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Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence

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Abstract

Hydrophobins are small, cysteine-rich, secreted proteins, ubiquitously produced by filamentous fungi that are speculated to function in fungal growth, cell surface properties, and development, although this has been rigorously tested for only a few species. Herein, we report identification of three hydrophobin genes from the entomopathogenic fungus, Metarhizium brunneum, and functional characterization of strains lacking these genes. One gene (HYD1/ssgA) encodes a class I hydrophobin identified previously. Two new genes, HYD3 and HYD2, encode a class I and class II hydrophobin, respectively. To examine function, we deleted all three separately, from the M. brunneum strain KTU-60 genome, using Agrobacterium tumefaciens-mediated transformation. Deletion strains were screened for alterations in developmental phenotypes including growth, sporulation, pigmentation, colony surface properties, and virulence to insects. All deletion strains were reduced in their ability to sporulate and showed alterations in wild-type pigmentation, but all retained wild-type hydrophobicity, except for one individual hyd3 mutant. Complementation with the wild-type HYD3 gene restored hydrophobicity. Each gene, present as a single copy in the genome, showed differential expression patterns dependent on the developmental stage of the fungus. When Spodoptera exigua (beet armyworm) larvae were treated with either conidia or blastospores of each hyd mutant, reductions in virulence and delayed mortality were observed as compared to WT. Together, these results suggest that hydrophobins are differentially expressed and may have distinct, but compensating roles, in conidiation, pigmentation, hydrophobicity, and virulence.

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Acknowledgments

A. Sevim was supported by the Council of Higher Education of Turkey (YOK) and performed the majority of the research in the Turgeon laboratory at Cornell. We thank Dr. Chengshu Wang (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China) for providing the HYD3 gene sequence. We also thank Dr. Richard Humber (Biological Integrated Pest Management Research Unit, Robert W. Holley Center for Agriculture and Health, USDA-ARS, Tower Road, Ithaca, NY 14853, USA) for help in assessing some morphological characters of the WT and mutant strains, and J. Bischoff (USDA, APHIS, Beltsville, MD) for molecular identification of KTU-60 as M. brunneum.

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  1. Department of Plant Pathology and Plant-Microbe Biology, Cornell University, 344 Plant Science Building, Ithaca, NY, 14853, USA

    Ali Sevim, Bruno G. G. Donzelli, Dongliang Wu & B. Gillian Turgeon

  2. Department of Biology, Faculty of Arts and Sciences, Rize University, Rize, Turkey

    Ali Sevim

  3. Department of Biology, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey

    Ali Sevim & Zihni Demirbag

  4. Biological Integrated Pest Management Research Unit, Robert W. Holley Center for Agriculture and Health, USDA, ARS, 538 Tower Road, Ithaca, NY, 14853, USA

    Donna M. Gibson

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  1. Ali Sevim
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  2. Bruno G. G. Donzelli
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Correspondence to B. Gillian Turgeon.

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Sevim, A., Donzelli, B.G.G., Wu, D. et al. Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence. Curr Genet 58, 79–92 (2012). https://doi.org/10.1007/s00294-012-0366-6

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