Identification of pH-signaling pathway-related genes in mycopasasitic fungus Coniothyrium minitans

Abstract:

[Objectives] To identify pH-signalling pathway (Pal)-related genes in mycoparasitic fungus Coniothyrium minitans and to understand the role of these genes in interaction between C. minitans and its host Sclerotinia sclerotiorum.[Methods] Six Pal-related homologues were obtained from the whole genome of C. minitans and designated as CmpalA, CmpalB, CmpalC, CmpalF, CmpalH and CmpalI. PEG-mediated protoplast transformation was used to create the deletion mutants of Pal-related genes. Five Pal-related genes were knocked out individually and the mutants designated as ΔCmpalA-33, ΔCmpalB-13, ΔCmpalC-5, ΔCmpalF-50 and ΔCmpalH-26. The biological characteristics, including colony morphology, mycoparasitism, oxalate degradation and antifungal activity,were compared between knock-out mutants and the wild-type strain.[Results] Compared to the wild type strain, five Pal-related genes-deletion mutants showed significantly reduced mycelia growth between pH 6 and 8. These results indicated that the disruption of these Pal-related genes increases sensitivity to neutral or alkaline pH. The sclerotia-infection assay showed that the parasitic activities of the five Pal-related genes-deletion mutants were dramatically reduced. qRT-PCR results showed that these Pal-related genes-deletion mutants suppressed expression levels of three mycoparasitism-associated genes Cmch1, Cmg1 and Cmsp1. Meanwhile, expression of CmpacC, the pH signaling pathway downstream gene, was also reduced in the Pal-related genes-deletion mutants. The oxalate degradation of the five Pal-related genes-deletion mutants at pH 6 were increased under pH 8, and the antifungal activity of those mutants were also increased at pH 8 comparison with the wild type.[Conclusion] Disruption of the Pal-related genes resulted in impaired C. minitans responses to ambient pH. The pH-signalling pathway (Pal) plays an important role in interaction between C. minitans and S. sclerotiorum, including mycoparasitism, oxalate degradation and antifungal activity in C. minitans against S. sclerotiorum.

Key words:Coniothyrium minitans;Sclerotinia sclerotiorum;pH-signalling pathway;mycoparasitism;oxalate degradation;antifungal activity