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Modulation of ethanol stress tolerance by aldehyde dehydrogenase in the mycorrhizal fungus Tricholoma vaccinum

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Abstract

We report the first mycorrhizal fungal aldehyde dehydrogenase gene, ald1, which was isolated from the basidiomycete Tricholoma vaccinum. The gene, encoding a protein Ald1 of 502 amino acids, is up-regulated in ectomycorrhiza. Phylogenetic analyses using 53 specific fungal aldehyde dehydrogenases from all major phyla in the kingdom of fungi including Ald1 and two partial sequences of T. vaccinum were performed to get an insight in the evolution of the aldehyde dehydrogenase family. By using competitive and real-time RT-PCR, ald1 is up-regulated in response to alcohol and aldehyde-related stress. Furthermore, heterologous expression of ald1 in Escherichia coli and subsequent in vitro enzyme activity assay demonstrated the oxidation of propionaldehyde and butyraldehyde with different kinetics using either NAD+ or NADP+ as cofactors. In addition, overexpression of ald1 in T. vaccinum after Agrobacterium tumefaciens-mediated transformation increased ethanol stress tolerance. These results demonstrate the ability of Ald1 to circumvent ethanol stress, a critical function in mycorrhizal habitats.

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Acknowledgments

This research was supported by Max Planck Society, Germany, and by the German Research Foundation with Graduate School 214. We are grateful to Dr. Matthias Gube and Dr. Susann Erdmann for phylogenetic analyses and technical assistance with real-time RT-PCR experiments, respectively. We thank Petra Mitscherlich for general technical help.

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  1. Institute of Microbiology, Friedrich Schiller University, Neugasse 25, 07743, Jena, Germany

    Theodore Asiimwe, Katrin Krause, Ines Schlunk & Erika Kothe

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  1. Theodore Asiimwe
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  2. Katrin Krause
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  3. Ines Schlunk
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  4. Erika Kothe
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Corresponding author

Correspondence to Erika Kothe.

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T. Asiimwe and K. Krause contributed equally to this work.

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Supplemental Table S1

Oligonucleotides used for isolating ald1 and generating gene expression constructs (DOC 49 kb)

Supplemental Table S2

Analyses of ALDH superfamily and fungal ALDH family members retrieved from fungal genomes of basidiomycota, ascomycota, zygomycota, and chytridiomycota reveal fungal ALDH duplication (DOC 54 kb)

Supplemental Fig. S1

Nucleotide and amino acid sequence of ald1 and its flanking sequences. The ald1 ORF is 2,448 bp in length, interrupted by 16 introns. Amino acids and nucleotides are represented by bold and non-bold letters, respectively. Seven putative stress response elements (black boxes), one GC-box-like element (dark gray boxes), two CAAT box-like elements (gray boxes), and four TATA box-like element (light gray box) were putatively identified in the promoter region. The putative polyadenylation signal is marked with an arrow in 3′ non-coding sequence. Start and stop codons are underlined (JPEG 220 kb)

High resolution image (TIFF 413 kb)

Supplemental Fig. S2

Alignment of the deduced Ald1 amino acid sequence (1) with other fungal ALDH sequences: Laccaria bicolor (2: EDQ99417), Coprinopsis cinerea (3: EAU87113), Agaricus bisporus (4: O74187), Ustilago maydis (5: EAK83639), and Aspergillus niger (6: P41751). The horizontal lines represent 10 conserved motifs of ALDH superfamily while the conserved amino acid residues are shaded. One putative NAD-binding domain proposed for ALDHs (gray box) and two general NAD(P)-binding domains (black boxes) were identified. The intron positions are marked with vertical lines (JPEG 225 kb)

High resolution image (TIFF 340 kb)

Supplemental Fig. S3

Alignment of the deduced Ald1 amino acid sequence with a broader range of other specific fungal ALDHs. A total of 53 specific fungal ALDHs and four ALDH sequences of other organisms (outgroup) were aligned using MAFFT v6 (Katoh and Toh 2008) and are listed in the same order as shown in Fig. 2 (JPEG 1826 kb)

High resolution image (TIFF 335 kb)

Fig. S4

Nucleotide and amino acid sequence of partial sequences of ald2 (upper sequence), a 1,289-bp genomic DNA fragment including eight introns, and ald3 (lower sequence), a 721-bp cDNA fragment with stop codon in bold (JPEG 295 kb)

High resolution image (TIFF 564 kb)

Supplemental Fig. S5

Southern blot analysis of T. vaccinum wild type and transformants. Genomic DNA was digested with restriction enzymes, and hybridized, using stringent conditions, with a 1-kb hph probe. No signal was obtained from untransformed T. vaccinum (lane 1). Transformants T1–T5 (lanes 2–6) showed single copy and multicopy integration (JPEG 52 kb)

High resolution image (TIFF 1785 kb)

Supplemental Fig. S6

PCR analysis of T. vaccinum transformants. PCR targeting the 1.0-kb hph gene (a) was carried out both on gDNA of transformants with (pBGaldh1SeGFP: lanes 2–5) and without egfp gene fusion (pBGaldh1: lanes 10–12). Lanes 1 and 9 show DNA size maker λPstI, lane 6 wild type, lane 7 negative control without DNA, and lane 8 positive control with vector pBGgHg. PCR targeting the 0.7-kb egfp gene (b) was carried out only on transformants with egfp gene fusion (pBGaldh1SeGFP: lanes 2–5). Lane 1 shows DNA size maker λPstI, lane 6 wild type, lane 7 negative control without DNA, and lane 8 positive control with vector pBGgHg (JPEG 14 kb)

High resolution image (TIFF 642 kb)

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Asiimwe, T., Krause, K., Schlunk, I. et al. Modulation of ethanol stress tolerance by aldehyde dehydrogenase in the mycorrhizal fungus Tricholoma vaccinum . Mycorrhiza 22, 471–484 (2012). https://doi.org/10.1007/s00572-011-0424-9

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