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Catalase overexpression reduces the germination time and increases the pathogenicity of the fungus Metarhizium anisopliae

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Abstract

Catalases and peroxidases are the most important enzymes that degrade hydrogen peroxide into water and oxygen. These enzymes and superoxide dismutase are the first lines of cell defense against reactive oxygen species. Metarhizium anisopliae displays an increase in catalase–peroxidase activity during germination and growth. To determine the importance of catalase during the invasion process of M. anisopliae, we isolated the cat1 gene. cat1 cDNA expression in Escherichia coli and the subsequent purification of the protein confirmed that the cat1 gene codes for a monofunctional catalase. Expression analysis of this gene by RT-PCR from RNA isolated from fungus grown in liquid cultures showed a decrease in the expression level of the cat1 gene during germination and an increase during mycelium growth. The expression of this gene in the fungus during the infection process of the larvae of Plutella xylostella also showed a significant increase during invasive growth. Transgenic strains overexpressing the cat1 gene had twice the catalase activity of the wild-type strain. This increase in catalase activity was accompanied by a higher level of resistance to exogenous hydrogen peroxide and a reduction in the germination time. This improvement was also observed during the infection of P. xylostella larvae. M. anisopliae transgenic strains overexpressing the cat1 gene grew and spread faster in the soft tissue of the insect, reducing the time to death of the insect by 25% and the dose required to kill 50% of the population 14-fold.

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Acknowledgments

This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT), Consejo Estatal de Ciencia y Tecnología del Estado de Guanajuato (CONCyTEG), Secretaria de Educacion Publica (SEP), and the University of Guanajuato. CEMH and IEPG were recipients of a fellowship from CONACyT, México.

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Authors and Affiliations

  1. Department of Biology, University of Guanajuato, Noria Alta s/n, Col. Noria Alta, Guanajuato, Guanajuato, CP. 36050, Mexico

    Claudia Erika Morales Hernandez, Israel Enrique Padilla Guerrero, Gloria Angelica Gonzalez Hernandez & Juan Carlos Torres Guzman

  2. Department of Agronomy, University of Guanajuato, Guanajuato, Mexico

    Eduardo Salazar Solis

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  1. Claudia Erika Morales Hernandez
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  2. Israel Enrique Padilla Guerrero
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  3. Gloria Angelica Gonzalez Hernandez
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  4. Eduardo Salazar Solis
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  5. Juan Carlos Torres Guzman
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Corresponding author

Correspondence to Juan Carlos Torres Guzman.

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Fig. S1

Comparison of Metarhizium anisopliae CAT1 with the most similar monofunctional catalases and catalase/peroxidases. M. anisopliae, Macat1* was aligned with catalases from: Aspergillus fumigatus, Af: catA (accession number P78574), catB (Q92405); A. nidulans, An: catA (P55305), catB (P78619), katG (Q96VT4); A. niger, Ani: catA (A2QT57), katG (A2Q7T1); A. oryzae, Ao: catA (Q877A1), catB (Q877A8), katG (Q2TW34); Botrytis cinerea, Bc: cat2 (G96WN1), catA (P55304); Candida albicans, Ca: CTA1 (O13289); Caenorhabditis elegans, Ce: ctl-1 (O61235), ctl-2 (Q27487), ctl-3 (Q8MYL7); Cladosporium fulvum, Cf: cat-2 (Q9C476); Cochliobolus heterostrophus, Ch: CAT2 (Q6UJ33); Claviceps purpurea, Cp: cat1 (O60038); Escherichia coli, Ec: katE (P21179), katG (P13029); Gibberella moniliformis, Gm: cat2 (B8XX03); Magnaporte grisea, Mg: katG1 (A4R559), MGG_10061 (A4R6C5), MGG_06442 (A4R8L1); Neurospora crassa, Nc cat-1: (Q9C168), cat-2 (Q8X182), cat-3 (Q9C169); Podospora anserina, Pa: catA (Q9HDP6), catB (Q9HDD5); Paracoccidioides brasiliensis, Pb: catA (Q6RSH8); Penicillium marneffei, Pm: katG (Q8NJN2); Saccharomyces cerevisiae, Sc: CTA1 (P15202), CTT1 (PO6115); Sclerotinia sclerotiorum, Ss: SS1G-02784 (A7EBU8); and Ustilago maydis, Um: kat G (Q4P914) http://www.ebi.ac.uk/. a Conserved amino acid that forms part of the active site (asterisk), heme coordination (empty circle), as well as the residues involved in forming the heme pocket (filled circle) are indicated. The conserved amino acid sequence FDHERVPERAVHARGAG containing the conserved H-102 and present in all heme catalases for the proper binding and reduction of the H2O2 molecule, and the heme binding site composed of amino acid residues RIFSYLDTQL are indicated (boxes). b Phylogenetic tree of monofunctional catalases and catalase/peroxidases. The alignment was performed using the Clustal W method from Lasergene 8.0 program MegAlign (DNASTAR, Inc.) with its default parameters. (DOC 187 kb)

Fig. S2

Expression and purification of recombinant catalase in E. coli. a Twenty microliters of each fraction was loaded into each lane of a 10% SDS-PAGE gel and stained with Coomassie blue. Lane 1 Crude extract from E. coli expressing CAT1 without IPTG; lane 2 Crude extract from E. coli expressing CAT1 with 1 mM IPTG induction for 3 h; lane 3 Flow-through proteins from Ni-NTA column; lane 4 Proteins eluted with 20 mM imidazole; lane 5 Proteins eluted with 40 mM imidazole; lane 6 Purified CAT1 protein eluted with 100 mM imidazole; lane M corresponds to molecular weight markers in kDa. b In-gel assay for catalase (lanes 1 and 2) or catalase and peroxidase (lanes 3 and 4). Fifty microliters of crude extract from E. coli expressing CAT1 with 1 mM IPTG induction for 3 h (lanes 1 and 3) or purified recombinant CAT1 (lane 2 and 4) was loaded into each lane of a non-denaturing 8% polyacrylamide gel, and then catalase (seen as clear zone, lanes 1 and 2) and peroxidase (seen as dark band on a clear zone, lanes 3 and 4) were visualized as described in “Materials and methods.” c In-gel assay for catalase (lane 1) or catalase and peroxidase (lane 2). Fifty micrograms of protein from cellular crude extracts of M. anisopliae Ma10 grown for 48 h in minimal medium with P. xylostella cuticle was loaded into each lane of a non-denaturing 8% polyacrylamide gel, and then catalase (lane 1) and peroxidase (lane 2) were visualized as described in “Materials and methods.” (PDF 4999 kb)

Fig. S3

In vitro expression of chit1 and cat1 from M. anisopliae. Conidia from M. anisopliae Ma10 were grown in liquid minimal media containing 2% glucose for 48 h (lane 1), liquid minimal media containing 1% chitin (lane 2) for 12 h or 1% P. xylostella cuticle (lane 3) for 12 h. The cultures were incubated in Erlenmeyer flasks at 28°C as described in “Materials and methods.” The fungal material was collected by filtration and was used to compare transcript abundance by RT-PCR analysis. a Relative RT-PCR of chit1 and AJ274118 (loading control) using total RNA. b Relative RT-PCR analysis of cat1 and AJ274118 (loading control) using total RNA (DOC 292 kb)

Fig. S4

UV/vis spectrum of purified CAT1. The UV/vis spectrum of purified CAT1 was obtained in solution with 50 μg of CAT1 in 50 mM phosphate buffer, pH 7.0. The spectrum was recorded with a spectrophotometer Ultrospec 4300 Pro (Amersham Bioscience) (DOC 79 kb)

Table S1

Primers designed for use in gene cloning, overexpression, DNA quantification, and gene expression by RT-PCR analysis (DOC 43 kb)

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Morales Hernandez, C.E., Padilla Guerrero, I.E., Gonzalez Hernandez, G.A. et al. Catalase overexpression reduces the germination time and increases the pathogenicity of the fungus Metarhizium anisopliae . Appl Microbiol Biotechnol 87, 1033–1044 (2010). https://doi.org/10.1007/s00253-010-2517-3

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