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

Abstract

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.

Ethanol Dehydrogenase I Contributes to Growth and Sporulation Under Low Oxygen Condition via Detoxification of Acetaldehyde in Metarhizium acridum

“…Bioassay results against locusts were in accordance with the transcription profiles of MaADH1 , showing no contribution of ADH1 to virulence (data now shown). However, our results were not consistent with the findings in M. anisopliae , which ADH1 is expressed during insect colonization and required for full virulence ( Callejas-Negrete et al, 2015 ). ADH1 is also involved in virulence in plant pathogenic fungus F. oxysporum ( Corrales Escobosa et al, 2011 ) and human pathogenic fungus C. albicans ( Ku et al, 2017 ) and A. fumigatus ( Grahl et al, 2011 ).…”

“…Our data revealed a decrease in ethanol production and an increase in acetaldehyde production in Δ MaADH1 culture, indicating a block in ethanol production from acetaldehyde under hypoxic condition when the MaADH1 gene was impaired. This physiological function was consistent with earlier reports in some fungi, such as S. cerevisiae ( Leskovac et al, 2002 ; de Smidt et al, 2008 ), Candida maltose ( Lin et al, 2010 ), F. oxysporum ( Corrales Escobosa et al, 2011 ), M. anisopliae ( Callejas-Negrete et al, 2015 ), and M. circinelloides ( Rangel-Porras et al, 2005 ).…”

“…Few past studies were reported on the function of alcohol dehydrogenase in entomopathogenic fungi. In M. anisopliae, ADH1 is expressed during infection process and required for virulence ( Callejas-Negrete et al, 2015 ). In this study, we explored the function of the MaADH1 gene by targeted disruption in M. acridum .…”

Genome‐wide identification of pathogenicity, conidiation and colony sectorization genes in Metarhizium robertsii

“…Consistent with an important role for anaerobic respiration during infection, disruption of a transketolase (X797_002094), involved in the pentose phosphate pathway, and zinc‐dependent alcohol dehydrogenase (X797_0117835) and pyruvate decarboxylase (anaerobic fermentation) (X797_002478) also attenuated virulence. A zinc‐dependent alcohol dehydrogenase has already been shown to be required for full virulence in M. anisopliae presumably because a fermentative metabolism facilitates colonisation of the oxygen‐poor haemolymph (Callejas‐Negrete et al ., ).…”

A novel zinc‐binding alcohol dehydrogenase 2 from Arachis diogoi, expressed in resistance responses against late leaf spot pathogen, induces cell death when transexpressed in tobacco

“…A zinc-binding alcohol dehydrogenase is induced in Oomycete plant pathogenic fungus, Phytophthora infestans during the infection process [7]. Recently, another member of the MDR protein superfamily, an alcohol dehydrogenase gene of the fungus Metarhizium anisopliae, was found to be expressed during insect colonization and invasion [8].…”