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

## TL;DR

This study explores how the rice blast fungus adapts to osmotic stress through experimental evolution, revealing that glycerol metabolism is not the main driver of this adaptation.

## Contribution

The study shows that osmoregulation in fungal suppressor strains evolved independently of glycerol metabolism-associated genes.

## Key findings

- Two suppressor types emerged from HOG pathway mutants: reversible and irreversible.
- Both suppressor types produce glycerol instead of arabitol as a stress response.
- Glycerol metabolism genes are not the main drivers of adaptive evolution in these mutants.

## Abstract

The world’s population is rising rapidly, and a major problem is global food security. Magnaporthe oryzae is placed first on a list of the world’s top ten plant pathogens with the highest scientific and economic importance since it causes blast, which is the most devastating disease of cultivated rice—the major food source for more than half of the world’s population. In this study, we demonstrate directed experimental adaptive evolution in this fungus and give insights into this evolutionary phenomenon with regard to the molecular mechanisms behind it. These results will help us better understand the molecular basis of evolutionary events of the rice blast fungus and may open the door to developing new strategies for plant protection and food security in the future. In addition, our study is a valuable contribution to the basic research area of signaling mechanisms in filamentous pathogenic fungi and will thus be of considerable interest to a broad readership within the scientific community.

Directed experimental adaptive evolution in fungal pathogens is largely unexplored. In the phytopathogenic fungus Magnaporthe oryzae, 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 ∆Mohik1, ∆Moypd1, ∆Mossk1, ∆Mossk2, ∆Mopbs2, and ∆Mohog1: 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.