were engineered, to investigate their role in shaping intracellular Ca2+ dynamics. Finally, the molecular cloning of a novel highly fluorescent Ca2+ indicator (mNeonGreen-GECO1) was carried out, as a complementary tool useful for Ca2+ imaging studies in plant-microbe interactions.