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 Δ MaChsII and Δ MaChsV 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 Δ MaChsV and Δ MaChsVII mutants showed high sensitivity to Congo red and calcofluor white, suggesting that the three genes are required for cell wall integrity. In addition, Δ MaChsIII and Δ MaChsVII 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, Δ MaChsIII and Δ MaChsV mutants were reduced in virulence by topical inoculation, while the Δ MaChsVII 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.
Homeodomain transcription factor Ste12 is a key target activated by the pathogenic mitogen-activated-protein kinase pathway, and the activated Ste12p protein regulates downstream gene expression levels to modulate phenotypes. However, the functions of Ste12-like genes in entomopathogenic fungi remain poorly understood and little is known about the downstream genes regulated by Ste12. In this study, we characterized the functions of a Ste12 orthologue in Metarhizium acridum, MaSte12, and identified its downstream target genes. The deletion mutant (ΔMaSte12) is defective in conidial germination but not in hyphal growth, conidiation, or stress tolerance. Bioassays showed that ΔMaSte12 had a dramatically decreased virulence in topical inoculations, but no significant difference was found in intrahemolymph injections when the penetration process was bypassed. The mature appressorium formation rate of ΔMaSte12 was less than 10% on locust wings, with the majority hyphae forming appressorium-like, curved but no swollen structures. Digital gene expression profiling revealed that some genes involved in cell wall synthesis and remodeling, appressorium development, and insect cuticle penetration were downregulated in ΔMaSte12. Thus, MaSte12 has critical roles in the pathogenicity of the entomopathogenic fungus M. acridum, and our study provides some explanations for the impairment of fungal virulence in ΔMaSte12. In addition, virulence is very important for fungal biocontrol agents to control insect pests effectively. This study demonstrated that MaSte12 is involved in fungal virulence but not conidial yield or fungal stress tolerance in M. acridum. Thus, MaSte12 and its downstream genes may be candidates for enhancing fungal virulence to improve mycoinsecticides.
Flo8/Som1, which functions downstream from the cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway, plays important roles in hyphal development, spore formation, and virulence in yeast and several filamentous fungi. However, the functions of Som1 in entomopathogenic fungi are still a mystery. In this study, MaSom1, a Flo8/Som1 homolog, was identified and functionally characterized in a model entomopathogenic fungus Metarhizium acridum. Similar to Flo8/Som1 in other fungi, MaSom1 mainly localized to the nucleus in M. acridum. Disruption of MaSom1 reduced conidial yield, delayed conidial germination, and impaired the fungal tolerances to heat and UV-B. The expression levels of some genes involved in defenses of heat shock and UV-B radiation were significantly reduced in ΔMaSom1. MaSom1 is also important for cell wall integrity and conidial surface structures in M. acridum. Some genes related to fungal cell wall synthesis were downregulated in ΔMaSom1. Bioassays showed that ΔMaSom1 had a dramatically decreased virulence after both topical inoculation and intrahemocoel injection of the fungus in locusts. Moreover, inactivation of MaSom1 reduced appressorium formation, diminished fungal growth in locust hemolymph in vitro, and enhanced insect immune responses. Taken together, these results indicate that disruption of MaSom1 leads to a decline of fungal virulence because of impairments in conidial germination and appressorium formation, reduction of fungal growth in host hemolymph, and enhancement of insect immune responses owing to the changes in conidial surface structures.
Entomopathogenic fungi play important roles in the control of populations of agricultural and disease vector pests in nature. The shortcomings of mycoinsecticides for pest management in the field cannot be completely overcome by improving single biocontrol properties of fungi. Therefore, enhancing the biocontrol potential of entomopathogenic fungi in multiple respects by genetic engineering is desirable. Transcription factors are usually involved in various important processes during fungal growth and pathogenesis via regulating a series of genes, and are important candidates for fungal improvement via genetic engineering. Herein, overexpression of MaSom1, a key transcription factor gene in the cAMP/PKA pathway, improves the biocontrol traits of Metarhizium acridum in multiple respects. When compared with WT, the MaSom1-overexpression strains exhibit enhanced tolerances to UV-B and heat shock, with increased mean 50% inhibition times by 66.9% and 155.2%, respectively. Advanced conidiation emerged accompanied by increased conidial yield up to 3.89 times after 3-day incubation for the MaSom1-overexpression strains compared to WT. Furthermore, when compared with WT, the virulence of the MaSom1-overexpression strains was also increased with the mean 50% lethality times reduced by 21.8% to 23.8%. Taken together, the MaSom1-overexpression improved the biocontrol potential of M. acridum in multiple respects. Our results provide insights into the application of key transcription factors for genetic engineering and offer a credible way to further improve the biocontrol potential of entomopathogenic fungi.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.