Accepted Manuscript Plant biomass degradation by fungi Miia R. Mäkelä, Nicole Donofrio, Ronald P. de Vries PII: DOI: Reference:

S1087-1845(14)00153-4 http://dx.doi.org/10.1016/j.fgb.2014.08.010 YFGBI 2722

To appear in:

Fungal Genetics and Biology

Received Date: Revised Date: Accepted Date:

10 July 2014 19 August 2014 25 August 2014

Please cite this article as: Mäkelä, M.R., Donofrio, N., de Vries, R.P., Plant biomass degradation by fungi, Fungal Genetics and Biology (2014), doi: http://dx.doi.org/10.1016/j.fgb.2014.08.010

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Plant biomass degradation by fungi Miia R. Mäkeläa, Nicole Donofriob, Ronald P. de Vriesc,d# a

Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, 00014

Helsinki, Finland b c

Department of Plant and Soil Sciences, University of Delaware, Newark, DE, 19716, U.S.A.

Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht,

The Netherlands d

Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT Utrecht, The

Netherlands #

Corresponding author. Fax: +31 302152097; E-mail address: [email protected]

ABSTRACT Plant biomass degradation by fungi has implications for several fields of science. The enzyme systems employed by fungi for this are broadly used in various industrial sectors such as food & feed, pulp & paper, detergents, textile, wine, and more recently biofuels and biochemicals. In addition, the topic is highly relevant in the field of plant pathogenic fungi as they degrade plant biomass to either gain access to the plant or as carbon source, resulting in significant crop losses. Finally, fungi are the main degraders of plant biomass in nature and as such have an essential role in the global carbon cycle and ecology in general. In this review we provide a global view on the development of this research topic in saprobic ascomycetes and basidiomycetes and in plant pathogenic fungi and link this to the other papers of this special issue on plant biomass degradation by fungi.

Keywords: plant biomass degradation; ascomycetes; basidiomycetes; plant pathogens; industrial applications

1. Introduction Plant biomass degradation has been a topic of research in fungi for many decades. Fungi are highly efficient degraders of plant biomass due to the fact that for many fungi this is the 1

predominant carbon source in their natural biotope. Plant biomass is the most abundant source of carbon on earth, but is highly varied in composition depending on plant species and tissue, season and geographical location. It consists mainly of polymers, in particular polysaccharides and lignin. As fungi cannot take up polymeric compounds they produce extracellular enzymes that degrade the polymers to mono- and short oligomers that are imported and metabolized in the cells. Applications of plant biomass degrading enzymes in industry date back to the early 1900’s (e.g. hydrolysis of starch and maltose (Okada, 1916)) accompanied by an increasing research effort into their characterization and production. Research into these enzymes has been mainly driven by societal interest. Plant biomass is not only the main carbon source for many fungi, but it is also the starting point of many foods and other products that are used in human society. Industrial processing of plant biomass involves modification or (partial) degradation of the plant polymers and the microbial enzymes capable of doing this have therefore received significant interest in industry. Fungi are especially attractive producers of such enzymes due to their ability to secrete large amounts of proteins and their broad enzyme sets related to plant biomass degradation. Fungal enzymes have applications related to for example baking, pulp & paper, animal feed, bio-fuels and bio-chemicals, textiles, detergents, beverages and alternative sweeteners (de Vries et al., 2010). Production of fungal enzyme cocktails by the large enzyme producers such as Novozymes, DSM and DuPont (formerly Danisco-Genencor) is mainly performed using a small number of ascomycete fungi (Trichoderma reesei, several Aspergillus species) (Pariza and Johnson, 2001), which have shown to possess good fermentation characteristics. Substantial strain improvement programs have resulted in strains that can produce enzymes at more than 30 g/l scale (Demain and Vaishnav, 2009). The use of these fungi is mainly aimed at the polysaccharide fraction of plant biomass. In contrast, lignin is a more recalcitrant polymer that has been mostly considered a waste product and a hindrance to efficient polysaccharide degradation. Most ascomycete fungi have no or very little ability to depolymerize lignin, but several basidiomycete fungi, in particular the white rot fungi, are very efficient in degrading this aromatic polymer (Hatakka, 2001). For many years the industrial view was that you can make anything you want out of lignin but money, but lately new approaches are changing this view.

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A second area of fungal research in which plant degradation has received attention, although to a lesser extent is plant pathogenicity. Fungal plant pathogens can cause devastating crop-losses. For instance, the rice pathogen Magnaporthe oryzae alone can annually cause a loss of 30% of rice (Talbot, 2003). Pathogenicity of plants by fungi often involves degradation or modification of the plant cell wall, and therefore this process is an important factor in this field (Faulkner and Robatzek, 2012). In this issue a comparative study of the plant cell wall degrading machinery of four oomycetes demonstrates significant differences between them in terms of genome content and ability to use different plant biomass components as a substrate (Brouwer et al., 2014). Recent developments in fungal research (genomics, transcriptomics, proteomics) have provided a much deeper insight into the mechanisms of fungi related to plant biomass degradation. In the past, studies often emphasized the plant biomass converting enzymes. However, now it is possible to link these enzymes to the metabolic pathways employed by fungi to convert the monomers that were released, and to the regulatory systems that drive this process. Recently, also other aspects of plant biomass degradation by fungi are attracting attention, such as in ant-fungus symbiosis (Lange and Grell, 2014) and the use of mixed cultivations to produce more efficient enzyme cocktails (Hu et al., 2011; Qi-He et al., 2011). Also non-enzymatic functions can affect plant biomass degradation. For example, one of the fungal metabolites, oxalate, has several roles in fungal physiology, including the promotion of lignocellulose decay (Kuan and Tien, 1993; Shimada et al., 1997; Urzúa et al., 1998; Varela and Tien, 2003). To ensure sufficient oxalate to enhance biomass decay without getting to toxic levels, its production and conversion by fungal enzymes is highly controlled (Mäkelä et al., 2009; Mäkelä et al., 2010). For this, fungal genomes often contain several genes encoding oxalate-converting enzymes that can be differentially regulated (Mäkelä et al., 2014b). Due to the highly complex nature of this field, additional factors that affect the degradation of plant biomass will be discovered. For example, starvation (van Munster et al., 2014) and the role of loosenins (Suzuki et al., 2014), signal transduction (Brown et al., 2014), the link between substrate structure and enzyme production (Peciulyte et al., 2014), the analysis of different glycoforms of the enzymes (Qu et al., 2014) and the fungal phosphoproteome (Yi et al., 2014) are among those addressed in this special issue.

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This review provides an introduction to the various aspects of this topic that are addressed in the other papers in this special issue, but also presents the development and current status of this highly important field of fungal research.

2. Plant biomass components and the enzymes acting on them. The main plant biomass components are polysaccharides and the aromatic polymer lignin. Plant polysaccharides can be divided into plant cell wall polysaccharides and storage polysaccharides (de Vries and Visser, 2001). The main forms of storage polysaccharides are starch and inulin, although also a variety of gums with different structures can be found. Starch is a polymer of α1,4-linked D-glucose residues that also contains some α-1,6-linked D-glucose branches (Mischnick and Momcilovic, 2010). Inulin consists of β-2,1-linked D-fructose residues and contains a terminal D-glucose residue (Roberfroid, 2005). Plant cell wall polysaccharides are divided into cellulose, several hemicelluloses and pectins and can form up to 70% of the biomass (Jorgensen et al., 2007) and contain highly varied monomeric compounds and linkages (Kowalczyk et al., 2014). Cellulose is a linear polysaccharide consisting of β-1,4-linked D-glucose residues (Carpita and Gibeaut, 1993). The polymeric chains are organized in ordered structures called microfibrils that provide the skeleton of the plant cell wall. Xylan is the most common hemicellulose structure in monocots and dicots as well as in hardwoods (Ebringerová and Heinze, 2000). It consists of a backbone of β-1,4-linked D-xylose residues that is decorated with a variety of side groups, such as L-arabinose, (4-O-methyl-)Dglucuronic acid, D-galactose, acetyl, feruloyl and p-coumaroyl groups. In softwood, the most common hemicellulose is galactoglucomannan, which consists of a β-1,4-linked D-mannose residues that is interrupted by β-1,4-linked D-glucose residues (Aspinall, 1980). Depending on the origin it contains a variable amount of α-1,6-linked D-galactose and acetyl residues (Timell, 1967). The third hemicellulose, xyloglucan, has the same backbone as cellulose, but is decorated with α-1,6-linked D-xylose residues (Vincken et al., 1997). In addition it can contain Larabinose, D-galactose and L-fucose residues linked either to the backbone or the D-xylose residues. Pectin is a heterogenic polymer that consists of four defined substructures (Perez et al., 2000). The main structure is homogalacturonan, a polymer consisting of α-1,4-linked D-galacturonic acid residues that can be methylated and/or acetylated. Xylogalacturonan is a modified form of 4

homogalacturonan in that it contains D-xylose side groups β-1,3-linked to the galacturonic acid backbone. In rhamnogalacturonan I the backbone consists of alternating α-1,4-linked Dgalacturonic acid and α-1,2-linked L-rhamnose residues. The galacturonic acid residues in all pectin substructures can be acetylated, while in rhamnogalacturonan the L-rhamnose residues can have side chains of L-arabinose (arabinan), D-galactose (galactan) or both (arabinogalactan) (Guillon and Thibault, 1989). These chains can contain terminal feruloyl residues. Rhamnogalacturonan II has a galacturonic acid backbone and four diverse side-chains containing several uncommon sugars such as 3-deoxy-D-manno-2-octulosonic acid (Mazeau and Perez, 1998). Lignin is a highly heterogenic aromatic polymer that is built from three phenylpropanoid precursors, p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol (Higuchi, 2006). The exact structure of lignin varies depending on plant species and tissue and includes a large variety of linkages of which the β-O-4 bond is the most common (Adler, 1977). Covalent and non-covalent linkages between the polysaccharides and between the polysaccharides and lignin create the intricate structure that provides strength to the plant cell wall and also acts as a defense against microbial attack (Grabber et al., 2000; Jeffries, 1990; Sun et al., 2001).

Due to the heterogeneity of plant polymers, a large variety of enzymes is needed to degrade them into the monomers, which serve as carbon sources for the fungi that produce these enzymes (Mäkelä et al., 2014a). These enzymes have been divided into families based on modules in their amino acid sequences in the Carbohydrate Active enZyme database (www.cazy.org) (Lombard et al., 2014), which has become an indispensable resource in the field. CAZy is a classification system of enzymes that is based on amino acid sequence modules and consists of six main groups: the glycoside hydrolases (GH), glycosyltransferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE), auxiliary activities (AA) and carbohydrate binding modules (CBM). Within each of these groups a multitude of families can be found, and a subset of these contain enzymes involved in plant biomass degradation. While some families contain only a single (known) enzyme activity (e.g. GH67), others contain several activities (e.g. GH43) and are therefore not an immediate indication of enzyme function. Recently, a subfamily system has been initiated within CAZy, addressing initially GH5 that contains endoglucanases, endomannanases and other enzyme activities (Aspeborg et al., 2012), to aid in enzyme function 5

assignment. Due to the rapidly growing set of fungal genome sequences, the number of CAZy entries without functional confirmation by far outnumbers the biochemically characterized entries. It is therefore not unlikely that new activities will be discovered in existing CAZy families when more family members are biochemically characterized. Other features in the CAZy database are links to enzyme structures and to GenBank and UniProt.

The complex and intricate structure of plant biomass not only forms a challenge for fungi to degrade and obtain their carbon source, but it also causes several experimental challenges. Traditional methods for the isolation of fungal genomic DNA, RNA and proteins from cultures grown on plant biomass often do not provide the required quality and/or quantity for genomics, transcriptomics and proteomics. Optimization of the protocols is needed in many cases and can even be species dependent. Availability of more universal methods would strongly enhance the field and one paper in this issue describes such a method for RNA isolation (Patyshakuliyeva et al., 2014).

3. Saprobic ascomycetes By far the largest research effort into plant biomass degradation has been performed with saprobic ascomycetes. The main industrial workhorses with respect to enzyme production (Aspergillus niger, Aspergillus oryzae and T. reesei) fall into this group of fungi and their long use in industry as well as intensive strain improvement has resulted in a wealth of information regarding their plant biomass depolymerizing enzymes. T. reesei was first identified in the pacific during World War II, as it was degrading various cellulose based army materials, such as clothing, tents and sandbags. Improvement strategies resulted in strains with increased cellulase production (Peterson and Nevalainen, 2012). T. reesei has been the benchmark fungus with respect to cellulase production for several decades (Kubicek, 1993) and is still leading in this area today. Aspergillus species, in particular A. niger, have received more attention for enzymes that degrade other plant polysaccharides, such as starch, xylan, pectin and inulin and also the extensive strain improvements have resulted in production levels in the gram per liter scale (Archer and Peberdy, 1997). A large number of enzymes involved in plant polysaccharide degradation has been purified and characterized from

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these fungi and to a lesser extent from other saprobic ascomycetes, and in many cases the corresponding enzymes have been cloned (de Vries and Visser, 2001).

While initial improvement strategies were mainly aimed at fermentation conditions and random mutagenesis of the fungal strains, later emphasis was given to molecular genetics as a tool to improve the production of the enzymes. This included the identification of regulators controlling the expression of the enzyme-encoding genes (de Vries, 2003; Kowalczyk et al., 2014; Tsukagoshi et al., 2001) as well as the construction of genetically modified strains with additional copies of the desired gene(s). The use of strong constitutive or inducible promoters to drive the expression of both regulator- and enzyme-encoding genes was also introduced (Culleton et al., 2014; Gouka et al., 1996; Hanegraaf et al., 1991). These strategies gave strongly improved strains for industrial enzyme production, but also resulted in a very narrow group of fungi that were being applied for industrial production of these enzymes. Research into discovery of new natural isolates that produced high levels of desired enzymes continued (e.g. (Benoliel et al., 2013; Robl et al., 2013)), but few of those were established as industrial producers. This is in part due to the significant investments made by the enzyme producers to optimize their production strains not only with respect to increased enzyme production, but also with respect to fermentation behavior, ease of handling and genetic tools. In addition, the existing producers have GRAS (generally regarded as safe) status, while these new isolates will require extensive testing to exclude that they produce mycotoxins or other undesired compounds.

Species with strong industrial interest often became the species of choice for academic studies as well, due to the availability of genetic tools and the larger amount of data on their physiology. It soon became apparent that many fundamental topics of fungal physiology are also highly relevant for industrial use of these fungi and T. reesei and Aspergillus species therefore not only became the organisms of choice in studies on plant biomass degrading enzymes, but also for topics like protein folding and secretion (Nevalainen and Peterson, 2014), stress response (Duran et al., 2010), primary and secondary metabolism (Blumenthal, 2004; Khosravi et al., 2014) and morphology. Although they are rarely used in industrial applications, Aspergillus nidulans (Wortman et al., 2009) and Neurospora crassa (Dunlap et al., 2007) hold a special position in the more fundamental studies into fungal biology, due to their ability to cross sexually. For many 7

saprobic ascomycetes, and in particular those with industrial applications, a sexual cycle was not available, which limits the speed and ease with which to make and analyze genetic mutants.

The availability of fungal genomes had a tremendous impact on fungal biology as a whole and also on research in plant biomass degradation by fungi. The first thing fungal genomes revealed on this topic was that the plant biomass modifying enzyme sets encoded in fungal genomes are much larger than suspected. More than 150 genes encoding putative plant biomass degrading enzymes could be identified in most filamentous ascomycetes, in some cases even more than 300. This has encouraged studies into understanding the need for such a large diversity of enzymes and the variations in strategies employed by these fungi. The genus Aspergillus is at the forefront of fungal genomics with the first three genomes already published in 2005 (Galagan et al., 2005; Machida et al., 2005; Nierman et al., 2005), rapidly followed by genomes for an additional five species (Fedorova et al., 2008; Payne et al., 2006; Pel et al., 2007) and more recently the publication of two additional draft genomes (Futagami et al., 2011; Sato et al., 2011) and genomes for 11 additional species available at the MycoCosm site of the Joint Genome Institute of the United States Department of Energy (Grigoriev et al., 2012). Through the Aspergillus Genome Database (AspGD) (Cerqueira et al., 2014) 19 Aspergillus genome sequences can now be queried (Benoit et al., 2013), including synteny analysis. The availability of genomes has also strongly stimulated regulatory studies into plant biomass degradation in ascomycetes. These studies still strongly focus on a small set of well-studied ascomycetes, mainly T. reesei and several Aspergilli, but homologs of the regulators are found in many other species. A strong emphasis is on the regulators involved in xylan and cellulose degradation, for which 10 regulators have already been identified (reviewed in (Tani et al., 2014)). However, regulators have also been identified that are involved in pectin degradation (Battaglia et al., 2011; Gruben et al., 2014). Significant variations between the presence of these positively acting regulators in filamentous ascomycetes has been observed, while none of them have homologs in basidiomycetes (Todd et al., 2014). The only universally present carbon utilization related regulator is the negatively acting carbon catabolite repression regulator CreA (Ruijter and Visser, 1997). Homologs of this regulator are maintained in all tested species across the fungal kingdom (Todd et al., 2014), suggesting a crucial role for this regulator in fungal physiology. 8

Transcriptomic and proteomic studies have revealed much about the strategies employed by different fungi and demonstrated that the process of plant biomass degradation by fungi is even more complex than was previously assumed. Examples of this are the studies on Myceliophthora thermophila (Kolbusz et al., 2014) and Botrytis cinerea (Zhang et al., 2013) in this issue. It has also pointed to some other ascomycetes with interesting abilities, such as the dung fungus Podospora anserina, that has a plant polysaccharide-degrading ability similar to many basidiomycetes (Espagne et al., 2008). As a late colonizer of herbivore dung it degrades predominantly lignocellulose, which is reflected in its genome with a strong expansion of genes involved in cellulose and xylan degradation (the main polysaccharides in lignocellulose), while the number of genes involved in pectin degradation is strongly reduced. It is significantly different from the more cosmopolitan Aspergillus species that encode a broader range of enzymes in their genome (Pel et al., 2007).

Release of monomers from plant biomass is strongly linked to the conversion of these monomers inside the fungal cell. This makes sense in a natural environment where fungi and bacteria are competing for nutrient sources. If the monomers are not immediately used by the organism that produced the enzymes that releases them, then they are likely to be taken by some of the other microorganisms that are present. In some cases it has been shown that the extracellular and metabolic enzymes are co-regulated. For instance, the (hemi-)cellulolytic regulator XlnR and the arabinolytic regulator AraR from Aspergillus not only control release of D-xylose and Larabinose, but also the pentose catabolic pathway (PCP) that converts these two sugars (Battaglia et al., 2011), as well as part of the pentose phosphate pathway that links the PCP to glycolysis (Battaglia et al, unpublished results). The increasing interest in plant biomass degradation by fungi has also resulted in a more detailed investigation of carbon metabolism and the identification of the genes of catabolic pathways for various monomeric components of plant biomass (Khosravi et al., 2014). Research in ascomycete saprobes is now moving more towards systems biology, where the degradation of the polymeric substrate, the uptake and conversion of the monomers and regulation of the process are studied as a complex network.

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4. Saprobic basidiomycetes Studies into plant biomass degradation by saprobic basidiomycetes have strongly focused on the decay of woody and litter biomass and in particular degradation of the aromatic polymer lignin (Crawford and Crawford, 1976; Kirk, 1971; Kirk et al., 1975; Kirk and Farrell, 1987). White rot fungi are the only organisms on earth that can fully mineralize lignin and have therefore been the subject of many studies in this field. In addition, litter-decomposing basidiomycetes also contribute to lignin degradation using largely the same strategy as white rot fungi. A more specialized life style is found for fungi that live together with termites, such as the species Termitomyces. These fungi are being fed plant biomass by the termites, who consume the resulting fungal biomass (Ohkuma, 2003).

White rot fungi degrade both lignin and polysaccharides leaving behind a white residue consisting mainly of cellulose (Hatakka and Hammel, 2010). A specific feature of these fungi is the production of various classes of peroxidases that are able to oxidize lignin (see below). In contrast, brown rot fungi degrade plant cell wall polysaccharides, but only modify lignin leaving behind a brown wood residue that has lost its strength properties (Hatakka and Hammel, 2010). Brown rot fungi lack the lignin-modifying peroxidases and a recent study suggested that they have diverged from the white rot lifestyle several times during the evolution of basidiomycete species (Floudas et al., 2012). Another characteristic of brown rot fungi is the absence of most cellulose depolymerizing enzymes and the use of Fenton chemistry as a major mechanism for polysaccharide degradation (Jensen et al., 2001). A recent study questioned the typical white rot / brown rot division based on the presence or absence of lignin-related peroxidases, suggesting a need for a more nuanced characterization of these lifestyles (Riley et al., 2014). A third basidiomycete life style is that of the litter and straw decomposers, who also contribute significantly to the global carbon cycle and their degradation pattern resembles that of the white rot fungi (Lundell et al., 2014). The main enzymes implicated in lignin degradation are class II peroxidases, namely lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP). LiP is an H2O2dependent oxidative enzyme with a wide substrate range of both phenolic and non-phenolic 10

aromatic compounds that includes the cleavage of propyl side chains of lignin and the aromatic rings of lignin model compounds (Cullen, 1997). MnP catalyzes the specific oxidation of Mn2+ to Mn3+ using H2O2.The formed Mn3+ ions are chelated by organic acids, such as oxalate, and are able to oxidize e.g. various phenolic compounds (Kuan and Tien, 1993). The non-phenolic lignin substructures can be oxidized through MnP-catalyzed lipid peroxidation reactions (Jensen et al., 1996; Kapich et al., 1999). A paper in this issue describes the degradation of non-phenolic lignin model compound by Bjerkandera adusta VP via lipid peroxidation (Reina et al., 2014). Also, the oxidation of non-phenolic lignin model compounds by MnP was shown to be enhanced by phenolic mediators in a study presented in this issue (Nousiainen et al., 2014). Different groups of MnPs on the basis of their gene structure are found in fungi (Floudas et al., 2012). In a paper in this special issue, a variation on the Mn2+-binding site was described for a MnP from Agrocybe praecox, confirming the biochemical activity of a novel group of atypical short-MnP enzymes (Hildén et al., 2014). The third type of lignin-modifying peroxidase, VP, combine the activity of MnP and LiP (Hammel and Cullen, 2008). Variations in the catalytic properties of LiPs, MnPs and VPs have also been reported thus making the classification of these enzymes more complicated (Fernández-Fueyo et al., 2012a; Fernández-Fueyo et al., 2012b; Ruiz-Duenas et al., 2011). Also enzymes from other peroxidase superfamilies have been suggested to participate in lignin degradation or modification. Dye-decolorizing peroxidases (DyPs) oxidize recalcitrant dyes and aromatic compounds. Although the biological function of DyP is still unclear, the enzyme from Auricularia auricular-judae has been shown to oxidize non-phenolic lignin model compounds and may therefore be part of the lignin oxidizing system of fungi (Liers et al., 2010). Two classes of heme-thiolate peroxidases (HTPs), chloroperoxidases (CPOs) and unspecific peroxygenases (UPOs), have also received attention, but their role in plant biomass modification is not yet understood (Hofrichter and Ullrich, 2014). Also various H2O2-producing enzymes play an essential role in lignin modification, by supplying the H2O2 required by the peroxidases (Kersten and Cullen, 1993). These oxidoreductases include glucose-methanol-choline (GMC) superfamily enzymes as well as copper radical oxidases (CROs) that are reviewed in this issue (Kersten and Cullen, 2014). Laccases are multi-copper oxidases that have diverse roles in fungal physiology and have continuously been connected to lignin depolymerization. Laccases catalyze the oxidation of 11

various phenolic compounds, but are not able to directly oxidize non-phenolic lignin model compounds (Bourbonnais and Paice, 1990; Eggert et al., 1996). In a paper in this issue the dissimilar secretion patterns of laccase and peroxidases on recalcitrant dyes containing agar plates was suggested to correlate with the colonization strategies of white rot fungi representing different ecophysiological groups (Barrasa et al., 2014). The most detailed studies into fungal lignin degradation have been performed with the model white rot basidiomycete Phanerochaete chrysosporium (Kersten and Cullen, 2007). It was the first species for which several of the lignin-modifying peroxidases were found and characterized (e.g. (Glenn et al., 1983; Gold et al., 1984; Kuwahara et al., 1984; Tien and Kirk, 1983; Tien and Kirk, 1984)), and culture conditions for the production of these enzymes were examined (Kirk et al., 1978). It was also the first species to have crystal structures for its lignin-modifying enzymes (Piontek et al., 1993; Poulos et al., 1993; Sundaramoorthy et al., 1994), have the regulation of the corresponding genes studied in detail (MacDonald et al., 2012), and to have a public genome sequence (Martinez et al., 2004). Studies of other white-rot fungi are more limited, although the availability of (post-)genomic tools has provided more in-depth knowledge on several species (see below). Initially, other white rot fungi were mainly examined from an interest in finding species that produce effective enzyme cocktails (e.g. (Elisashvili, 2009; Kiiskinen et al., 2004; Tekere et al., 2001))

Polysaccharide degrading enzymes from basidiomycetes have received less attention than their ligninolytic systems, which is likely due to the fact that such enzyme systems are also produced by the more industrially favored ascomycetes. In addition, growth of basidiomycetes in largescale fermentations remains a challenge. However, e.g. white rot fungi have all the enzymes required for complete depolymerization of cellulose, and the active expression of both classical cellulases and oxidatively cellulose depolymerizing CDH and LPMOs by Dichomitus squalens during growth on solid state wood cultures was shown in a paper in this issue (Rytioja et al., 2014a). Cellulolytic systems of basidiomycetes were studied already in the early 1950’s (Reese and Levinson, 1952) and a thorough overview of the enzymes involved in plant polysaccharide degradation from basidiomycetes has recently been published (Rytioja et al., 2014b). Some studies into improving cellulase production in basidiomycetes were performed (e.g. (Eriksson and Johnsrud, 1983) and several enzymes were produced in ascomycete hosts to evaluate their 12

biochemical properties (Casado López et al., 2014). Mutation of the genes encoding these enzymes can improve their properties for industrial applications, as was demonstrated for a Pleurotus ostreatus α-L-arabinofuranosidase in a paper in this issue (Marcolongo et al., 2014). The availability of genome sequences and post-genomic technologies (proteomics, transcriptomics), have provided a much better understanding of the plant biomass degrading strategies of basidiomycetes, similar to the developments in ascomycetes (see above). While some studies mainly addressed the genome of single species, although often in comparison to earlier published species (Duplessis et al., 2011; Eastwood et al., 2011; Fernández-Fueyo et al., 2012a; Martin et al., 2008; Martinez et al., 2009; Martinez et al., 2004; Morin et al., 2012; Ohm et al., 2010; Olson et al., 2012; Suzuki et al., 2012), larger studies involving several new genomes have also been performed (Binder et al., 2013; Floudas et al., 2012; Riley et al., 2014). These have provided a better view of the diversity of basidiomycetes with respect to plant biomass degradation and resulted in many follow-up studies. Some of those are presented in papers in this issue, such as studies into the variation of genome content with respect to genes encoding plant biomass degrading enzymes (Kersten and Cullen, 2014; Ohm et al., 2014) and the expression or regulation of these genes (Fernandez-Fueyo et al., 2014; Rytioja et al., 2014a; Suzuki et al., 2014) and production of the corresponding proteins (Reina et al., 2014). Comparisons have also been made to basidiomycete species that have a mutualistic life style and clear differences from saprobes could be observed (Martin et al., 2008). A study into the expression of plant biomass degradation related genes in the ectomycorrhizal fungus Laccaria bicolor is also presented in this issue (Veneault-Fourrey et al., 2014). This new level of knowledge on the wealth of enzymes involved in plant biomass degradation that are encoded in basidiomycete genomes has increased the interest in these enzymes. The major obstacles so far are the complications in producing these enzymes in industrial hosts that are mainly ascomycetes. While some enzymes have been produced successfully, their levels are still too low for industrial applications (Rytioja et al., 2014b), so improvements need to be made to fully apply the basidiomycete potential.

5. Plant pathogenic ascomycetes and basidiomycetes

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While most studies to date on cell wall-degrading enzymes in plants have striven to elucidate the role of individual genes in virulence, genome-wide studies are now revealing plant pathogens to be an important source of plant cell wall degrading enzymes. In 2011, one study examined the hydrolytic capabilities of 156 non-pathogenic and pathogenic fungi and determined that plant pathogens, such as Colletotrichum graminicola (corn anthracnose), Sclerotinia sclerotiorum (white mold) and Botrytis cinerea (gray mold) to name a small few, grouped among the top 86 enzymatically active fungi tested, compared to their standard, T. reesei (King et al., 2011). Curiously, the authors found that pathogenic species were more active on non-treated biomass, such as corn stover, switchgrass and soybean stems. These species included well known plant pathogens in the genus Fusarium, as well as S. sclerotorium (various molds and rots) and Macrophomina phaseolina (charcoal rot). They point out that while the utility of T. reesei is well-known it is not efficient at degrading untreated plant biomass, in comparison to the aforementioned plant pathogenic ascomycetes. In 2013, genomes of about 215 fungi and oomycetes were analyzed for genes encoding plant cell wall-degrading enzymes (Choi et al., 2013). They identified 6682 genes that grouped into 22 families they determined, based upon mode of action and substrate. Similar to the above study, they found that plant pathogenic fungi possessed more plant cell wall degrading enzymes than wood-decaying fungi. Plant pathogenic fungi are enriched in genes encoding pectin lyases and polygalacturanases compared to woodrotting fungi, belying their requirement to penetrate and interact with living host tissues, rather than degrade dead tissue. It remains to be determined whether supplementation with enzymes from these species will enhance overall performance of fungal-based biomass degradation. The importance of plant cell wall degrading enzymes in pathogenicity cannot be understated. In order for many fungi to gain access to cellular contents, they must first breach the plant’s fortress-like cuticular layer, followed by the cell wall via coordinated production of enzymes to target cutin, cellulose, hemi-cellulose and pectin (Faulkner and Robatzek, 2012; Skamnioti and Gurr, 2007). The first plant layer of defense is the cuticle, and for several fungi, cutinase genes are known to play a vital role in penetrating this substrate. M. oryzae, for example, requires the cutinase 2 (CUT2) gene in order to not only effectively utilize cutinase as a carbon source, but also in proper formation of the main penetration structure, the appressorium (Skamnioti and Gurr 2007). In Fusarium solani, cutinase activity was directly proportional to virulence of field isolates (Morid et al., 2009). The second plant layer of defense is the cell wall, 14

a sturdy structure primarily comprised of cellulose, pectin and hemi-cellulose. In the aforementioned study by Choi et al. (2013), they examined the genomes of about 210 fungi grouped into five categories based on their lifestyles that included animal pathogens, opportunistic animal pathogens, plant pathogens, saprobes and wood-rotting fungi. Of these five groups, the plant pathogen genomes were specifically enriched for polygalacturonases and pectin lyases, and overall, these 31 genomes contained more plant cell wall degrading enzymes than any other group, including cellulose-degrading enzymes. It has been known for some time that pectin lyases are both important virulence factors, and that fungi usually possess multiple copies of them. In Fusarium solani (teleomorph Nectria haematococca) for example, it was demonstrated that deletion of the two of the four pectin lyase genes together resulted in a drastic reduction in pathogenicity (Rogers et al., 2000). Choi et al. also examined cellobiohydrolases, known to degrade cellulose in the plant cell wall. Again, they show that these enzymes are found more predominantly in the genomes of plant pathogenic fungi, as compared to other groups. In this issue, Klaubauf et al. demonstrate how the production of cellobiohydrolases as well as other important plant cell wall degrading enzymes is regulated (Klaubauf et al., 2014). They take a genetic approach by examining a master regulator, XlnR of xylan and cellulose degradation, in five different fungi, including the plant pathogens Fusarium graminearum and M. oryzae. Interestingly, when XlnR is deleted in M. oryzae, it shows marked differences from the other fungi tested, including growth on different carbon sources and a dramatically reduced cellulolytic enzyme profile. In the preceding section, A. niger was introduced as one of the workhorses of biomass degradation. This ascomycete also groups with pathogenic fungi, in that it causes pre- and postharvest diseases on important crops such as soybean, onions, grapes and peanuts (Susca et al., 2010). Even more important however, is the ability of this fungus to produce several mycotoxins including ochratoxin A and fumonisin B2, which can cause serious human health concerns. A. niger has a rich history of being used at the industrial scale in fermentations, and now in biomass degradation, and as being “generally regarded as safe” (GRAS) by the United States Food and Drug Administration (FDA) (Nielsen et al., 2009). Recent studies have raised concerns about the GRAS status of A. niger, finding both wines, table grapes and raisins contaminated with fumonisins (Mogensen et al., 2010). This extremely useful, yet potentially toxic, plant

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pathogenic fungus underscores the need to continue exploration into plant pathogenic fungi as new sources of plant cell wall degrading enzymes. Surprisingly, one such newly-found source is the phytopathogenic basidiomycete Ustilago maydis, the causal agent of corn smut. In combination with a T. reesei cocktail, enzymes from U. maydis caused a 57% and 22% increase in release of total sugars and glucose, respectively (Couturier et al., 2012). An in silico secretome analysis of U. maydis yielded two oxidoreductases as among the most abundant proteins. The authors speculated that oxidoreductases could contribute to biomass instability through the formation of reactive oxygen species, or through interaction with CAZy family AA9 (formerly GH61) proteins, as their T. reesei cocktail contained such enzymes.

6. Concluding remarks Plant biomass degradation by fungi is a booming field which attracts interests in both academia and industry. The new technologies available for biological research (genomics, transcriptomics, proteomics, metabolomics), but also improved visualization techniques (e.g. microscopy) enable researchers to study this topic in much more detail and reveal the mechanisms that drive this process. This special issue provides a view on the width of the field and also demonstrates that the fungal functions involved in plant biomass degradation go beyond the enzymes that cleave the linkages in the polymers. It can be expected that future research will reveal many other aspects that affect the degradation of plant biomass. In addition, it will provide understanding into the complex relationships between microorganisms in nature resulting in the highly efficient decay that is an essential part of the earth’s carbon cycle.

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