Copper tolerance of the soft-rot fungus Phialophora malorum grown in-vitro revealed by microscopy and global protein expression

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Highlights

  • The soft rot fungus Phialophora malorum was studied to understand its mechanisms of high Cu-tolerance.

  • P. malorum mycelia grown in 0.064% CuSO4 media bound Cu intracellularly in deposits.

  • In 0.64% Cu cultures, Cu adsorbed as extracellular precipitates on hyphal surfaces.

  • Proteomics showed up-regulation of proteins in Cu-media indicating their likely involvement in Cu-tolerance.

Abstract

In this study, we used proteomics in conjunction with microscopy to study differences in the proteome and hyphal morphology of the copper tolerant soft rot fungus Phialophora malorum grown in media containing 0.064, 0.64% Cu as CuSO4. Unique proteins were found in the control and the copper-treated (0.064% CuSO4) samples. Of five unique proteins found in the 0.064% CuSO4 treated cultures, ATP synthase subunit alpha is considered to play an important role in copper tolerance as it is involved in the biosynthesis of fatty acids and steroids and may relate to morphological changes associated with hyphal cell walls of the fungus when grown in the presence of copper. ICP-AES analyses showed total mycelial Cu to increase with media Cu with 5246- and 16535 μg Cu/g dry wt mycelia respectively found in 0.064 and 0.64% Cu-cultures after 6 weeks growth. Rubeanic acid staining of 0.064% mycelia showed Cu bound in intracellular bodies while most Cu was found as extracellular precipitates on the surfaces of hyphae in 0.64% Cu. SEM showed hyphal surfaces enrobed in fibrillar polysaccharides to which Cu was bound.

Introduction

The anamorphic Phialophora genus and closely related genera Cadophora and Lecythophora include some of the most well-known fungal species causing soft rot decay of copper-treated wood exposed in contact with soil or submerged in water. Studies have shown several Phialophora spp. isolated from preservative treated wood (i.e. containing Cu/Zn/Cr/As; Cu/Cr/As (Henningsson and Nilsson, 1976; Nilsson and Henningsson, 1978) to possess considerable tolerance to copper (as CuSO4 or micronized copper) (Daniel and Nilsson, 1988; Karunasekera and Daniel, 2013). Copper tolerance has frequently been considered as providing certain wood degrading fungi with a competitive advantage for colonizing and degrading copper-treated wood in-service (Green and Clausen, 2005). Previous ultrastructural and x-ray microanalytical studies of Phialophora mutabilis have shown both intracellular- and extracellular localization of copper (i.e. bound to fungal cell wall and extracellular slime) (Daniel and Nilsson, 1989) suggesting varied mechanisms for tolerating external copper when grown in-vitro and when degrading copper-treated wood. The actual mechanism(s) of copper tolerance (biochemical/structural or both) involved and how Cu-detoxification may occur is however, poorly understood.

Brown rot fungi also exhibit copper tolerance when exposed to CuSO4 or copper-based preservatives in laboratory assays (Clausen et al., 2000; Green and Clausen, 2001; Green and Clausen, 2005). Studies on brown rot fungi have indicated that excess production of oxalic acid to be a primary means employed by the organisms for inactivating copper in preservative treated wood (Green and Clausen, 2005). In contrast to brown rot fungi however, copper tolerant soft rot fungi like P. malorum are not known to produce excess oxalic acid when grown on copper-treated wood.

During wood decay, fungi can express a variety of proteins involved in general fungal metabolism as well as secrete a variety of cell wall breakdown enzymes (e.g. hydrolases, oxidoreductases; Kang et al., 2009; Daniel, 2014, 2016). A number of proteins have also been implicated in copper detoxification of accumulated metal ions by intracellular complexation/immobilization processes by binding to intracellular protein chelators like metallothionein (e.g. Schwartz et al., 2013). Changes in the dynamics of protein turn-over can be studied using proteomics which provides information (both qualitative/quantitative) on the global expression of proteins produced by organisms at the time of extraction (Philips and Bogyo, 2005; Kang et al., 2009). Proteomics has for example, been used previously for providing insights on the changes in proteins produced during wood decay by white rot fungi (Martinez et al., 2004).

Proteomic approaches have been used previously to investigate copper response proteins in plants. For example, studies with rice showed most of the proteins effected by copper were also involved in photosynthesis (Ahsan et al., 2009). A significant number of antioxidant and defense-related proteins were also involved in Cu-induced oxidative stress responses. In a study on copper and herbicide stress on Arabidopsis subjected to copper and herbicide stress, Smith et al. (2004) found a number of glutathione S-transferase (GST) proteins that may have specific functions in detoxifying copper in plants cells. Proteomic studies on Cannabis sativa under copper stress have shown aldo/keto reductases upregulated and possibly the first proteins to interact with the reduction of CuII to CuI making them available for partner proteins to interact or induce sequestration in intracellular vacuoles (Ahsan et al., 2009).

In the present study, we used a correlated light microscopy, staining, electron microscopy, ICP-AES (Inductively Coupled Plasma-Atomic Emission Spectrometry) and proteomic approach to observe morphological changes in fungal hyphae and investigate changes in protein metabolism in P. malorum under normal- and copper supplemented growth conditions. P. malorum was chosen because of its strong copper tolerance when grown in-vitro with copper-containing media (CuSO4 in agar/liquid cultures) (Nilsson and Henningsson, 1978; Daniel and Nilsson, 1988; Karunasekera and Daniel, 2013). P. malorum was grown at non-lethal levels in CuSO4 supplemented media and studied using LC-MS-MS to determine whether changes in global proteins were induced through copper stress. Since the genome of P. malorum is unknown, both the Swissprot (includes all fungal species) and genome sequence database of Chaetomium globosum, a ubiquitous and well-known soft rot wood degrading fungus were also used for comparison.

Section snippets

Fungal cultures and culture development

Phialophora malorum (strain 211-C-15-1) [M. N. Kidd & A. Beaumont] McColloch was obtained from the culture collection maintained at the Department of Forest Products/Wood Science (now Department of Biomaterials and Technology/Wood Science), Uppsala, Sweden where it is routinely grown on 2.5% w/v malt extract agar (MEA) at 20 °C on Petri dishes. The strain was previously isolated from 2% w/v chromated copper arsenate (CCA) treated poles. P. malorum liquid cultures were established using Abrams

Observations on fungal mycelia

Typical examples of shake flasks with mycelia development (after 6 weeks) in control, 0.064 and 0.64% Cu are shown in Fig. 1. Most characteristic was the green colour developed in the 0.064% Cu cultures and filtered mycelia (Fig. 1b, e) in comparison to the control (creamy-orange mycelia; Fig. 1a, d) and blue-green colour of the 0.64% Cu (Fig. 1c, f) cultures; the colour already developing after 2–3 weeks of culture. Copper was present in both the culture media (i.e. in solution) and bound to

Conclusions

  • Studies on the Cu-tolerant soft rot fungus P. malorum confirmed its ability to grow in-vitro at 0.064 and 0.64% Cu;

  • Using rubeanic acid, copper was visualized intracellularly as green-black deposits in low Cu-containing cultures as well as extracellular precipitates coating hyphae in high Cu-media;

  • SEM confirmed abundant precipitates on the surface of P. malorum hyphae associated with extracellular fibrillar polysaccharide materials;

  • Total Cu levels of P. malorum fungal mycelia increased by ca.

Acknowledgements

This study was supported by the Swedish Research Council for Environment, Agriculture Sciences and Spatial Planning (FORMAS Grants: 2011-416; 2011-6383-19675 and (2008-1399) The SRC (VR) grant 621-2015-4870 (JB) and the staff at MS Proteomics facility at Uppsala University Magareta Ramstöm, Katarina Hörnaesus are acknowledged.

References (23)

  • G. Daniel
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