The Adh1 gene of the fungus Metarhizium anisopliae is expressed during insect colonization and required for full virulence.

Zymography of alcohol dehydrogenase (ADH) activity in the entomopathogenic fungus Metarhizium anisopliae grown under various conditions revealed that micro-aerobic growth was associated with increased ADH activity. The major ADH protein, AdhIp, was purified to homogeneity by affinity chromatography and has an estimated molecular weight of 41kDa and an isoelectric point (pI) of 6.4. Peptide mass fingerprint analysis allowed the identification and cloning of the gene that encodes this protein, Adh1, as annotated in the M. anisopliae genome database. AdhIp is related to the medium-chain dehydrogenase/reductase (MDR)/zinc-dependent alcohol dehydrogenase-like family and contains conserved ADH sequence motifs, such as the zinc-containing ADH signature, the FAD/NAD binding domain and amino acid residues that are conserved in most microbial ADHs. Semi-quantitative RT-PCR analysis revealed that Adh1 gene expression occurs at low levels during early Plutella xylostella infection and that the Adh1 gene was primarily expressed at larval death and as mycelia emerge from the insect cuticle before conidiation. Antisense-RNA experiments indicated that NAD(+)-dependent ADH activity was diminished by 20-75% in the transformants, and the transformants that had lower ADH activity showed allyl alcohol resistance, which indicates that reduction in ADH activity also occurs in vivo. Bioassays performed using antisense adh1 transformants, which have lower ADH activity, showed that LC50 values were two to five times higher than the wild-type, indicating that AdhIp is required for full capability of the fungus to penetrate and/or colonize the insect.

Isolation, characterization and expression analysis of the ornithine decarboxylase gene (ODC1) of the entomopathogenic fungus, Metarhizium anisopliae.

The gene ODC1, which codes for the ornithine decarboxylase enzyme, was isolated from the entomopathogenic fungus, Metarhizium anisopliae. The deduced amino acid sequence predicted a protein of 447 amino acids with a molecular weight of 49.3 kDa that contained the canonical motifs of ornithine decarboxylases. The ODC1 cDNA sequence was expressed in Escherichia coli cells; radiometric enzyme assays showed that the purified recombinant protein had ornithine decarboxylase activity. The optimum pH of the purified Odc1 protein was 8.0-8.5, and the optimum reaction temperature was 37°C. The apparent K(m) for ornithine at a pyridoxal phosphate concentration of 20mM was 22 µM. The competitive inhibitor of ODC activity, 1,4-diamino-2-butanone (DAB), at 0.25 mM inhibited 95% of ODC activity. The ODC1 mRNA showed an increase at the beginning of appressorium formation in vitro. During the M. anisopliae invasion process into Plutella xylostella larvae, the ODC1 mRNA showed a discrete increase within the germinating spore and during appressorium formation. The second expression peak was higher and prolonged during the invasion and death of the insect. The ODC1 gene complements the polyamine auxotrophy of Yarrowia lipolytica odc null mutant.

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

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.