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. The fungal cell wall is a dynamic and flexible organelle that modulates the interaction of the pathogen with its host and acts as a critical recognition and evasion interface with host defenses. Chitin is a hallmark constituent of the fungal cell wall and all fungi contain multiple chitin synthase (Chs) genes. However, systematic characterization of chitin synthase genes has not yet been reported in entomopathogenic fungi. By using the insect pathogen Metarhizium acridum as a model, we performed a systematic genetic analysis of the seven member Chs family (ChsI-VII) in the insect pathogenic fungus. Construction of strains bearing targeted single gene mutations revealed differential contributions of specific Chs genes to growth, cell wall integrity, and stress responses. In addition, 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 ultimately to virulence. These data provide new insights into the roles of Chs genes and chitin as critical components affecting fungal membrane structure and function.

Most fungi, including entomopathogenic fungi, have two different conidiation patterns, normal and microcycle conidiation, under different culture conditions, eg, in media containing different nutrients. However, the mechanisms underlying the conidiation pattern shift are poorly understood. In this study, Metarhizium acridum undergoing microcycle conidiation on sucrose yeast extract agar (SYA) medium shifted to normal conidiation when the medium was supplemented with sucrose, nitrate, or phosphate. By linking changes in nutrients with the conidiation pattern shift and transcriptional changes, we obtained conidiation pattern shift libraries by Solexa/Illumina deep-sequencing technology. A comparative analysis demonstrated that the expression of 137 genes was up-regulated during the shift to normal conidiation, while the expression of 436 genes was up-regulated at the microcycle conidiation stage. A comparison of subtractive libraries revealed that 83, 216, and 168 genes were related to sucrose-induced, nitrate-induced, and phosphate-induced conidiation pattern shifts, respectively. The expression of 217 genes whose expression was specific to microcycle conidiation was further analyzed by the gene expression profiling via multigene concatemers method using mRNA isolated from M. acridum grown on SYA and the four normal conidiation media. The expression of 142 genes was confirmed to be up-regulated on standard SYA medium. Of these 142 genes, 101 encode hypothetical proteins or proteins of unknown function, and only 41 genes encode proteins with putative functions. Of these 41 genes, 18 are related to cell growth, 10 are related to cell proliferation, three are related to the cell cycle, three are related to cell differentiation, two are related to cell wall synthesis, two are related to cell division, and seven have other functions. These results indicate that the conidiation pattern shift in M. acridum mainly results from changes in cell growth and proliferation. The results indicate that M. acridum shifts conidiation pattern from microcycle conidiation to normal conidiation when there is increased sucrose, nitrate, or phosphate in the medium during microcycle conidiation. The regulation of conidiation patterning is a complex process involving the cell cycle and metabolism of M. acridum. This study provides essential information about the molecular mechanism of the induction of the conidiation pattern shift by single nutrients.