Evolutionary adaptation of living organisms is commonly thought to be the result of processes that have taken place over long periods of time. By contrast, we found that the filamentous rice blast fungus Magnaporthe oryzae rapidly suppresses the osmosensitive "loss of function" (lof) phenotype in knockout mutants of the high-osmolarity glycerol (HOG) pathway. That suppression occurs highly reproducibly after 4 weeks of continuous growth upon salt stress. Stable mutants reestablished in osmoregulation arise independently out of individual osmosensitive lof mutants of the HOG pathway. The major compatible solute produced upon salt stress by these suppressor strains was found to be glycerol, whereas it is arabitol in the wildtype strain. We aim to address the molecular or biochemical mechanisms behind this rapid suppression and characterize the associated factors and signaling pathways which enable or prevent suppression. Therefore, we present a protocol to generate these suppressor mutants in M. oryzae easily to study the molecular basis of evolutionary processes or even epigenetic modulation. This protocol may be applicable to many other fungi and will open a door for researchers worldwide since the HOG pathway is worked on intensively in many different model organisms.
Keywords: Adaptation; Arabitol; Bypass suppressor; Glycerol; High-osmolarity glycerol (HOG) pathway; Magnaporthe oryzae; Osmolyte production; Polyol metabolism; Rewiring; Suppressor mutation.