The Molecular Mechanism of Fludioxonil Action Is Different to Osmotic Stress Sensing

Abstract

The group III two-component hybrid histidine kinase MoHik1p in the filamentous fungus Magnaporthe oryzae is known to be a sensor for external osmotic stress and essential for the fungicidal activity of the phenylpyrrole fludioxonil. The mode of action of fludioxonil has not yet been completely clarified but rather assumed to hyperactivate the high osmolarity glycerol (HOG) signaling pathway. To date, not much is known about the detailed molecular mechanism of how osmotic stress is detected or fungicidal activity is initiated within the HOG pathway. The molecular mechanism of signaling was studied using a mutant strain in which the HisKA signaling domain was modified by an amino acid change of histidine H736 to alanine A736. We found that MoHik1p^H736A is as resistant to fludioxonil but not as sensitive to osmotic stress as the null mutant ∆Mohik1. H736 is required for fludioxonil action but is not essential for sensing sorbitol stress. Consequently, this report provides evidence of the difference in the molecular mechanism of fludioxonil action and the perception of osmotic stress. This is an excellent basis to understand the successful phenylpyrrole-fungicides' mode of action better and will give new ideas to decipher cellular signaling mechanisms.

Keywords: HIK1; Magnaporthe oryzae; MoHIK1; fludioxonil; fungicide; high osmolarity glycerol (HOG) pathway; histidine kinase; mode of action; phenylpyrrole; signal transduction.

Figure 1

(A) Comparison of a protein sequence from the HisKA domain of the group III HK in different fungi. The putative histidine phosphotransfer sites are highlighted in blue. The differences between the sequences are highlighted in red. (B) Schematic illustration of the amino acid exchange from histidine (blue) to alanine (yellow).

Figure 2

(A) Vegetative growth of Magnaporthe oryzae wild-type strain (MoWT), the loss-of-function mutant ∆Mohik1 and the MoHik1p^H736A mutant under fludioxonil stress. Mycelium growth was measured on MM supplemented with different concentrations of the fungicide fludioxonil. The experiment was conducted for 7 days at 26 °C. (B) Pictures of the cultures grown for 7 days on MM + 20 µg/mL.

Figure 3

(A) Vegetative growth of MoWT, the loss-of-function mutant ∆Mohik1 and the MoHik1p^H736A mutant under osmotic stress. The growth was measured on complete media (CM) and minimal media (MM) supplemented with different concentrations of sorbitol. The incubation time was 7 days at 26 °C. (B) Pictures of the cultures grown for 7 days on CM + 1.2 M sorbitol.