Expression of extracellular peptidases
Transcriptional analyses revealed a large number of peptidases and transporters expressed during the degradation of protein substrates by P. involutus (Fig. 6). Considering extracellular peptidases, we identified nine genes encoding extracellular aspartate endopeptidases (Fig. 4a). Based on sequence homology, they were classified into three MEROPS A1 subfamilies: the polyporopepsins, the CnAP1 peptidases and the A01 unassigned peptidases. Most upregulated in media containing pollen, gliadin and BSA were members of the polyporopepsin subfamily (described by Kobayashi, 2012). In the litter extracts, the most upregulated aspartate endopeptidases displayed close sequence homology to A01 unassigned peptidases, including AmProt1 from A. muscaria (Nehls et al., 2001). Seven of the aspartate endopeptidases had a putative MW in the range 41–49 kDa. Two of them were predicted to encode significantly larger proteins with a MW of 75.9 kDa (Pi:181372) and 58.8 kDa (Pi:72240). Each sequence contained one predicted transmembrane helix, suggesting membrane-spanning properties. The activity of an atypical, larger aspartic endopeptidase (AmProt2; c. 100 kDa) has been reported in A. muscaria (Nehls et al., 2001). In addition, there are two other MEROPS families of acidic extracellular endopeptidases identified: the serine-carboxyl peptidase and the glutamic peptidase families. Two genes encoding members in the sedolisin S53 family of the serine-carboxyl peptidases were significantly upregulated during protein degradation, including members of the grifolisin and the scytalidosin subfamilies (Suzuki et al., 2005; Takahashi & Oda, 2008). The secreted protease activity of P. involutus was slightly suppressed by the metalloprotease inhibitor EDTA and the serine-protease inhibitor PMSF. Two members of these families were found among the upregulated transcripts, including a fungalysin (M36 subfamily) and a subtilisin (S8 subfamily; Graycar et al., 2012; Kolattukudy & Sirakova, 2012).
Figure 6. The molecular components of the protein degradation pathways of Paxillus involutus. Organic nitrogen (N) in soil solution is degraded by the combined action of endo- (polyporopepsin, CnAP1, A01 unassigned peptidases, fungalysin, scytalidoilsin and grifolisin) and exo-peptidases (aminopeptidases, carboxypeptidases) into peptides, amino acids and ammonium (see text for details). Peptides and oligopeptides assimilation is facilitated by Peptide transporter family (PTR) and Oligopeptide transporter family (OPT), amino acids are transported by Yeast amino acid transporter (YAT) and L-type amino acid transporter (LAT) while ammonium is internalized using Amt transporters. Intracellular peptidases further degrade oligopeptides and peptides while amino acids and ammonium are incorporated into the amino acid metabolic pathway. Intercellular transfer of urea and polyamine are facilitated by DHA1 (Drug:H+ antiporter) and PiDur3 (P. involutus degradation of urea transporter). Vacuolar peptidases are active on proteins transported through amino acid/choline transporter (ACT) and amino acid/auxin porter (AAAP).
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It has been proposed that the ability of ECM fungi to utilize protein as N source depends on the synergistic action of secreted endo- and exopeptidases, because only amino acids and smaller peptides can be assimilated via membrane transporters (Chalot & Brun, 1998). In agreement with this suggestion, the transcriptional analysis revealed that P. involutus expressed both types of peptidases during digestion of proteins. Five genes predicted to encode extracellular exopeptidases were upregulated during degradation of the protein sources, including three aminopeptidases (M28 family), a dipeptidase (S9 family), and a carboxypeptidase (S28 family). Such exopeptidases are secreted by A. fumigatus during protein degradation at an acidic pH (Sriranganadane et al., 2010). The upregulation of multiple endo- and exopeptidases suggests that P. involutus is an efficient organic N degrader.
Assimilation of amino acids and peptides
During growth on protein-containing substrates, P. involutus expresses a large diversity of N transporters that are presumably involved in the assimilation of released amino acids and peptides (Fig. 5). Among the highly upregulated transcripts were two members of the yeast amino acid transporter (YAT) family, and show a high sequence homology to the general amino acid transporter AAT1 of A. muscaria (Nehls et al., 1999), which has been characterized as a high-affinity amino acid transporter with broad substrate specificity. Within the L-type amino acid transporter (LAT) family, two transcripts were upregulated that show significant sequence similarity to a high-affinity methionine permease in L. bicolor (Lucic et al., 2008). Upregulated peptide transporters were found within two families: the peptide transporter (PTR) family and the oligopeptide (OPT) family. The PTR gene has close sequence homology to the high-affinity peptide transporter PTR2A of Hebeloma cylindrosporum, which is involved in the assimilation of di- and tripeptides (Benjdia et al., 2006). Three phylogenetic clusters of OPT transporters have been identified in the genome of L. bicolor (Lucic et al., 2008). The upregulated P. involutus transcripts were found in all three clusters implying active assimilation of peptides and amino acids.
Intracellular metabolism and compartmentalization of assimilated nitrogen
Internalized peptides have to be hydrolysed into amino acids prior being metabolized or used as precursors in proteins synthesis. So far, no intracellular peptidases have been characterized in ECM fungi, but several candidates were found among upregulated transcripts in P. involutus, including dipeptidyl peptidases and unassigned endo- and exopeptidases (Fig. 4b). The upregulated transcripts also represent peptidases with specific cellular functions, including stress response (bleomycin hydrolase, metacaspases; Kambouris et al., 1992), the glutathione degradation pathway (Dug1; Kaur et al., 2009), and protein processing (Stp1, Ste24, Map2; Li & Chang, 1995; Tam et al., 2001; Bien et al., 2009).
Studies particularly in yeasts and plants have shown that only a small fraction of the amino acids assimilated is metabolized and used for protein synthesis. The remaining fraction is metabolically inert and compartmentalized in vacuoles (Sekito et al., 2008). Two of the upregulated transcripts in P. involutus showed high sequence homology to vacuolar amino acid transporters previously characterized in S. cerevisiae. A transporter (Pi:13733) of the amino acid/auxin porter (AAAP) family is a homologue to Avt4 that has a role in vacuolar amino acid export (Russnak et al., 2001). The second putative vacuolar transporter (Pi:8342) is a member of the amino acid/choline transporter (ACT) family. This protein displays sequence homology to the S. cerevisiae UGA4 gene, encoding a vacuolar permease involved in the transport and utilization of GABA and putrescine (Uemura et al., 2004; Lucic et al., 2008). Vacuoles also have important roles in degrading peptides and proteins. The molecular components of this pathway have been well characterized in S. cerevisiae (Li & Kane, 2009). Several homologues of these peptidases were found among upregulated transcripts in P. involutus, including carboxypeptidase Y and carboxypeptidase S (Knop et al., 1993). The upregulated subtilisin shows close sequence homology to proteinase B, which suggests that it is localized to the vacuole. Together with the regulation of transcripts encoding vacuolar transporters, data suggest that the vacuoles have an active role in the storage and metabolism of the N compounds that are formed during protein degradation by P. involutus.
Glutamate/glutamine, alanine and aspartate/asparagine have been shown to be main sinks for N assimilated by ECM fungi (Finlay et al., 1988; Morel et al., 2005). Several of the most upregulated transcripts in P. involutus during the assimilation of organic N are representing enzymes involved in the amino acid metabolism, including glutamine synthetase (EC 220.127.116.11) and glutamate synthase (EC 18.104.22.168; Table S1; Morel et al., 2006). The upregulation of glutamine-fructose-6-phosphate transaminase (EC 22.214.171.124), amidophosphoribosyltransferase (EC 126.96.36.199) and carbamoyl-phosphate synthase (EC 188.8.131.52) suggests that the N of glutamine and glutamate is utilized for the synthesis of amino sugars, purines and pyrimidines. Moreover, the upregulation of transcripts encoding aminotransferases, such as aspartate transaminase (EC 184.108.40.206) and alanine transaminase (EC 220.127.116.11) indicates an active transfer of amino groups between glutamate and other amino acids.
In a previous study it was shown that urea is one of the major N compounds found in the extraradical mycelium of P. involutus during mycorrhizal symbiosis with the Betula pendula (Morel et al., 2005). Polyamines have also been found in high amounts in the mycelium of P. involutus grown axenically in synthetic medium (Fornalé et al., 1999). Both of these N compounds are derivatives of the urea cycle, and transcripts encoding for urea and polyamine transporters were upregulated in the extraradical mycelium of P. involutus (Morel et al., 2005). PiDur3 was described as an urea import protein, involved in the transfer of urea in the extraradical mycelium (Morel et al., 2005, 2008), whereas TPO3 was suggested to be a vacuolar transporter which allows long-distance translocation of N compounds along the mycelium, such as polyamines (Morel et al., 2005). In agreement with these previous studies, we found several homologues for transporters and enzymes involved in the urea/polyamine metabolism that were significantly upregulated during protein degradation in P. involutus, including PiDur3, a putative polyamine transporter of the Drug:H+ antiporter (DHA1) family which includes the TPO1-TPO4 polyamine transporters (Tomitori et al., 1999; Albertsen et al., 2003; Lucic et al., 2008) and the UGA4 vacuolar transporter (Uemura et al., 2004; Lucic et al., 2008). In addition, our study also showed significant upregulation of transcripts encoding arginase, urease and ornithine decarboxylase, enzymes responsible for the synthesis of intracellular urea and polyamines (Table S1, Fig. S5). The synthesis of urea and polyamine is probably stimulated during C starvation. In accordance to our previous work (Rineau et al., 2012), glucose is not detectable in the medium after 7 d of incubation with P. involutus. Carbon-rich compounds such as ornithine are formed by arginine degradation, which also generates urea (KEGG map00330; www.genome.jp/kegg/)), leading to high concentrations of urea and polyamines. Ornithine and urea are, however, degraded further in a catabolic arm of the urea cycle, to form polyamines such as putrescine/spermine and ammonium (Bago et al., 2001). The expression of the metabolic enzymes in coordination with the expression of the urea and polyamine transporters suggests an active synthesis and transportation of urea and polyamine across the hyphae.
Nitrogen catabolite repression
In agreement with findings in several other basidiomycetes, including both mycorrhizal and saprophytic species (Kalisz et al., 1987; Leake & Read, 1991; Zhu et al., 1994), the total extracellular proteolytic activity of P. involutus was mainly regulated by protein induction and only partially by ammonium repression. The experiments with the other basidiomycetes were conducted by growing the mycelia for several days in liquid media containing a protein inducer and ammonium at various concentrations. Similar experiments in P. involutus resulted in a variable responses ranging from stimulation to repression of proteolytic activity (not shown). To measure the nitrogen catabolite repression (NCR) response more precisely, ammonium, glutamic acid or nitrate were added to cultures at a time point when the mycelium was secreting high levels of proteolytic activity. Results demonstrated that the extracellular protease activity of P. involutus is repressible by ammonium (Fig. S4). The NCR has been studied in detail in filamentous ascomycetes (Cohen, 1973; Cohen & Drucker, 1977; Jarai & Buxton, 1994). In comparison with the response observed in these fungi, the repression in P. involutus was much less and it was restricted to a rather short period of time (< 24 h). In Aspergillus nidulans, ammonium, glutamine and glutamate have been identified as the signals for NCR (Margelis et al., 2001). Nitrate is also used as an N source by Aspergillus, although it will not be utilized unless the cells are depleted for the favored compounds ammonium, glutamine or glutamate (Marzluf, 1996). In accordance with the role of these N sources in Aspergillus, NCR in P. involutus was induced by ammonium and glutamate, but not by nitrate. However, in contrast to ascomycetes (Marzluf, 1996), the repression of the protease activity in P. involutus did not correlate with a decreased level of transcripts encoding peptidases and amino acid/peptide transporters (Table S2). Accordingly, NCR of protease activity in P. involutus appears to be operating at the protein level.
The lessened NCR response agrees with a recent study showing that the addition of ammonium had relatively minor effects on the decomposition and assimilation of N by P. involutus from plant litter organic material (Rineau et al., 2013). Moreover, the effects were only observed when glucose was added to the litter material. By contrast, numerous studies have shown that the decomposition of litter material by saprophytic fungi is repressed by high availability of N (Fenn & Kirk, 1981; Edwards et al., 2011) and glucose (Aro et al., 2005). However, the effects of N-availability on the expression of enzymes (including peptidases) involved in the degradation of litter material have only been considered for a small number of species. Hence, further studies are needed to reveal whether there are any consistent differences between saprophytic and ECM fungi in the regulation of such enzymes in response to more favourable N sources such as ammonium.
The mobilization of protein N by ECM fungi is a biochemical process involving several stages including the breakdown of proteins, the uptake of the released mono- and oligomers, and the internal transformation of amino acids and peptides. Identifying the molecular components of this process is an important step towards identifying the mechanisms that control the use of organic N by mycorrhizal plants and how it may vary by species and environment. Our study shows that P. involutus expressed a large diversity of extracellular peptidases, in particular aspartate endopeptidases during the degradation of proteins. The relative expression levels of the genes encoding these enzymes varied depending on the protein source and the availability of the protein substrate (Fig. 4a). A number of other fungi are known to express a battery of aspartate endopeptidases during the breakdown of protein substrates. For example, the secreted aspartic proteases (SAP) gene family of the human pathogen Candida albicans has 10 members and some of them are differentially regulated in response to specific environmental conditions (Naglik et al., 2003). Moreover, heterologously expressed Sap peptidases can be clustered into three distinct groups based on their substrate specificity, indicating that they target different protein substrates during the infection (Aoki et al., 2011). It remains to be determined whether the secreted aspartate peptidases of P. involutus differ in biochemical properties including substrate specificities.
The ability to assimilate N from range of different protein sources and environmental conditions will depend on the synergistic action of secreted endo- and exopeptidases and the expression of a matching set of N transporters. Indeed, the expression levels of N transporters in P. involutus varied extensively with the protein source (Fig. 5). Further studies are however, needed to identify how the components of the extracellular peptidase machinery, the transporters and the internal metabolism are coordinately regulated in ECM fungi during the mobilization of different organic N sources available in forest soils (Fig. 6).