Abstract
Chitin is an important component of the fungal cell wall with a family of chitin synthases mediating its synthesis. Here, we report on the genetic characterization of the full suite of seven chitin synthases (MaChsI-VII) identified in the insect pathogenic fungus, Metarhizium acridum. Aberrant distribution of chitin was most evident in targeted gene knockouts of MaChsV and MaChsVII. Mutants of MaChsI, MaChsIII, MaChsIV showed delayed conidial germination, whereas ΔMaChsII and ΔMaChsV mutants germinated more rapidly when compared to the wild-type parent. All MaChs genes impacted conidial yield, but differentially affected stress tolerances. Inactivation of MaChsIII, MaChsV, MaChsVII resulted in cell wall fragility, and ΔMaChsV and ΔMaChsVII mutants showed high sensitivity to Congo red and calcofluor white, suggesting that the three genes are required for cell wall integrity. In addition, ΔMaChsIII and ΔMaChsVII mutants showed the highest sensitivities to heat and UV-B stress. Three of seven chitin synthase genes, MaChsIII, MaChsV, MaChsVII, were found to contribute to fungal virulence. Compared with the wild-type strain, ΔMaChsIII and ΔMaChsV mutants were reduced in virulence by topical inoculation, while the ΔMaChsVII mutant showed more severe virulence defects. Inactivation of MaChsIII, MaChsV, or MaChsVII impaired appressorium formation, affected growth of in insecta produced hyphal bodies, and altered the surface properties of conidia and hyphal bodies, resulting in defects in the ability of the mutant strains to evade insect immune responses. These data provide important links between the physiology of the cell wall and the ability of the fungus to parasitize insects and reveal differential functional consequences of the chitin synthase family in M. acridum growth, stress tolerances, cell wall integrity and virulence.
Highlights
The fungal cell wall is a dynamic and flexible organelle that modulates the interaction of the organism with its environment and, in the case of pathogens, acts as a critical recognition and evasion interface with host defenses [1]
We revealed that three chitin synthase genes MaChsIII, MaChsV and MaChsVII were shown to be important for fungal appressorium formation and evasion of insect cellular and/or humoral defenses, promoting the fungal dimorphic transition to the production of hyphal bodies that occurs within hosts, and to virulence
The chitin synthase genes in M. acridum were named as MaChsI-VII in accordance with their placement among orthologs (S1 Fig)
Summary
The fungal cell wall is a dynamic and flexible organelle that modulates the interaction of the organism with its environment and, in the case of pathogens, acts as a critical recognition and evasion interface with host defenses [1]. Chitin fibrils and cross-linked proteins help shape and maintain the overall mechanical strength of the fungal cell, contributing to the environmental survival and virulence in pathogenic fungi [3,4,5,6]. Fungal chitin biosynthesis is catalyzed by a family of chitin synthases grouped into seven classes (I to VII) based on amino acid homology, with variation seen in the number of chitin synthase (Chs) genes seen in different fungi [2, 7]. In the budding yeast Saccharomyces cerevisiae, three Chs genes Chs (class I), Chs (class II) and Chs (class IV) are present [7], whereas Chs III, V, VI and VII are found only in filamentous fungi [8]. Filamentous fungi often contain 6–10 Chs genes, e.g. the plant pathogen Ustilago maydis contains eight Chs genes (Chs1-Chs and Mcs1) in its genome [9]
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