Recent studies show the potential impacts of insect symbiont microbes on biofuel application with regard to their degradation capability of a recalcitrant plant cell wall. In this study, we describe a novel fungal isolate, D. decipiens oita, as a single symbiotic fungus from the Xiphydria woodwasp found in the northern forests of Japan. Our detailed secretome analyses of D. decipiens oita, together with activity measurements, reveal that this insect-associated fungus exhibits high and broad activities for plant cell wall material degradation, suggesting potential applications within the biomass conversion industry for plant mass degradation.
KEYWORDS: cellulolytic fungus, glycoside hydrolases, cellulose, hemicellulose, proteomics
ABSTRACT
Wood-devastating insects utilize their symbiotic microbes with lignocellulose-degrading abilities to extract energy from recalcitrant woods. It is well known that free-living lignocellulose-degrading fungi secrete various carbohydrate-active enzymes (CAZymes) to degrade plant cell wall components, mainly cellulose, hemicellulose, and lignin. However, CAZymes from insect-symbiotic fungi have not been well documented except for a few examples. In this study, an insect-associated fungus, Daldinia decipiens oita, was isolated as a potential symbiotic fungus of female Xiphydria albopicta captured from Hokkaido forest. This fungus was grown in seven different media containing a single carbon source, glucose, cellulose, xylan, mannan, pectin, poplar, or larch, and the secreted proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 128 CAZymes, including domains of 92 glycoside hydrolases, 15 carbohydrate esterases, 5 polysaccharide lyases, 17 auxiliary activities, and 11 carbohydrate-binding modules, were identified, and these are involved in degradation of cellulose and hemicellulose but not lignin. Together with the results of polysaccharide-degrading activity measurements, we concluded that D. decipiens oita tightly regulates the expression of these CAZymes in response to the tested plant cell wall materials. Overall, this study described the detailed proteomic approach of a woodwasp-associated fungus and revealed that the new isolate, D. decipiens oita, secretes diverse CAZymes to efficiently degrade lignocellulose in the symbiotic environment.
IMPORTANCE Recent studies show the potential impacts of insect symbiont microbes on biofuel application with regard to their degradation capability of a recalcitrant plant cell wall. In this study, we describe a novel fungal isolate, D. decipiens oita, as a single symbiotic fungus from the Xiphydria woodwasp found in the northern forests of Japan. Our detailed secretome analyses of D. decipiens oita, together with activity measurements, reveal that this insect-associated fungus exhibits high and broad activities for plant cell wall material degradation, suggesting potential applications within the biomass conversion industry for plant mass degradation.
INTRODUCTION
Recent studies show the potential impacts of insect symbiont microbes on biofuel applications with regard to their degradation capability of a recalcitrant plant cell wall, which consists of cellulose, hemicellulose, and lignin (1). Such microbes range from bacteria to fungi and help host insects to utilize simple sugars from plant cell wall materials, for example, the insect symbiont Streptomyces. Streptomyces species have been identified as symbionts of various insects, including woodwasps, bark beetles, leaf-cutter ants, honey bees, and others (2, 3). One of the two major families of woodwasps, Siricidae, has been considered as a highly invasive pest across the world and is thus an extensively studied model of symbiosis for wood devastation (4, 5). Female adult Siricidae carry symbiotic microbes in specialized organs called mycangia, which are used to inoculate host trees during oviposition (6), ultimately degrading lignocellulose and/or providing nutrition for Siricidae larvae. Previous reports detailed the association between Siricidae and their symbiotic fungus Amylostereum (5, 7), which belongs to Basidiomycota. Although the Amylostereum spp. are known to degrade plant polysaccharides (8), the enzymes responsible for plant cell wall degradation have yet to be determined, due in part to a lack of genomic information about insect-associated fungi. Recently, several bacteria were isolated from Siricidae woodwasps, and their polysaccharide degradation activities on plant cell wall components were investigated on a genomic level (9, 10). Among them, the Streptomyces sp. strain SirexAA-E, isolated from the Siricidae woodwasp Sirex noctilio, found in North American forests, was able to efficiently deconstruct plant biomass (11). This bacterium secreted various carbohydrate-active enzymes (CAZymes) (12) with the ability to degrade plant cell wall; furthermore, different subsets of CAZymes were secreted in response to different carbon sources in growth media (13).
Xiphydria is another major woodwasp family; fungal Daldinia spp. belonging to Xylariales, Ascomycota, are often found to be associated with this family and are considered to establish a symbiotic relationship to the host Xiphydria in the mycangium (14, 15). In contrast to the well-studied Siricidae woodwasp symbiosis, the cellulolytic potential of Daldinia spp. has not been well documented or characterized. Daldinia spp. appear to exhibit a wide range of host specificities, having been isolated from decayed wood, plant leaves, insect, marine algae, and humans (16). Recently, a whole-genome sequence of Daldinia eschscholtzii EC12, originally isolated as an endophyte, was released in order to elucidate its potential for a consolidated bioprocessing organism for biorefinery processes (17–19). Dozens of CAZymes were annotated within the D. eschscholtzii EC12 genome, and endo- and exocellulase activities in the culture supernatant from different feedstock grown conditions were measured (20). Despite this, proteome-based CAZyme profiles from neither endophytic nor insect-associated Daldinia spp. have been reported.
In this study, we describe a novel fungal isolate, D. decipiens oita, as a potential symbiotic fungus from a Xiphydria woodwasp found in the northern forests of Japan. To evaluate the cellulolytic ability of this Xiphydria woodwasp symbiont, D. decipiens oita was grown on different carbon source-containing cultures, and the secreted proteins were identified by a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic approach. Our detailed secretome analyses of D. decipiens oita identified a diverse set of CAZymes and a large variety of proteases. Taken together with activity measurements, this insect-associated fungus exhibits high and broad activities for plant cell wall decomposition, suggesting potential applications within the biomass conversion industry for plant biomass degradation.
RESULTS
Isolation and species determination of D. decipiens oita from Hokkaido woodwasp.
Symbiont microbes were isolated from one female adult Xiphydria albopicta captured in summer 2015 and two female adult X. albopicta captured in summer 2016 in Bibai City, Hokkaido, Japan (Fig. 1). Quite recently, the taxonomic status of the well-known woodwasp Xiphydria camelus was categorized into three different species, including X. camelus, X. eborata, and the new species X. albopicta (21), and our specimens were X. albopicta according to their body color. We dissected and isolated the mycangia from X. albopicta; they were then cultured on a series of agarose media, including Luria-Bertani agar, potato dextrose agar, yeast malt extract agar, and yeast dextran agar plates. From all three dissections, only D. decipiens oita, determined by the internal transcribed spacer (ITS) sequencing afterward, was isolated. A phylogenetic tree was built based on the ITS sequence of D. decipiens oita and the 17 reported Daldinia spp., of which four type strains were included (Fig. 1). D. decipiens oita was categorized in a clade including paratype strain of D. decipiens JX658441, and the closest reported D. decipiens species was one of the fungal symbionts of X. camelus in Moravia, Czech Republic (15).
FIG 1.
Picture of Xyphydria albopicta (formerly X. camelus) with dissected mycangium organ in the expanded image (A) and the tree of the novel fungus isolate and other phylogenetically related fungi by ITS sequences (B) are shown. The names shown are GenBank accession codes. The filled red star indicates the position of D. decipiens oita, and the filled black star indicates the fungal strain (D. eschscholtzii EC12) used as a source of genome information for proteomic analysis. T and PT, type strain and paratypes, respectively.
Fungal cultivation on seven different carbon sources.
D. decipiens oita was cultivated for 3 days in liquid medium variants, each containing one of the following seven different plant materials as carbon sources. The protein concentration (μg/ml) of each culture supernatant (secretome) was determined to be 14.8 ± 0.8, 28.8 ± 6.4, 34.9 ± 2.1, 25.8 ± 3.5, 51.2 ± 8.3, 29.3 ± 2.7, and 30.4 ± 1.9 for the glucose, cellulose, xylan, mannan, pectin, poplar, and larch cultures, respectively (see Fig. S1 in the supplemental material). Each secretome was analyzed by SDS-PAGE (Fig. S2). In the glucose secretome, the total protein was less than that of other carbon source-based secretomes according to t test results (P < 0.01) in Fig. S1, and the band pattern on SDS-PAGE was quite different from others (Fig. S2). In the cellulose secretome, protein bands were distributed from 20 to 130 kDa, and two major bands were found with molecular weights between 40 and 50 kDa, which could be potential cellulases (20). In addition, the band pattern of the cellulose secretome was similar to the poplar and larch secretomes (Fig. S2). The secretomes from xylan, mannan, and pectin differed from the cellulose secretome. These results suggest that the D. decipiens oita secretes different sets of enzymes depending on the source of carbon provided in the growth medium.
Proteomic analysis of secretomes.
To identify the protein components present in each secretome, we performed proteomic analysis using the previously reported D. eschscholtzii EC12 protein data sets and CAZyme annotation (20). In Fig. 2 and Data Set S1 in the supplemental material, the identified proteins (unique peptides >2) and the putative CAZyme classification were shown for each secretome. We identified a total of 128 CAZymes, including domains of 92 glycoside hydrolases (GHs), 15 carbohydrate esterases (CEs), 5 polysaccharide lyases (PLs), 17 auxiliary activities (AAs), and 11 carbohydrate-binding modules (CBMs). Neither a glycosyl transferase (GT) nor expansin was found in any secretome tested in this study. Of the 15 detected CEs, there were four CE1s, one CE3, four CE4, one CE5, two CE12s, two CE15s, and one CE16. The five detected PLs consisted of four PL4s and one PL20. The 17 detected AA domains were as follows: two cellobiose dehydrogenases (CDHs) consisting of AA3 and AA8 domains, one AA3 GMC oxidase, one AA5_1 glyoxal oxidase (GLOX), one AA7 gluco-oligosaccharide oxidase (GOOX), and ten AA9 lytic polysaccharide monooxygenases (LPMOs). The vast majority (10 of 11) of identified CBMs are annotated as domains associated with the following catalytic domains: two AA9-CBM1s, two GH15-CBM20s, four GH43-CBM35s, and two GH72-CBM43s. Although CBM13 was identified in our secretome analyses, there are no associated additional domains that possess a reported function. Other proteins identified in the secretomes include extracellular peptidases, a limited number of intracellular proteins and secreted proteins with currently unknown function (see Data Set S1 in the supplemental material).
FIG 2.
Overall results for detected proteins secreted by D. decipiens oita grown in seven different cultures by LC-MS/MS (unique peptides > 2, n = 3).
In Fig. 2, the composition of proteins determined in each secretome is shown. The total number of proteins identified in the glucose secretome was 132, a relatively smaller number than that in the other secretomes (150 to 197). The percentage of CAZymes identified in the glucose secretome was around 20%, which was lower than that of other culture conditions (41% to 47% CAZymes). To note, most of the CAZymes identified in the glucose secretome were found in other secretomes (Fig. S3), suggesting that they are constitutively secreted enzymes from D. decipiens oita. These enzymes include GH3 β-glucosidases, GH16 and GH17 β-1,3 glucanases, GH18 chitinases, GH55 laminarinases (β-1,3-glucanase), and GH71 α-1,3-glucanase (Table S1), which are considered to be involved in fungal cell wall degradation (22, 23). In addition, constitutively secreted GH47 α-1,2-mannosidase, GH76 α-1,6-mannosidase, and GH125 α-mannosidase are possibly involved in protein glycan processing in the fungal endoplasmic reticulum (Table S1). Few CAZymes involved in plant polysaccharide degradation were constitutively secreted, including GH3 glycosidases, a GH28 polygalacturonase, a GH93 exo-α-l-1,5-arabinase, a GH15 glucoamylase with a CBM20 starch-binding domain, and an AA3-AA8 CDH (see Table S1 in the supplemental material), each of which is considered to break down easily digestible polysaccharides such as pectin, arabinan, and starch.
To compare a set of secreted enzymes in different cultures, the ten most abundant proteins in each culture were ordered based on the exponentially modified protein abundance index (emPAI) value (n = 3) (Table 1). Most of them have N-terminal secretion signals, and peptidases and hypothetical proteins were secreted in all the cultures. In the cellulose and lignocellulose (poplar and larch) cultures, the CAZymes involved in cellulose and xylan degradations were highly abundant. The GH6 and GH7 cellobiohydrolase (CBH)/endo-glucanase (EG) (OTB16487.1 and OTB15818.1) have theoretical molecular weights of 42.3 and 48.1 kDa, respectively (Table 1), which is consistent with the SDS-PAGE (Fig. S2). A GH11 endo-xylanase (EX), CE1 acetyl xylan esterase (AXE), and CE15 4-O-methyl-glucuronyl methylesterase, which function in xylan degradation, were relatively abundant. The GH5_subfamily 5 EG was an abundant cellulase in both cellulose and mannan cultures (Fig. S4). GH47 α-mannosidase was also enriched in mannan culture. Pectin strongly induced PL4 rhamnogalacturonan endolyases, which are involved in pectin degradation. In contrast, xylan induced various CAZymes, including GH43 arabinan endo-α-1,5-arabinosidase, CE1 AXE, and PL4 rhamnogalacturonan endolyases. These results indicate that growth media containing different plant polysaccharides can greatly influence secretion of CAZymes involved in plant cell wall degradation. In Fig. S5, the secreted 128 CAZymes in different carbon sources were illustrated in the heatmap with a dendrogram based on peptide counts. Among four purified polysaccharides, cellulose, xylan, mannan, and pectin, CAZyme expression patterns seemed distinguishable from each other. In contrast, the patterns between two lignocelluloses, poplar and larch, were similar. In addition, CAZymes found in the culture supernatants of two lignocelluloses seemed to be a combination of the CAZymes abundant in cellulose, xylan, and mannan cultures. To provide better insight into the pattern of secreted CAZymes in different carbon sources, a total of 128 secreted CAZymes were mapped in the Venn diagrams based on the associated Table S2 (Fig. 3). In Fig. 3A, a total of 126 CAZymes were detected in the four plant polysaccharide secretomes and glucose secretome, of which 41 CAZymes were shared among cellulose, xylan, mannan, and pectin secretomes. There were 20 cellulose-specific, 7 xylan-specific, 6 mannan-specific, and 7 pectin-specific CAZymes, respectively, in accordance with their annotated functions in Table S2. In Fig. 3B, we also compared two lignocellulose secretomes with three pure polysaccharide secretomes. Strictly between the poplar secretome (including 80 CAZymes) and larch secretome (including 84 CAZymes) comparison, 72 CAZymes were shared, which means that poplar and larch secretomes are similar. Except for two larch-specific CAZymes, a GH5_subfamily 16 β-1,6-glucanase and a GH2 mannanase, all of the CAZymes secreted in the two lignocellulose cultures were also detected in the cellulose-, xylan-, or mannan-combined secretome.
TABLE 1.
Ten most abundant proteins in culture supernatants from seven different carbon source-containing mediaa
| Medium and protein no. | Accession no. | Putative function (SS)b | Mol wt (kDa) | pI | Coverage (%) | No. of peptides | Area (n = 3) | emPAI |
|---|---|---|---|---|---|---|---|---|
| Glucose | ||||||||
| 1 | OTB15176.1 | Glutaminase (SS) | 75.1 | 4.6 | 25.7 | 11 | 5.8E+09 | 12.69 |
| 2 | OTB09986.1 | Carboxypeptidase (SS) | 66.4 | 4.9 | 19.3 | 8 | 9.0E+08 | 3.64 |
| 3 | OTB18686.1 | Serine protease/peptidase S28 (SS) | 60.3 | 4.8 | 31.3 | 12 | 2.2E+09 | 3.10 |
| 4 | OTB13697.1 | Hypothetical protein (SS) | 38.4 | 4.9 | 22.7 | 5 | 1.6E+08 | 2.98 |
| 5 | OTB11289.1 | Endo-peptidase (SS) | 33.5 | 6.8 | 21.1 | 5 | 1.9E+09 | 2.79 |
| 6 | OTB11810.1 | Ribonuclease (SS) | 13.8 | 5.2 | 22.2 | 2 | 3.8E+08 | 2.16 |
| 7 | OTB13532.1 | Serine protease/peptidase S28 | 56.4 | 5.0 | 15.3 | 6 | 4.0E+08 | 2.01 |
| 8 | OTB11189.1 | GH76 α-1,6-mannanase | 40.8 | 4.4 | 12.3 | 3 | 2.9E+08 | 1.89 |
| 9 | OTB12888.1 | Translation elongation factor alpha | 49.8 | 9.1 | 27.8 | 11 | 9.9E+07 | 1.89 |
| 10 | OTB18872.1 | Hypothetical protein | 13.6 | 5.4 | 36.7 | 4 | 7.3E+07 | 1.78 |
| Cellulose | ||||||||
| 1 | OTB11552.1 | Serine protease (SS) | 39.8 | 6.5 | 43.9 | 9 | 4.3E+09 | 50.80 |
| 2 | OTB16487.1 | GH6 cellobiohydrolase/endoglucanase (SS) | 42.3 | 4.9 | 50.9 | 18 | 2.9E+10 | 23.04 |
| 3 | OTB13329.1 | CE1 carbohydrate esterase (SS) | 31.5 | 7.5 | 25.6 | 6 | 1.0E+10 | 17.48 |
| 4 | OTB15818.1 | GH7 cellobiohydrolase/endoglucanase (SS) | 48.1 | 4.9 | 39.0 | 17 | 3.0E+10 | 16.01 |
| 5 | OTB19514.1 | CE15 4-O-methyl-glucuronoyl methylesterase (SS) | 36.8 | 7.5 | 39.5 | 8 | 1.7E+09 | 13.13 |
| 6 | OTB10635.1 | Hypothetical protein (SS) | 20.0 | 5.0 | 24.6 | 4 | 1.9E+08 | 12.90 |
| 7 | OTB13000.1 | GH5_5 endo-β-1,4-glucanase | 36.3 | 5.1 | 40.7 | 6 | 3.6E+09 | 7.73 |
| 8 | OTB12538.1 | Hypothetical protein (SS) | 37.8 | 7.0 | 33.0 | 10 | 1.7E+09 | 7.58 |
| 9 | OTB17511.1 | CE15 4-O-methyl-glucuronoyl methylesterase (SS) | 42.0 | 7.1 | 41.3 | 8 | 9.6E+09 | 7.19 |
| 10 | OTB12194.1 | GH11 endo-β-xylanase | 23.4 | 8.8 | 27.2 | 6 | 7.4E+09 | 7.11 |
| Xylan | ||||||||
| 1 | OTB11552.1 | Serine protease (SS) | 39.8 | 6.5 | 43.4 | 8 | 5.1E+09 | 83.83 |
| 2 | OTB15176.1 | Glutaminase (SS) | 75.1 | 4.6 | 25.7 | 9 | 5.2E+09 | 5.58 |
| 3 | OTB09891.1 | Hypothetical protein (SS) | 23.3 | 8.1 | 36.0 | 7 | 2.6E+09 | 4.88 |
| 4 | OTB14661.1 | Metallopeptidase M36 (SS) | 69.6 | 4.9 | 22.3 | 6 | 3.3E+09 | 4.62 |
| 5 | OTB11810.1 | Ribonuclease (SS) | 13.8 | 5.2 | 22.2 | 2 | 1.6E+09 | 4.62 |
| 6 | OTB13329.1 | CE1 carbohydrate esterase (SS) | 31.5 | 7.5 | 23.9 | 5 | 6.8E+08 | 4.41 |
| 7 | OTB15726.1 | Metallopeptidase M28 (SS) | 53.0 | 5.3 | 28.1 | 12 | 3.3E+08 | 3.92 |
| 8 | OTB18670.1 | GH43 endo-α-1,5-l-arabinosidase | 34.2 | 7.7 | 28.3 | 7 | 1.4E+09 | 3.44 |
| 9 | OTB10580.1 | PL4 rhamnogalacturonan endolyase (SS) | 57.6 | 9.0 | 22.5 | 7 | 1.7E+09 | 3.37 |
| 10 | OTB11289.1 | Endo-peptidase (SS) | 33.5 | 6.8 | 19.1 | 4 | 3.4E+09 | 3.28 |
| Mannan | ||||||||
| 1 | OTB11552.1 | Serine protease (SS) | 39.8 | 6.5 | 43.9 | 8 | 2.5E+09 | 116.88 |
| 2 | OTB09891.1 | Hypothetical protein (SS) | 23.3 | 8.1 | 46.7 | 8 | 1.1E+09 | 7.38 |
| 3 | OTB15176.1 | Glutaminase (SS) | 75.1 | 4.6 | 25.7 | 9 | 6.1E+09 | 7.11 |
| 4 | OTB14661.1 | Metallopeptidase M36 (SS) | 69.6 | 4.9 | 23.1 | 7 | 2.7E+09 | 5.63 |
| 5 | OTB11810.1 | Ribonuclease (SS) | 13.8 | 5.2 | 22.2 | 2 | 1.6E+08 | 4.62 |
| 6 | OTB13252.1 | GH47 α-mannosidase | 58.0 | 4.6 | 24.1 | 8 | 1.1E+09 | 3.64 |
| 7 | OTB15726.1 | Metallopeptidase M28 (SS) | 53.0 | 5.3 | 28.1 | 12 | 2.1E+08 | 3.51 |
| 8 | OTB13532.1 | Serine protease (SS) | 56.4 | 5.0 | 15.3 | 6 | 3.1E+09 | 3.49 |
| 9 | OTB13000.1 | GH5_5 endo-β-1,4-glucanase | 36.3 | 5.1 | 35.2 | 5 | 1.1E+09 | 3.44 |
| 10 | OTB11194.1 | Ribosomal protein, including ubiquitin | 17.5 | 9.7 | 30.1 | 5 | 1.7E+08 | 3.33 |
| Pectin | ||||||||
| 1 | OTB14661.1 | Metallopeptidase M36 (SS) | 69.6 | 4.9 | 28.0 | 9 | 8.2E+09 | 11.80 |
| 2 | OTB11552.1 | Serine protease (SS) | 39.8 | 6.5 | 35.4 | 5 | 9.8E+08 | 9.00 |
| 3 | OTB09891.1 | Hypothetical protein (SS) | 23.3 | 8.1 | 46.7 | 8 | 3.1E+09 | 7.38 |
| 4 | OTB15176.1 | Glutaminase (SS) | 75.1 | 4.6 | 21.6 | 8 | 5.5E+09 | 5.58 |
| 5 | OTB09986.1 | Carboxypeptidase (SS) | 66.4 | 4.9 | 19.3 | 8 | 3.0E+09 | 4.78 |
| 6 | OTB11810.1 | Ribonuclease (SS) | 13.8 | 5.2 | 22.2 | 2 | 8.6E+08 | 4.62 |
| 7 | OTB11289.1 | Endo-peptidase (SS) | 33.5 | 6.8 | 22.4 | 6 | 7.3E+09 | 4.46 |
| 8 | OTB20031.1 | PL4 rhamnogalacturonan endolyase (SS) | 75.8 | 5.0 | 24.5 | 11 | 1.5E+09 | 3.25 |
| 9 | OTB10580.1 | PL4 rhamnogalacturonan endolyase (SS) | 57.6 | 9.0 | 28.8 | 9 | 1.8E+09 | 2.98 |
| 10 | OTB15726.1 | Metallopeptidase M28 (SS) | 53.0 | 5.3 | 28.1 | 12 | 1.5E+08 | 2.78 |
| Poplar | ||||||||
| 1 | OTB11552.1 | Serine protease (SS) | 39.8 | 6.5 | 43.9 | 8 | 3.1E+09 | 42.94 |
| 2 | OTB15818.1 | GH7 cellobiohydrolase/endoglucanase (SS) | 48.1 | 4.9 | 37.2 | 16 | 3.3E+09 | 7.38 |
| 3 | OTB09891.1 | Hypothetical protein (SS) | 23.3 | 8.1 | 46.7 | 8 | 1.1E+09 | 6.02 |
| 4 | OTB15176.1 | Glutaminase (SS) | 75.1 | 4.6 | 25.7 | 9 | 1.2E+09 | 5.58 |
| 5 | OTB16487.1 | GH6 cellobiohydrolase/endoglucanase (SS) | 42.3 | 4.9 | 33.4 | 12 | 4.4E+09 | 5.45 |
| 6 | OTB14661.1 | Metallopeptidase M36 (SS) | 69.6 | 4.9 | 23.1 | 7 | 1.7E+09 | 5.11 |
| 7 | OTB11810.1 | Ribonuclease (SS) | 13.8 | 5.2 | 22.2 | 2 | 8.3E+08 | 4.62 |
| 8 | OTB12194.1 | GH11 endo-β-xylanase | 23.4 | 8.8 | 27.2 | 6 | 3.6E+08 | 4.34 |
| 9 | OTB16996.1 | Hypothetical protein (SS) | 25.1 | 5.6 | 18.6 | 4 | 5.0E+08 | 3.33 |
| 10 | OTB13329.1 | CE1 carbohydrate esterase (SS) | 31.5 | 7.5 | 23.9 | 5 | 6.2E+08 | 2.98 |
| Larch | ||||||||
| 1 | OTB11552.1 | Serine protease (SS) | 39.8 | 6.5 | 45.0 | 10 | 3.7E+09 | 315.23 |
| 2 | OTB16487.1 | GH6 cellobiohydrolase/endoglucanase (SS) | 42.3 | 4.9 | 40.5 | 16 | 9.8E+09 | 9.00 |
| 3 | OTB09891.1 | Hypothetical protein (SS) | 23.3 | 8.1 | 48.1 | 8 | 1.3E+09 | 6.02 |
| 4 | OTB12194.1 | GH11 endo-β-xylanase | 23.4 | 8.8 | 27.2 | 6 | 9.6E+08 | 5.58 |
| 5 | OTB15818.1 | GH7 cellobiohydrolase/endoglucanase (SS) | 48.1 | 4.9 | 37.2 | 16 | 9.0E+09 | 5.42 |
| 6 | OTB13329.1 | CE1 carbohydrate esterase (SS) | 31.5 | 7.5 | 23.9 | 5 | 1.5E+09 | 5.31 |
| 7 | OTB19514.1 | CE15 4-O-methyl-glucuronoyl methylesterase (SS) | 36.8 | 7.5 | 35.4 | 7 | 5.6E+08 | 4.62 |
| 8 | OTB11810.1 | Ribonuclease (SS) | 13.8 | 5.2 | 22.2 | 2 | 8.7E+08 | 4.62 |
| 9 | OTB11289.1 | Endo-peptidase (SS) | 33.5 | 6.8 | 22.4 | 6 | 2.1E+09 | 4.46 |
| 10 | OTB16996.1 | Hypothetical protein (SS) | 25.1 | 5.6 | 38.1 | 5 | 1.8E+09 | 4.34 |
The theoretical molecular weight and pI were calculated based on the corresponding amino acid sequences, and emPAI values were estimated based on 3 independent proteomic results for each protein.
SS, secretion signal.
FIG 3.
Venn diagram comparing CAZymes from different secretomes. Secretomes from glucose, cellulose, xylan, mannan, and pectin cultures (A) and from cellulose, xylan, mannan, poplar, and larch cultures (B) are compared.
Polysaccharide-degrading activities of the D. decipiens oita secretomes.
To support the proteomic results described in the previous sections, polysaccharide-degrading activities of each secretome were measured. We used the same amount of proteins from each secretome and tested their ability to degrade eight different pure polysaccharides, including Avicell, carboxymethyl cellulose (CMC), oat-spelt xylan, beech xylan, mannan, pectin, xyloglucan, and arabinan (Fig. 4). The specific activity (U/mg) of each reaction was evaluated, except for the Avicell hydrolysis (U/g), since Avicell is relatively less reactive than other polysaccharides due to the high crystalline cellulose structure (Fig. S6). Among the seven secretomes, the glucose secretome showed the lowest activity throughout the substrate panel, consistent with its proteomic results (Fig. 2). With the cellulose secretome, CMC, oat-spelt xylan, beech xylan, and xyloglucan were well hydrolyzed, and yet pectin, mannan, and arabinan were hydrolyzed less effectively. Although the pectin secretome had significantly high carboxymethyl cellulase (CMCase), mannanase, pectinase, and arabinase activities, the xylanase and xyloglucanase activities were somewhat low. Both xylan and mannan secretomes possessed a broad range of activities, and there was no apparent difference found in the current results. For lignocellulose secretomes (poplar and larch), their activities were similar to each other, and CMCase and xylanase activities were comparable to those of cellulose secretome. In contrast, mannanase, pectinase, xyloglucanase, and arabinase activities in the lignocellulose secretomes were comparable to those of xylan or mannan secretomes. To provide different views of secretome activities, we also assessed polysaccharide activity measurements (U/liter) directly from the culture supernatant without filter concentration (Fig. S7). Altogether with the results from proteomic and biochemical analyses, it is thought that the poplar and larch secretome activities are the sum of the cellulose, xylan, and mannan secretomes.
FIG 4.
Specific activities (U/mg) of seven different secretomes toward the following pure polysaccharides: CMC (A), oat-xylan (B), beech xylan (C), mannan (D), pectin (E), xyloglucan (F), and arabinan (G). The averages and standard deviations were estimated using three biological replicas from each secretome and reaction.
DISCUSSION
We isolated a Japanese woodwasp-associated symbiotic fungus from specimens of Xiphydria albopicta (formerly X. camelus) captured in northern Japan (Fig. 1). ITS sequencing assigned this fungus as a well-characterized Xiphydria-associated fungus, Daldinia decipiens (14, 15), and we named a new isolate D. decipiens oita. In Daldinia, belonging to Xylariales, Ascomycota, 47 taxa have been assessed and are widely distributed over the world, most of which are traditionally regarded as saprotrophic fungi that cause white-rots (16). Among them, D. decipiens was found in Northern Europe and recognized to be a woodwasp-associated species (14–16). To determine the CAZyme composition of D. decipiens oita, LC-MS/MS was used to analyze the secretomes of this fungus independently grown on either glucose, one of the four major purified plant polysaccharides (cellulose, xylan, mannan, and pectin), or two lignocellulose materials (poplar and larch) (Fig. 2 and Table S1). Currently, a total of 437 CAZyme-coding genes are annotated in the D. eschscholtzii EC12 genome, including 229 GHs, 37 CEs, 8 PLs, 22 CBMs, 79 AAs, and others (20). Using this database, we identified a total of 128 CAZymes, including domains of 92 GHs, 15 CEs, 5 PLs, 17 AAs, and 11 CBMs, which correspond to 27.4% (40.2, 40.5, 62.5, 21.5, and 55% of total GHs, CEs, PLs, AAs, and CBMs, respectively) of total CAZymes encoded in the genome. In the previous study, the secretome composition of a white rot fungus, Phlebiopsis gigantea, was determined by using the in-house sequenced genome information, and the following CAZymes were identified in the P. gigantea proteome: 64 of 197 GHs (32.5%), 7 of 19 CEs (36.8%), 0 of 5 PLs (0%), 20 of 94 AAs (21.3%), and 4 of 17 CBMs (23.5%) (24). Thus, overall our proteomic analyses of D. decipiens oita using the genome of the same genus D. eschscholtzii EC12 had provided a fair number of identified CAZymes, though we should account for the possibility that some CAZymes encoded in the genome D. decipiens oita could be missing in our analyses.
In the secretome profiles of D. decipiens oita, the classical fungal cellulases, cellobiohydrolases (CBHs), and endoglucanases (EGs), belonging to GH7, GH6, GH5_subfamily 5, GH12, and GH45, were secreted to decompose cellulose synergistically. For hemicellulose degradation, a variety of GHs that target different types of sugar linkages in hemicellulose was detected, including GH2 β-1,4-mannosidase, GH3 glycosidase, GH5_subfamily 7 β-mannanase, GH10 endo β-1,4-xylanases (EXs), GH11 EXs, GH29 α-l-fucosidase, GH28 polygalacturonase, GH30 EX/EG, GH35 β-galactosidase GH43 hemicellulase, GH51 α-l-arabinosidase, GH67 α-glucuronidase, GH74 xyloglucanase, GH78 α-l-rhamnosidase, and GH105 unsaturated rhamnogalacturonoyl hydrolase. In addition, carbohydrate esterases belonging to CE1, CE3, CE4, CE5, CE12, and CE16 were identified in the secretomes, a group of which are predicted to remove acetyl groups from hemicellulose in the plant cell wall. Another CE enzyme, CE15 4-O-methyl-glucuronyl methylesterase, was also identified, which may cleave hemicellulose-lignin linkages (25). Finally, four PL4 rhamnogalacturonan endolyases for pectin degradation and one PL20 glucuronan lyase were detected. Altogether, these results indicate that D. decipiens oita possesses broad activity for plant polysaccharide degradation. Indeed, our reported polysaccharide-degrading activities of D. decipiens oita (Fig. 4) were higher and broader than that of another lignocellulose-degrading sordaiomycete, Podospora anserina (26), suggesting the CAZymes used by D. decipiens oita may have significant potential for industrial use.
The GH6 and GH7 CBHs and EG (OTB16487.1 and OTB15818.1) were identified as major secreted CAZymes of D. decipiens oita (Fig. S2 and Table 1), but we found that these cellulases lacked the canonical CBM1 domain (Fig. S8). Generally, conventional cellulases belonging to the GH5_subfamily 5, GH6, and GH7 possess the CBM1 domain as the associated domain to support crystalline cellulose degradation. However, three GH6 genes, five GH7 genes, and three GH5_subfamily 5 genes carried in the D. eschscholtzii EC12 genome all lacked a CBM1 domain. The only exception was GH6 (OTB18636.1), which was not identified in the secretomes analyzed in this study. Despite the general lack of CMB1 domain-containing cellulases, some of secreted AA9 LPMOs possessed N-terminal CBM1 domains (OTB13872.1 and OTB18238.1). Compared to some other lignocellulose-degrading fungi (27, 28), CAZymes lacking the associated CBM1 could be unique to the Daldinia spp.
In the case of 13 detected auxiliary activity enzymes (AAs), 10 AA9 LPMOs were found, together with one or two AA3-AA8 CDHs, suggesting that LPMOs and their redox partner, CDH(s), function in oxidative cleavage of cellulose or hemicellulose chains (29). In the glucose, mannan, pectin, and lignocellulose (poplar and larch) secretomes, one AA7 gluco-oligosaccharide oxidase was identified, whose function is oxidization of cello- and xylo-oligosaccharides (30, 31), although a precise physiological function is currently unclear. In the case of lignin degradation, either AA5_subfamily 1 glyoxal oxidase or AA3 GMC oxidoreductase, known as an H2O2 supply enzyme to class II peroxidases (PODs) such as lignin peroxidase (32), was secreted in the cellulose, xylan, mannan, and lignocellulose secretomes but not found in the glucose or pectin secretome. No AA2 POD was detected in the tested supernatants. These results suggested that the H2O2-producing enzymes may not supply peroxide to POD, but it may supply other mechanism(s), such as Fenton chemistry and LPMOs in D. decipiens oita (29). The genome used for the database possesses eight AA1 families, including six multicopper oxidases and two ferroxidases, though neither laccases nor other AA1 proteins were found in our proteomic analysis. Thus, consistent with other lignocellulose-degrading ascomycetes (27), D. decipiens oita seems to utilize not oxidative lignin degradation but the LPMO-CDH system.
Most of the lignocellulose (poplar and larch) secretomes can be explained by a combination of the CAZymes found in the cellulose, xylan, and mannan secretomes (Fig. 3B and Fig. S5). Consistently, the specific degradation activities of poplar and larch secretomes toward seven different purified substrates seemed to be a combination of reactivities of cellulose, xylan, and mannan secretomes (Fig. 4). The major polysaccharide compositions of softwoods (conifer) are cellulose, mannan, and xylan, whereas hardwoods are mainly composed of cellulose and xylan (33). Taken with the degradation activity of the supernatants, we reason that D. decipiens oita recognizes different plant polysaccharide compositions contained in these lignocellulose materials and, in turn, secretes a suite of CAZymes for efficient degradation. Within the purified plant polysaccharides, cellulose strongly induced cellulose- and xylan-degrading enzymes, while pectin strongly induced pectin-degrading enzymes with various hemicellulases (Fig. 3A and Fig. 4). This finding is consistent with the previous study of a lignocellulose-degrading ascomycete that utilizes cellulose and sugar beet pulp (arabinan) substrates very actively (26). Also, the secretomes of cellulose, xylan, mannan, and pectin differed from each other (Fig. 3A and Fig. S5), which indicates that D. decipiens oita senses the unique sugar components of each polysaccharide such as cello-oligosaccharides, xylose, arabinose, mannose, galacturonic acids, l-rhamnose, d-galactose, and other products in the plant cell wall to regulate secretions of CAZymes. In filamentous fungi, several CAZyme induction mechanisms via sensing signal molecules from plant polysaccharides derivatives of mono- and oligo-sugars have previously been proposed (34). Further research will investigate the polysaccharide-responsive signaling mechanisms used by D. decipiens oita to regulate CAZyme production and secretion.
Various types of peptidases were constitutively secreted in the tested secretomes (Table 1 and Data Set S1). In addition, CAZymes involved in the degradation of the fungal cell wall and easily degradable polysaccharides (arabinan, pectin, and starch) were constitutively secreted, although their assayed specific activities for arabinan and pectin degradation in the glucose-induced secretome were negligible (Fig. 4 and Table S1). The constitutively secreted peptidases and a limited number of CAZymes under the growth of sufficient glucose have been reported for other filamentous fungi, including Ascomycetes and Basidiomycetes (35, 36). Thus, with regard to constitutively secreted peptidases, D. decipiens oita may extract N sources from accessible polypeptides in the plant cell wall and also recycle them from secreted enzymes. Meanwhile, identified constitutively secreted fungal cell wall-degrading CAZymes might be important for D. decipiens oita to outcompete other microbial contaminants in the symbiotic environments, consistent with several studies on mycoparasitic fungi (22, 35). In future studies, the physiological roles of this fungus can be directly assessed by the meta-omics approach. For example, one of the well-characterized insect-associated interactions was reported in the symbiotic gardens of leaf-cutter ants by means of metagenomic and metaproteomic approaches (37–39). Series of enzymes, such as glycoside hydrolases, prominent laccases, and peptidases, were secreted by the fungus Leucoagaricus gongylophorus to supply C and N sources from the plant biomass to the members in the symbiotic garden.
Altogether, we report here the detailed secretomic analyses of the new insect associated fungus, D. decipiens oita, isolated from a woodwasp in a northern Japan forest, grown in the defined media. Results indicated that wood wasp-associated D. decipiens oita secretes diverse CAZymes and peptidases to efficiently extract energy in the wood-devastating insect-associated symbiotic environment.
MATERIALS AND METHODS
General reagents.
Polysaccharides used for culture medium were as follows: glucose (Sigma-Aldrich, St. Louis, MO), beech xylan (Megazyme, Ireland), mannan (Megazyme), pectin (Sigma-Aldrich), larch wood, and poplar wood. Larch wood and poplar wood powder were collected and ground (<0.5 mm) in this study. Polysaccharides used for enzyme assays were Avicell (Sigma-Aldrich), CMC (Megazyme), oat-spelt xylan (Megazyme), beech xylan (Megazyme), mannan (Megazyme), pectin (Wako Pure Chemical Industries, Tokyo, Japan), xyloglucan (Megazyme), and arabinan (Megazyme).
Fungal species classification.
Mycangia, organs to maintain symbiont microbes, were suspended in a saline solution then spread on potato dextrose agar (Wako Pure Chemical Industries). The colonies were separated and grown several times until single filamentous fungus was isolated, then isolates were grown in a potato dextrose broth. The genomic DNA was extracted from the collected mycelium by using DNeasy plant kit (Qiagen, Germany). The ITS region, including 5.8 rRNA, was amplified by using a primer set ITS1 and ITS4 as previously described (40). The ITS sequence was searched on the NCBI blastn site (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome), and the best hit showed 100% identity to Daldinia decipiens isolated from Xyphydria woodwasp in the Czech Republic (HM192910).
Proteomic database construction using the draft genome of D. eschscholtzii EC12.
The genome of D. eschscholtzii EC12 was reported previously (20), and the draft genome is available in the NCBI data bank (accession no. PRJNA196027), of which 11,161 putative protein-coding genes were annotated. The FASTA file that contains all the putative protein sequences and previously assigned CAZy functions was used in the proteomic analysis (https://www.ncbi.nlm.nih.gov/protein/?term=txid1001832[Organism:noexp]).
Phylogenetic tree analysis within Daldinia spp.
To build a phylogenetic tree, 13 ITS sequences of the reported Daldinia spp. were collected from the GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi): D. placentiformis, AM749939; D. loculata, AF176959; D. fissa, AF176976; D. childiae, AM292044; D. petriniae, AF176970; D. decipiens, HM192910, HM192911, AM292040, and AM292042; D. clavata, AM749931; D. eschscholtzii, MF029744; D. eschscholtzii strain EC12, JQ424940; and D. caldariorum, AM749934. In addition, the sequences of the related type strains were collected as follows: D. decipiens (JX658441), D. barkalovii (JX658537), D. carpinicola (JX658442), and D. govorovae (JX658443). The Hypoxylon macrocarpum (AY616705) was used as an outgroup. These sequences were aligned using MAFFT (https://mafft.cbrc.jp/alignment/server/) (41) using default parameters and RAxML (42) was used to build phylogenetic tree (bootstrap = 1,000) using default parameters.
Fungal cultivation.
D. decipiens oita was grown in modified Highley medium (43) containing 1% (wt/wt) glucose, 1% (wt/wt) Avicell (Sigma-Aldrich, MO), 1% (wt/wt) beechwood xylan, 1% (wt/wt) mannan, 1% (wt/wt) pectin, 1% (wt/wt) poplar, or 1% (wt/wt) larch as a carbon source with supplementation of 1% (wt/wt) yeast extract and 2% (wt/wt) polypeptone for 3 days at 25°C at 150 rpm. Three days of cultivation was exponential growth phase under these cultivation conditions. The culture supernatant was collected by filtration (Vivaspin 20, 5,000 MWCO; Sartorius Stedim, Germany), and the protein concentration was estimated by a protein assay (Bio-Rad, Hercules, CA). Portions (10 μg) of the crude protein were analyzed by SDS-PAGE prior to the enzyme assay and proteome analysis to confirm that there was not any detectable level of unwanted proteolytic digestion.
Plant polysaccharide degradation activity assays.
For enzyme activity assays of each secretome from three biological replicas with three technical replicas, both soluble and insoluble polysaccharides were adjusted to 10 mg/ml in 0.1 M sodium acetate (pH 5.0). In the assay, 50 μl of the crude protein sample (10 μg/ml) was mixed with 50 μl of each substrate in a total reaction volume of 100 μl. The reaction was performed for 16 h at 37°C. After completion of the reaction, the reducing end product was measured by the dinitrosalicylic acid assay at an optical density at 540 nm (44), and the molar equivalent of reducing sugar was estimated by using a glucose standard (13). The averages and standard deviations were estimated based on technical triplicates with three biological replicas.
Proteomic analysis of the secretome.
One hundred micrograms of crude secreted proteins of each culture condition from three independent biological replicas was precipitated by 20% (vol/vol) trichloroacetic acid and then washed with ice-cold acetone two times. The precipitated protein sample was dissolved in 10 μl of 8 M urea, and then 70 μl of 25 mM ammonium bicarbonate buffer was added. The sample was reduced by adding 5 mM dithiothreitol for 30 min at 50°C, followed by alkylation by adding 15 mM iodoacetamide for 30 min at room temperature in the dark. Tryptic digestion was performed by adding 1.0 μg of proteomic-grade trypsin (Roche, Germany) to each sample, followed by incubation overnight at 37°C. The trypsin-digested peptides were separated by using a nanoLC1000 (Thermo Fisher Scientific, Rockford, IL) equipped with a C18 column (NTCC-360/75-3-125; Nikkyo Technos, Tokyo, Japan) connected to a hybrid linear ion trap-orbitrap mass spectrometer (Q-Execative Plus; Thermo Fisher Scientific) and operated by using an Xcalibur software v3.1 (Thermo Fisher Scientific). Peptide fragments separated by a linear gradient raised from 5 to 30% acetonitrile in 0.1% formic acid for 120 min were monitored at a scan range of 300.0 to 2,000.0 m/z and a resolution of 70,000. Raw MS/MS data were analyzed by using Proteome Discoverer software v2.1 (Thermo Fisher Scientific) under the following settings: the peptide mass tolerance was set to 10 ppm, and the fragment mass tolerance was set to 0.8 Da. Fixed modifications of carbamidomethyl at Cys and dynamic modifications of oxidation at Met, phosphorylation at Glu, His, Lys, Arg, Ser, Thr, and Tyr, and acetylation at His, Lys, Ser, Thr, and Tyr were used for the database search. The probable significance of the identifications was carried out using the same proteome discoverer software. For the stringent screening of proteins determined by proteomics in each secretome, we omitted the proteins with the unique peptides of less than two fragments. Based on acquired LC-MS/MS spectra, the abundance of each protein was semiquantitatively calculated using emPAI value (45). All detected protein sequences were searched by blastp with an E value of 10–15 using the default parameter of the blast2go v5.2 software (46) to annotate putative functions, as previously described (47). Secretion signal peptide was predicted using SignalP v5.0 (http://www.cbs.dtu.dk/services/SignalP/) (48). The theoretical Mw and pI values of the corresponding proteins were calculated using ExPASy (https://web.expasy.org/compute_pi/). All raw data have been uploaded to and are freely available at the Chorus website (ID 207108 to ID 207128, https://chorusproject.org/pages/index.html). The averages and standard deviations of area values were estimated from three biological replicas obtained by LC-MS/MS, and the emPAI represents the values of three biological replicates.
Phylogenetic analyses of GH5 protein family.
To determine the subfamily of 15 GH5 genes carried in the D. decipiens genome, we prepared a phylogenetic tree of GH5 genes with the amino-acid sequences belonging to Eukaryote subfamilies 1, 2, 4, 5, 7, 8, 9, 11, 12, 15, 16, 22, 23, 24, 27, 30 31, 49, 50, and 51 (49). As previously described (50), the multiple alignment was performed with MAFFT software (https://mafft.cbrc.jp/alignment/software/) using default parameters, and phylogenetic trees were built by RAxML with bootstrapping (n = 1,000) also using default parameters.
Data availability.
The ITS sequence of D. decipiens oita was deposited in the NCBI data bank (DDBJ/EMBL/GenBank accession no. LC376942).
Supplementary Material
ACKNOWLEDGMENTS
This study was supported by the JSPS grants 16K18727 and 19K15881 (to C.H.), an Inamori Foundation grant (to T.E.T.), an NSF grant 1514923 (to B.B.M.), and JSPS KAKENHI grants 15KK0269 and 15K18812 (to T.E.T.). The authors declare no conflict of interest.
C.H. and T.E.T. designed the experiments. C.H., R.S., B.B.M., S.O., and H.H. performed the experiments. C.H., K.M., R.M., B.B.M., and T.E.T. analyzed the results. C.H., B.B.M., and T.T. wrote the manuscript. All the authors agreed on the manuscript.
Footnotes
Supplemental material is available online only.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The ITS sequence of D. decipiens oita was deposited in the NCBI data bank (DDBJ/EMBL/GenBank accession no. LC376942).