The evolution of RNA interference system, blue light sensing mechanism and circadian clock in Rhizophagus irregularis give insight on Arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) symbiosis is formed by plant roots and arbuscular mycorrhizal fungi (AMF) which are the oldest symbiotic partners of plants and have evolved at least 460 million years ago with the emergence and evolution of land plants. AMF are also the most ubiquitous symbiotic partner of plants as they can colonize more than 80% of vascular plant species. AMF are an early diverged fungal lineage whose phylogenetic position is still under debate. AMF are obligatory plant root symbionts which depend on a source of carbon from the photosynthesis of host plants. In exchange, AMF help plants to absorb various essential soil nutrients, such as phosphorus and transfer these nutrients through their hyphae which have grown into and colonized plant root cells in which they form a structure called an arbuscule. The nutrient allocation and interlocked metabolic pathways between the fungus and the host underwent selective pressure as symbiotic partners. Moreover, AMF hyphae act as an ecological niche for various soil bacteria and other fungi, thus forming the backbone of the rhizospheric part of the plant. AM symbiosis is an essential element to understand plant physiology and ecosystem. Despite the crucial roles of AMF in ecosystems, their genetics and evolution are far from being understood. The RNA interference (RNAi) system, the blue light sensing mechanism and the circadian clock are important mechanisms which regulate expression of various genes in fungi.  Although it has an acknowledged role in gene regulation, especially with symbiosis as well as reflected selective pressures on core proteins in the cases of other symbiotic organisms such as nematode worms, the RNAi system has never been considered in AMF. The same is true for the case of blue light sensing mechanisms. Only a few studies showed that blue light can affect spore germination and hyphae growth of AMF, but the mechanism was not addressed. In the case of the circadian clock, even though circadian rhythms are ubiquitous in fungi and a diurnal rhythm of hyphae growth was reported in AMF during a field level study, the mechanism was unknown. Currently, the genome and transcriptome of the model AM fungus Rhizophagus irregularis isolate DAOM 197198, are publicly available and they were used in my studies. The objective of my Ph.D. project was therefore to study the evolution of the RNAi system, blue light sensing mechanisms and the circadian clock in R. irregularis genome using both bioinformatic and molecular biological approaches. The specific objectives of my Ph.D. project were: 1) to investigate whether the RNAi system is conserved in the genome of R. irregularis and explore the evolutionary traits in its core proteins; 2) to describe the blue light sensing mechanism in the genome of R. irregularis; and 3) to search for a fungal circadian mechanism in the genome of R. irregularis.  I surveyed genomic and transcriptomic data to search for conserved elements of the RNAi system of R. irregularis and its relatives. Two life stages of the R. irregularis lifecycle (germination of spores without roots and established mycorrhizal symbiosis) were investigated for gene expressional profiles using reverse-transcriptase quantitative PCR (qPCR). I identified particular evolutionary traits in R. irregularis RNAi system, such as horizontal gene transfer (HGT) of its core gene coding ribonuclease III protein from autotrophic cyanobacteria, which has never been reported in any eukaryotes so far. I also found and identified an ancient mechanism of blue light sensing which is related to circadian clock. It was intriguing to find a conserved core gene (frequency) that responds to light exposure in the genome of this underground plant-root symbiont. At the same time, the circadian clock core component (FRQ) was not found in other basal fungal lineages including Mucoromycotina, a fungal subphylum which is considered as the closest relative of AMF.  The outcome of my Ph.D. project advanced our knowledge on important mechanisms which regulate the expression of various genes in the oldest and most ubiquitous symbiotic partner of plants. My results also provide new insight on the intimacy between cyanobacteria and AMF which resulted in a unique HGT in the RNAi system. It also expands the knowledge of evolution of the circadian frq gene in fungi. Furthermore, the presence of circadian clock and output genes in R. irregularis opens the door to the chronological study of AM symbiosis, which can be used as a model for the plant holobiont.

Keyphrases: Arbuscular mycorrhizal fungi (AMF), Holobiont, Evolution, Symbiosis, RNA interference (RNAi), Horizontal gene transfer (HGT), Light sensing, Circadian clock, Chronobiology, Horloge circadianne