International Biodeterioration & Biodegradation 105 (2015) 90e96 Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod Insights into the mechanism of copper-tolerance in Fibroporia radiculosa: The biosynthesis of oxalate * Katie M. Ohno a, , Carol A. Clausen a, Frederick Green III a, Susan V. Diehl b a USDA Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI 53726, USA b Mississippi State University, Department of Forest Products, 201 Locksley Way, Starkville, MS 39759, USA article info abstract Article history: Copper is currently used as the key component in wood preservatives despite the known tolerance of Received 18 May 2015 many brown-rot Basidiomycetes. Copper-tolerant fungi, like Fibroporia radiculosa, produce and accu- Received in revised form mulate high levels of oxalate when exposed to copper. To gain insight into the mechanism of oxalate 12 August 2015 production, four F. radiculosa isolates decaying untreated and 1.2% ammoniacal copper citrate-treated Accepted 24 August 2015 wood were evaluated for the differential expression of citrate synthase, isocitrate lyase, glyoxylate de- Available online xxx hydrogenase, a succinate/fumarate antiporter, and a copper resistance-associated ATPase pump. Samples were analyzed at 2, 4, 6, and 8 weeks for oxalate production and gene expression. ATPase pump Keywords: Copper-tolerance expression increased in the presence of copper when initial oxalate concentrations were low, suggesting Brown-rot decay it functions in helping the fungus adapt to the copper-rich environment by pumping toxic copper ions Oxalate out of the cell. A connection in expression levels between citrate synthase, the succinate/fumarate Fibroporia antiporter isocitrate lyase, and glyoxylate dehydrogenase for the four isolates was found suggesting the Antrodia production of oxalate originates in the mitochondrial TCA cycle via citrate synthase, shunts to the Gene expression glyoxysomal glyoxylate cycle via the succinate/fumarate antiporter, moves through a portion of the glyoxylate cycle (isocitrate lyase), and ultimately is made in the cytoplasm (gyloxylate dehydrogenase). Published by Elsevier Ltd. 1. Introduction in decayed wood treated with copper-based preservatives (Freeman and McIntyre, 2008). Another important factor related to The mechanism of copper-tolerance in brown-rot fungi has copper tolerance is the presence of a low pH environment, which is often been associated with the production of oxalate (Murphy and also stimulated by oxalate production (Clausen et al., 2000; Humar Levy, 1983; Sutter et al., 1983; Daniel, 1994; Leithoff et al., 1995; et al., 2001; Clausen and Green, 2003; Green and Clausen, 2003, Pohleven et al., 2002; Green and Clausen, 2003, 2005; Hastrup 2005). Choi et al. (2002) showed spores from copper-tolerant or- et al., 2006; Freeman and McIntyre, 2008; Arango et al., 2009; ganisms did not germinate in the presence of copper because the Schilling and Inda, 2011). High extracellular accumulation of oxa- spores lack the ability to actively produce oxalate (Freeman and late initiates the precipitation of copper into the insoluble form of McIntyre, 2008). Thus, oxalate accumulation and precipitation of copper oxalate forming crystals and renders the copper ion inert copper oxalate crystals play an important role in the mechanism of (Pohleven et al., 1999; De Groot and Woodward, 1999; Humar et al., copper tolerance. 2002). Both copper oxalate and calcium oxalate crystals are present The production of oxalate occurs by numerous classes of fungi and many accumulate it in high concentrations. The majority of brown-rot fungi produce oxalate in detectable amounts; however, Abbreviations: CS, citrate synthase; ICL, isocitrate lyase; GLOXDH, glyoxylate its accumulation is limited in white-rot fungi most likely due to dehydrogenase; ANTI, succinate/fumarate antiporter; ATPase, copper resistance- oxalate decarboxylase (Hastrup et al., 2006). In white-rot fungi, associated ATPase pump; TCA, tricarboxylic acid cycle; GLOX, glyoxylate cycle; intracellular oxalate decarboxylase breaks down oxalate to carbon OXA, oxaloacetase; UN, untreated wood; CC, 1.2% ammoniacal copper citrate treated dioxide and formate (Espejo and Agosin, 1991; Hastrup et al., 2006). wood; RH, relative humidity; SP, Southern pine; CDS, coding sequence; RT-qPCR, In brown-rot fungi, oxalate has the potential to accumulate as a Reverse Transcriptase Quantitative Polymerase Chain Reaction; HK, actin; CT, threshold values. function of fungal respiration (Schilling and Jellison, 2005). Some * Corresponding author. brown-rot fungi produce oxalate for initial decay but do not E-mail address: [email protected] (K.M. Ohno). http://dx.doi.org/10.1016/j.ibiod.2015.08.016 0964-8305/Published by Elsevier Ltd. K.M. Ohno et al. / International Biodeterioration & Biodegradation 105 (2015) 90e96 91 þ accumulate significant levels during the decay process, while other the cytoplasm and functions to pump copper ions (Cu2 ) out of the brown-rot fungi utilize oxalate throughout the decay process cell (Fig. 1). resulting in steady levels of accumulation (Goodell, 2003). Co-biocides are needed to prevent these copper-tolerant or- Several pathways have been proposed for the biosynthesis of ganisms from circumventing the copper-based preventative mea- oxalate in brown-rot fungi; however, in recent years, many re- sures in use today; therefore, it is critical that these mechanisms be searchers have linked it to the tricarboxylic acid (TCA) and glyox- investigated. Knowing the exact mechanisms and specific re- ylate (GLOX) cycles (Dutton and Evans, 1996; Munir et al., 2000; quirements of organisms involved in copper tolerance would give Munir et al., 2001; Yoon et al., 2002; Schilling and Jellison, 2005; insight into the physiological intricacies of copper tolerance. A Hastrup et al., 2006). The TCA cycle is localized in the mitochon- better understanding of this mechanism is crucial in developing dria while the GLOX cycle takes place in the glyoxysome (Dutton targeted methods to control these particular organisms. The pur- and Evans, 1996). Munir et al., (2001) investigated oxalate biosyn- pose of this study is to gain insight into the biosynthesis of oxalate thesis in the copper-tolerant fungus Tyromyces palustris. This study by which the brown-rot decay fungus, F. radiculosa, regulates hypothesized that 1 mol of glucose is converted to 2 mol of oxalic tolerance of copper-treated wood. Specific objectives of this study acid rather than producing the traditional CO2 from the TCA cycle. are to examine select F. radiculosa isolates during decay of un- Through their work, it was discovered that there are two major treated and copper-treated wood for the differential expression of oxalate producing enzymes, oxaloacetase (OXA) and glyoxylate genes associated with oxalate production, and to propose a po- dehydrogenase (GLOXDH). Munir et al. (2001) discovered quanti- tential pathway involved in the production of oxalate. fiable OXA and GLOXDH enzymatic activity, with OXA synthesizing more oxalate than GLOXDH in T. palustris. 2. Materials and methods Using this discovery as a model, we hypothesized that Fibroporia radiculosa, another copper-tolerant fungus, may use a similar 2.1. Sample preparation mechanism for oxalate production. Initial enzymatic analysis showed quantifiable GLOXDH, but not OXA, in four isolates of F. Four isolates of F. radiculosa (Peck) Gilb. & Ryvarden were used radiculosa (unpublished data). These results were related to the in this study: FP-90848-T, L-9414-SP, L-11659-SP, and TFFH 294 genome analysis of F. radiculosa isolate TFFH 294 showing up- (USDA Forest Products Laboratory, Madison, WI). Cultures were regulation of GLOXDH, not OXA, in the early stages of decay maintained on 2% Malt Extract Agar (BD, Fisher Scientific). Isolates (Tang, 2011). In addition to GLOXDH, Tang et al. (2012) found up- were subjected to an AWPA Standard E10-15 (2015) decay chamber regulation of citrate synthase (CS), a succinate-fumarate anti- at 26.7 C and 70% relative humidity (RH) for 2 weeks to allow porter (ANTI), isocitrate lyase (ICL), and a copper resistance- initial colonization (AWPA, 2015). Southern pine (SP) test wafers associated ATPase pump (ATPase) during the early stages of measuring 70 mm  22 mm  4mm(t r  l) were vacuum decay. Citrate synthase is found in the mitochondria as part of the treated with 1.2% ammoniacal copper citrate (CC) for 40 min TCA cycle and converts oxaloacetate to citrate (Fig. 1). The succi- at À172 kPa. After initial colonization, two CC-treated SP test wafers nate/fumarate antiporter, found in the cytoplasm, functions as a were placed directly on top of the colonized feeder strips and shuttle from isocitrate in the TCA cycle to isocitrate in the GLOX incubated at 26.7 C 70%RH for 8 weeks. Untreated SP test wafers cycle which is located in the glyoxysome. The ATPase pump is not were also exposed to the four isolates and incubated at 26.7 C, 70% associated with either the TCA or GLOX cycles; instead, it is found in RH for 8 weeks. Both untreated and CC-treated SP test wafers Fig. 1. Genes (italics) up-regulated in early decay. (A) TCA cycle; (B) GLOX cycle. Light gray box indicates TCA cycle in the mitochondria; dark gray box indicates GLOX cycle in the glyoxysome (Tang, 2011). 92 K.M. Ohno et al. / International Biodeterioration & Biodegradation 105 (2015) 90e96 exposed to the four isolates were harvested at weeks