A dehydration-inducible gene in the truffle Tuber borchii identifies a novel group of dehydrins

Abstract

Background: The expressed sequence tag M6G10 was originally isolated from a screening for differentially expressed transcripts during the reproductive stage of the white truffle Tuber borchii. mRNA levels for M6G10 increased dramatically during fruiting body maturation compared to the vegetative mycelial stage.

Results: Bioinformatics tools, phylogenetic analysis and expression studies were used to support the hypothesis that this sequence, named TbDHN1, is the first dehydrin (DHN)-like coding gene isolated in fungi. Homologs of this gene, all defined as "coding for hypothetical proteins" in public databases, were exclusively found in ascomycetous fungi and in plants. Although complete (or almost complete) fungal genomes and EST collections of some Basidiomycota and Glomeromycota are already available, DHN-like proteins appear to be represented only in Ascomycota. A new and previously uncharacterized conserved signature pattern was identified and proposed to Uniprot database as the main distinguishing feature of this new group of DHNs. Expression studies provide experimental evidence of a transcript induction of TbDHN1 during cellular dehydration.

Conclusion: Expression pattern and sequence similarities to known plant DHNs indicate that TbDHN1 is the first characterized DHN-like protein in fungi. The high similarity of TbDHN1 with homolog coding sequences implies the existence of a novel fungal/plant group of LEA Class II proteins characterized by a previously undescribed signature pattern.

Figure 1

Panel A, TbDHN1 deduced amino acid sequence and dehydrin 6 from Hordeum vulgare. New CSPs of TbDHN1 (see text for description) [GenBank:DQ308610] are red background coloured; Prosite plant dehydrin signature patterns are green background coloured in dehydrin 6 [GenBank:AAD02257]: S-segment in light green and K-segments in dark green. Sequence fragments aligned by blastx are in upper cases (see panel B). Panel B, Basic local alignment between TbDHN1 and dehydrin 6 from Hordeum vulgare. The best basic local alignment (E-value = 4e-25) was built by blastx between amino acids 75 and 305 of TbDHN1 and amino acids 82 and 324 of dehydrin 6 [GenBank:AAD02257]. New CSPs of TbDHN1 are red background coloured.

Figure 2

Distribution of the conserved signature pattern (CSP) within TbDHN1 and its homolog sequences. CSP is indicated with red boxes.

Figure 3

Bayesian posterior probability tree. Neighbor-Joining bootstrap values, grater than 50%, are shown above branches where available; Bayesian posterior probabilities are shown below branches. A full circle denotes the newly classified sequences, while the other LEA proteins were already classified by Wise [12]. Fungal LEA Class III proteins are marked with an asterisk. Newly generated TbDHN1 sequence is in bold. Bar = posterior probability.

Figure 4

Nucleotide and amino acid sequences of TbDHN1. Genomic and mRNA were sequenced by genomic and cDNA library screening. The coding sequence is shown in bold lower case; the 79-bp-long intron is underlined. The putative TATA box is indicated by an open box. indicates the transcription initiator. Stop codons are marked with an asterisk. AA amino acids that form the CSP; AA amino acids that form the repeated block of 46 amino acids.

Figure 5

Panel A, Southern Blot analysis of TbDHN1. T. borchii genomic DNA digested with HindIII (H, Lane 1), KpnI (K, Lane 2) and SmaI (S, Lane 3) was probed with an ECL-labelled TbDHN1 cDNA probe. Panel B, Gel-blot analysis of the TbDHN1 transcript under salinity and low temperature stress. Induction (left): mRNA extracted from mycelia grown for 30 days on control medium (CM; Lane 1); treated for 30 min in NaCl 0.4 M (NaCl 30 min; Lane 2); treated for 24 h in NaCl 0.4 M (NaCl 24 h; Lane 3); treated for 48 h at 4°C (cold shock 48 h; Lane 4). Repression (right): mRNA extracted from mycelia grown for 30 days on CM (CM; Lane 1), treated for 29 h in NaCl 0.4 M (NaCl 29 h; Lane 2); treated for 24 h in NaCl 0.4 M and then transferred on CM for 5 h (NaCl 24 h→ CM 5h; Lane 3). Signals obtained by ³²P -labeled TbDHN1 probe (top) were normalized using the rRNA 28 S probe as internal standard (bottom).