Directed Experimental Adaptive Evolution of Osmoregulation in Fungal Pathogen Magnaporthe oryzae Is Independent of Glycerol Metabolism-Associated Genes

Directed experimental adaptive evolution in fungal pathogens is largely unexplored. In the phytopathogenic fungus <i>Magnaporthe oryzae</i>, long-term cultivation under osmotic stress was found to lead to individuals arising as suppressor strains out of osmosensitive "loss-of-function" mutants, in which the high osmolarity glycerol (HOG) pathway was inactivated. The underlying mechanisms of reestablished osmoregulation in the suppressor strains are not known. Here, we found that two different types emerged from the mycelium parts of each <i>∆Mohik1</i>, <i>∆Moypd1</i>, <i>∆Mossk1</i>, <i>∆Mossk2</i>, <i>∆Mopbs2,</i> and <i>∆Mohog1</i>: reversible suppressors, which still struggle with osmotic stress, and irreversible suppressors, which can cope with the same stress situations. This phenomenon only takes place in lof mutants, which are related to the HOG pathway and are not in other osmosensitive mutants. Both suppressor types produce glycerol as a stress response instead of arabitol as it is in the wildtype strain. Glycerol production was found to be almost twice as high in the irreversible strains as compared to the reversible strains. Thus, glycerol metabolism (gm) was assumed to be involved in the molecular mechanism of this adaptive-driven evolution. We generated a set of double mutant strains in which we deleted different gm-related genes within the HOG lof-mutants. Since suppressors originate from these double lof-mutants upon long-term stress, we exclude gm-associated genes acting as drivers for adaptive-driven evolution.