The Myosin Motor Domain-Containing Chitin Synthases Are Involved in Cell Wall Integrity and Sensitivity to Antifungal Proteins in Penicillium digitatum.

Mónica Gandía,Paloma Manzanares,Jose F. Marcos,Sandra Garrigues,Begoña Bolós

doi:10.3389/fmicb.2019.02400

Abstract

Penicillium digitatum is the main postharvest pathogen of citrus fruit and is responsible for important economic losses in spite of the massive use of fungicides. The fungal cell wall (CW) and its specific component chitin are potential targets for the development of new antifungal molecules. Among these are the antifungal peptides and proteins that specifically interact with fungal CW. Chitin is synthesized by a complex family of chitin synthases (Chs), classified into up to eight classes within three divisions. Previously, we obtained and characterized a mutant of P. digitatum in the class VII gene (ΔchsVII), which contains a short myosin motor-like domain (MMD). In this report, we extend our previous studies to the characterization of mutants in chsII and in the gene coding for the other MMD-Chs (chsV), and study the role of chitin synthases in the sensitivity of P. digitatum to the self-antifungal protein AfpB, and to AfpA obtained from P. expansum. The ΔchsII mutant showed no significant phenotypic and virulence differences with the wild type strain, except in the production and morphology of the conidia. In contrast, mutants in chsV showed a more dramatic phenotype than the previous ΔchsVII, with reduced growth and conidial production, increased chitin content, changes in mycelial morphology and a decrease in virulence to citrus fruit. Mutants in chsVII were specifically more tolerant than the wild type to nikkomycin Z, an antifungal inhibitor of chitin biosynthesis. Treatment of P. digitatum with its own antifungal protein AfpB resulted in an overall reduction in the expression of the chitin synthase genes. The mutants corresponding to MMD chitin synthases exhibited differential sensitivity to the antifungal proteins AfpA and AfpB, ΔchsVII being more susceptible than its parental strain and ΔchsV being slightly more tolerant despite its reduced growth in liquid broth. Taking these results together, we conclude that the MMD-containing chitin synthases affect cell wall integrity and sensitivity to antifungal proteins in P. digitatum.

Highlights

Fungi are responsible for serious diseases in plants, animals and humans, resulting in a large number of deaths and millionaire economic losses (Brown et al, 2012; Fisher et al, 2012)
A recent re-evaluation showed that P. digitatum has a duplication of chsIII that would code for a second class III enzyme (ChsIIIb, PDIG_76160), as occurs with other fungi (Mellado et al, 1996; Choquer et al, 2004)
The role of chitin synthase genes in fungal development and pathogenesis has been studied through gene deletions in a wide number of fungi, and the general conclusions are common in many fungal species, with just minor differences (Morcx et al, 2013; Gandía et al, 2014; Muszkieta et al, 2014; Liu et al, 2016; Zhang et al, 2016)

Summary

Introduction

Fungi are responsible for serious diseases in plants, animals and humans, resulting in a large number of deaths and millionaire economic losses (Brown et al, 2012; Fisher et al, 2012). Numerous studies on the biological role of the distinct chs genes in fungal pathogens have been conducted, obtaining a repertoire of deletion mutants of these genes in different filamentous fungi such as Aspergillus fumigatus (Muszkieta et al, 2014), Fusarium graminearum (Cheng et al, 2015), Magnaporthe oryzae (Kong et al, 2012) or Botrytis cinerea (Morcx et al, 2013). Overall, these studies have shown specific roles for each class/division, including the response to CW stress and involvement in virulence

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