Differential stress-induced regulation of two quinone reductases in the brown rot basidiomycete Gloeophyllum trabeum

Abstract

Quinone reductases (QRDs) have two important functions in the basidiomycete Gloeophyllum trabeum, which causes brown rot of wood. First, a QRD is required to generate biodegradative hydroxyl radicals via redox cycling between two G. trabeum extracellular metabolites, 2,5-dimethoxyhydroquinone (2,5-DMHQ) and 2,5-dimethoxy-1,4-benzoquinone (2,5-DMBQ). Second, because 2,5-DMBQ is cytotoxic and 2,5-DMHQ is not, a QRD is needed to maintain the intracellular pool of these metabolites in the reduced form. Given their importance in G. trabeum metabolism, QRDs could prove useful targets for new wood preservatives. We have identified two G. trabeum genes, each existing in two closely related, perhaps allelic variants, that encode QRDs in the flavodoxin family. Past work with QRD1 and heterologous expression of QRD2 in this study confirmed that both genes encode NADH-dependent, flavin-containing QRDs. Real-time reverse transcription PCR analyses of liquid- and wood-grown cultures showed that qrd1 expression was maximal during secondary metabolism, coincided with the production of 2,5-DMBQ, and was moderately up-regulated by chemical stressors such as quinones. By contrast, qrd2 expression was maximal during fungal growth when 2,5-DMBQ levels were low, yet was markedly up-regulated by chemical stress or heat shock. The total QRD activity in lysates of G. trabeum mycelium was significantly enhanced by induction beforehand with a cytotoxic quinone. The promoter of qrd2 contains likely antioxidant, xenobiotic, and heat shock elements, absent in qrd1, that probably explain the greater response of qrd2 transcription to stress. We conclude from these results that QRD1 is the enzyme G. trabeum routinely uses to detoxify quinones during incipient wood decay and that it could also drive the biodegradative quinone redox cycle. However, QRD2 assumes a more important role when the mycelium is stressed.

FIG. 1.

Amino acid sequence comparison between G. trabeum QRD1 (Gt QRD1, AAL67860), G. trabeum QRD2 (Gt QRD2), P. chrysosporium QRD (Pc QRD, AAD21025), A. thaliana QRD (At QRD, BAA97523), and T. versicolor QRD (Tv QRD, AAG53945). Boxed areas indicate conserved sequences.

FIG. 2.

Native PAGE of recombinant QRD2. Lanes: A, 0.5 μg of enzyme stained with Coomassie blue; B, 0.5 μg of enzyme detected by immunoblotting against an anti-myc antibody; C, 2.5 μg of enzyme stained for QRD activity with NADH, menadione, and MTT. No staining occurred when menadione was omitted.

FIG. 3.

Expression of G. trabeum qrd1 and qrd2 as a function of growth conditions and time in culture. (A) Rate of 2,6-DMBQ reduction by one intact mycelial mat from a liquid culture (triangles), concentration of 2,5-DMBQ plus 2,5-DMHQ in one liquid culture (squares), and dry weight of one mycelial mat from a liquid culture (circles). Error bars show the standard deviations for triplicate samples. (B) Relative expression of qrd1 (black bars) and qrd2 (white bars) in liquid cultures and in spruce wood blocks, as determined by Taqman real-time RT-PCR. Gene expression was normalized to gpd expression and calibrated to the value for qrd1 on day 2, which was assigned a value of 1. Error bars show the standard deviations for triplicate samples. (C) Relative expression of qrd1, qrd2, and gpd in 4- and 10-day-old liquid cultures, as determined by Northern hybridization.

FIG. 4.

Induction of qrd1 (black bars) and qrd2 (white bars) in G. trabeum, as determined by Taqman real-time RT-PCR. Gene expression in treated samples is given as the fold change relative to expression in untreated controls. 2,6-DMBQ, 4-day-old liquid cultures were treated with 50 or 500 μM 2,6-DMBQ for 1 h; phase 2 inducers, 4-day-old liquid cultures were treated with 500 μM dl-sulforaphane (Slf) or 500 μM BHA for 1 h; heat shock, 4-day-old liquid cultures or 8-day-old wood block cultures were treated at 45°C for 1 h. The bars labeled Wood Rec (recovery) show relative gene expression after heat-shocked wood block cultures had been returned to the normal incubation temperature of 31°C for 2 h. Error bars show the standard deviations for triplicate samples.