Evolutionary conservation of host responses to arbuscular mycorrhizal symbiosis in liverworts

Arbuscular mycorrhizal symbiosis (AMS) arose in land plants more than 400 million years ago, perhaps acting as a major contributor to plant terrestrialization. The ability to engage in AMS is evolutionarily conserved across most clades of extant land plants, including early diverging bryophytes. Despite its broad taxonomic distribution, little is known about the molecular components that underpin AMS in early diverging land plants. Several AMS associated genes were recently shown to be conserved in liverworts and hornworts, but evidence of them being associated with symbiosis in bryophytes is scarce. The present study aimed to bridge this knowledge gap by investigating the evolutionary conservation of AMS molecular pathways in the liverwort Marchantia paleacea, with a combination of phylogenetic, comparative transcriptomic and reverse genetic approaches. The dynamic response of M. paleacea to colonization by the AM fungus Rhizophagus irregularis was investigated with time-resolved transcriptomics across progressive stages of symbiosis development. Gene orthology inference and comparative analysis of M. paleacea to two AMS angiosperm models -Medicago truncatula and Oryza sativa- revealed a deep conservation of transcriptional responses to AMS across distantly related land plants. In parallel, phylogenetic analysis of genes conserved for symbiosis across angiosperms identified a core set of symbiosis-associated genes that is ancestral to land plants. Some of the sequences belonging to this AMS gene set are repeatedly lost across land plants that have lost the ability to engage in AMS, suggesting that evolutionary pressure for their retention is strictly associated with AMS. The functional conservation of candidate bryophyte AMS genes identified with the above approaches was investigated by (1) functional complementation of rice mutants, (2) CRISPR/Cas9 targeted gene KO of two bryophyte AMS gene candidates in M. paleacea. Taken together, this thesis illustrates that the genetic machinery regulating key aspects of symbiosis in plant hosts is largely conserved and coregulated across distantly related land plants. Since bryophytes are a monophyletic sister clade of all other land plants, the genes analysed in this study likely acquired an AMS-associated function before the last common ancestor of land plants and provide an insight on the first molecular pathways associated with symbiosis at the dawn of land plant evolution.