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1
Supporting Information
I. Supplementary results
1. Genome features
Table S1. Genome completeness and assembly metrics of M. importuna SCYDJ1-A1 and CCBAS932
strains.
M. importuna SCYDJ1-A1
M. importuna CCBAS932
Genome assembly size
48.80 Mbp
48.21 Mbp
Sequencing read coverage depth
83.9×
67.8×
Number of contigs
1509
1793
Number of scaffolds
338
540
Number of scaffolds ≥ 2 kbp
294
423
Scaffold N50
27
29
Scaffold L50
0.61 Mbp
0.60 Mbp
Number of gaps
1171
1253
Scaffold length in gaps
2.2%
2.1%
Three largest scaffolds (Mbp)
1.99 Mbp, 1.58 Mbp and 1.24 Mbp
1.46 Mbp, 1.39 Mbp and 1.36 Mbp
2
Fig. S2. Pairwise synteny of scaffolds between the genomes of M. importuna strains SCYDJ1-A1 (X-axis) and CCBAS932 (Y-axis). The VISTA program (Martin et al.,
2004) integrated in the JGI Annotation Pipeline was used for pairwise alignment of scaffolds as well as visualization of the alignment results. Threshold of sequence length
with continuous high homology was set at 50 bp cut-off. The dot-plot figure was extracted from the JGI MycoCosm genome portal of M. importuna SCYDJ1-A1.
3
Table S2. Comparison of gene-model characteristics between M. importuna SCYDJ1-A1 (red) and
CCBAS932 (blue) strains.
Filtered gene model
Common gene
Strain-specific gene
gene counts
11971
9891
2080
11600
9873
1727
genes with PFAM hit
6607 (55.19%)
6456 (65.27%)
151 (7.26%)
6637 (57.22%)
6450 (65.33%)
187 (10.83%)
exons per gene
3.23
3.55
1.67
3.38
3.61
2.07
introns per gene
2.23
2.55
0.67
2.38
2.61
1.07
protein average length
(amino acids)
398
449
155
410
453
168
genes with predicted
signal peptide
1359 (11.35%)
1174 (11.87%)
185 (8.89%)
1318 (11.36%)
1130 (11.45%)
188 (10.89%)
genes of predicted
transmembrane protein
2480 (20.72%)
1853 (18.73%)
627 (30.14%)
2334 (20.12%)
1858 (18.82%)
476 (27.56%)
Table S3. 9783 common genes shared by the SCYDJ1-A1 and CCBAS932 strains of M. importuna,
determined by BlastP Best Reciprocal Hit analysis. The table is of big size, and is therefore provided as an
individual Excel file available online: TableS3.xls.
All files of the gene prediction and functional annotation information, including gene identification,
CDS, predicted transcripts and proteins, SignalP-predicted signal peptides, PFAM, KOG, InterPro, GO term,
KEGG pathway, and EC number, are downloadable from:
https://genome.jgi.doe.gov/portal/pages/dynamicOrganismDownload.jsf?organism=Morimp1. Account login
is required for download.
4
2. Physiochemical and physical conditions of morel cultivation in this study
Table S4. Initial state of physiochemical characteristics of the pre-homogenized soil used as the mushroom
bed for morel cultivation.
Physiochemical parameter
Value
Bulk density
1.14 ± 0.07 g cm-3
pH
8.17 ± 0.02
Total organic C
6.04 ± 0.05 g kg-1
Total N
1.05 ± 0.06 g kg-1
Ammonium N
123.90 ± 7.50 mg kg-1
Total P
1.20 ± 0.08 g kg-1
Mineral P
37.59 ± 2.38 mg kg-1
Total K
15.94 ± 0.55 g kg-1
Exchangeable Ca
42.55 ± 2.01 cmol kg-1
Exchangeable Mg
1.99 ± 0.15 cmol kg-1
Available Cu
3.60 ± 0.17 mg kg-1
Available Fe
8.29 ± 0.19 mg kg-1
Available Zn
0.81 ± 0.02 mg kg-1
Available Mn
3.72 ± 0.32 mg kg-1
Fig. S2. ENB temperature measured by electronic thermometer sensors inserted into three testing
ENBs.-values are mean of three replicates.
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3. Influence of ENB presence on the chemical components in fruiting bodies
Fig. S3. Time-course changes in the content of organic compounds and mineral elements in fruiting bodies. The coloured columns indicate mean of three biological replicates,
Chemical composition (mg g-1 dry weight)
0
100
200
300
400
500
600
700
Total C
Total N
Total P
Total K
Total carbohydrates
Reducing sugars
Total proteins
Free amino acids
Crude fats
Remove ENB at day15
Remove ENB at day45
Remove ENB at day75
Keep ENB for the entire course
0
50
100
Total C
Total N
Total P
Total K
Total carbohydrates
Reducing sugars
Total proteins
Free amino acids
Crude fats
Chemical composition (mg g-1 dry weight)
6
with standard deviations. Significance of difference was judged by one-way ANOVA. A full list of all p-values is provided in Table S8.
The duration of ENB contact with the mushroom bed influenced the content of several organic compounds and mineral elements in fruiting bodies. Besides increasing
fruiting body yield, keeping ENB over 45 days led to higher contents in total proteins (p-value = 0.023, by one-way ANOVA) and free amino acids (p-value = 0.035, by
one-way ANOVA) but lower total N (p-value = 0.006, by one-way ANOVA), in comparison to keeping for only 15 days. This suggests that using ENB for longer time
tended to facilitate the conversion of non-amino acid N into amino acids and proteins. Total C (p-values between 0.879 to 1.000, by one-way ANOVA), total P (p-values
between 0.515 to 1.000, by one-way ANOVA), total K (p-values between 0.484 to 0.996, by one-way ANOVA) and crude fats (p-values between 0.631 to 0.998, by one-
way ANOVA) were not significantly affected by ENB contact duration. Compared with two previously reported samples of wild-collected M. importuna (Vieira et al.,
2016), the cultivated fruiting bodies harvested in this study contained a higher content of crude fats but lower contents of total carbohydrates and total proteins.
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4. Changes in dry weight and major nutrient contents during ENB decomposition
Table S5. Content of chemicals in ENB at day 0, 15, 45 and 75.
Content (mg per ENB)
day 0
day 15
day 45
day 75
pH
6.26 ± 0.02
6.09 ± 0.03
6.18 ± 0.03
6.36 ± 0.04
Water content
48.29 ± 0.91%
50.61 ± 1.31%
56.97 ± 1.16%
62.70 ± 2.51%
Total C
12.55 ± 0.20 × 104
11.05 ± 0.84 × 104
8.07 ± 0.62 × 104
6.92 ± 0.18 × 104
Total N
3398.71 ± 85.95
3999.34 ± 91.68
3789.73 ± 259.41
3590.03 ± 264.69
C:N ratio
36.92
27.63
21.30
19.26
Organic N
3377.21 ± 85.62
3980.65 ± 91.42
3761.86 ± 257.49
3524.22 ± 258.79
Ammonium N
21.50 ± 0.38
18.69 ± 0.43
27.86 ± 1.94
65.80 ± 6.64
Nitrate N
11.31 ± 0.73
8.94 ± 0.90
13.69 ± 1.20
11.28 ± 1.24
Total P
462.43 ± 9.81
435.95 ± 6.30
352.87 ± 22.54
313.37 ± 31.40
Mineral P
72.13 ± 1.93
89.05 ± 2.22
54.31 ± 2.98
37.78 ± 2.75
Total K
764.22 ± 7.41
720.00 ± 31.34
504.20 ± 28.71
357.29 ± 23.02
Total carbohydrates
13.55 ± 0.48 × 104
11.12 ± 0.37 × 104
7.64 ± 0.81 × 104
3.94 ± 0.26 × 104
Total reducing sugars
958.36 ± 15.75
1885.00 ± 17.25
1994.01 ± 167.74
1112.20 ± 82.52
Amylose
28.73 ± 2.09 × 103
19.65 ± 0.43 × 103
7.34 ± 0.77 × 103
2.47 ± 0.08 × 103
Amylopectin
5.02 ± 0.13 × 104
4.47 ± 0.02 × 104
3.36 ± 0.30 × 104
1.41 ± 0.08 × 104
Cellulose
2.59 ± 0.03 × 104
2.02 ± 0.03 × 104
1.63 ± 0.10 × 104
1.06 ± 0.06 × 104
Hemicellulose
18.67 ± 0.06 × 103
12.51 ± 0.93 × 103
10.42 ± 0.16 × 103
5.41 ± 0.43 × 103
Protopectins
9096.87 ± 578.76
7547.25 ± 351.79
3114.69 ± 162.42
869.04 ± 92.38
Water-soluble pectins
2857.86 ± 101.10
3722.67 ± 26.08
7724.35 ± 536.53
3813.30 ± 275.68
Free maltose
64.75 ± 1.19
233.91 ± 4.11
553.28 ± 39.27
71.12 ± 8.50
Free cellobiose
21.89 ± 1.53
105.64 ± 9.08
1145.82 ± 72.03
815.28 ± 26.38
Free trehalose
522.21 ± 14.18
760.08 ± 13.92
1524.56 ± 104.14
229.85 ± 22.41
Free glucose
51.30 ± 1.93
2081.15 ± 46.36
6892.07 ± 476.91
2125.06 ± 195.48
Free fructose
502.01 ± 16.57
517.82 ± 10.08
330.12 ± 23.22
161.69 ± 9.82
Free galactose
200.18 ± 4.29
308.07 ± 4.50
510.63 ± 35.88
120.70 ± 11.35
Free mannose
905.85 ± 14.58
1426.55 ± 16.07
4012.08 ± 231.39
1792.38 ± 124.18
Free xylose
18.73 ± 0.43
29.80 ± 0.34
159.54 ± 11.08
14.64 ± 1.17
Free arabinose
56.11 ± 1.46
232.79 ± 5.82
568.50 ± 38.78
131.18 ± 10.74
Crude fats
2833.00 ± 77.23
2503.37 ± 71.89
3516.83 ± 23.60
2694.17 ± 138.24
Triglycerides
1009.51 ± 86.09
945.67 ± 61.21
2771.87 ± 254.90
1828.71 ± 180.38
Free fatty acids
37.68 ± 3.26
31.62 ± 1.05
32.10 ± 1.20
47.66 ± 3.66
Lignin
7537.50 ± 510.41
7371.11 ± 335.69
6005.28 ± 589.45
1379.84 ± 143.43
Total H units
103.26 ± 1.28
100.53 ± 1.55
62.54 ± 5.02
16.46 ± 0.99
Total S units
1149.62 ± 55.10
655.20 ± 20.03
500.01 ± 20.92
133.62 ± 8.71
Total G units
2426.67 ± 98.70
1515.27 ± 30.04
1493.81 ± 165.47
322.40 ± 31.54
Total H:S:G ratio
0.04:0.47:1
0.07:0.43:1
0.04:0.33:1
0.05:0.41:1
Free H monomer
3.60 ± 0.08
0.59 ± 0.02
0.52 ± 0.05
0.52 ± 0.04
Free S monomer
0.86 ± 0.02
2.47 ± 0.07
0.61 ± 0.04
0.41 ± 0.03
Free G monomer
4.34 ± 0.18
1.19 ± 0.02
0.68 ± 0.08
0.70 ± 0.05
Free H:S:G ratio
0.83:0.20:1
0.50:2.08:1
0.76:0.90:1
0.74:0.59:1
Free ferulic acid
38.24 ± 0.49
5.86 ± 0.14
1.34 ± 0.05
1.35 ± 0.10
Total proteins
3.61 ± 0.09 × 104
4.25 ± 0.09 × 104
4.03 ± 0.28 × 104
3.81 ± 0.28 × 104
Total soluble proteins
4.74 ± 0.22 × 103
8.31 ± 0.41 × 103
11.78 ± 1.12 × 103
10.69 ± 0.06 × 103
Free amino acids
6.60 ± 0.22 × 103
2.72 ± 0.15 × 103
7.07 ± 0.06 × 103
19.55 ± 0.15 × 103
The-values are mean of three biological replicates, with standard deviation. Significant difference in multi-group comparison of an
item at the three time-points was judged by one-way ANOVA. Significant difference in pairwise comparison of two items at the
same time-point, or during the same period, was judged by t test. A full list of all p-values is provided in Table S8.
8
5. Isotopic verification of ENB assimilating N from soil during days 0-15
Nitrogen assimilation from soil towards ENB was verified by a supplementary experiment on a mushroom
bed made of 15N-labeled, pre-homogenized soil. 15N relative abundance (percentage of 15N in total N) of the
soil was 0.371 ± 0.002% before labeling, and 1.371 ± 0.046% after labeling with (15NH4)2SO4. 15N relative
abundance in the ENB substrate increased (p-value = 0.001, by one-way ANOVA) from 0.368 ± 0.001%
(natural level) at day 0 to 0.550 ± 0.037% at day 15 (Fig. S3), indicating that N element in the 15N-labeled
soil was delivered into the ENBs. The soluble proteins extracted from ENB had an even higher level of 15N
at day 15 (1.721 ± 0.028%) (p-value = 1.68 × 10-6, by one-way ANOVA) (Fig. S3). It suggests that the
assimilated N was further enriched into the soluble proteins in ENB.
Fig. S4. Enrichment of 15N in ENB which were placed on the mushroom bed of a 15N-labeled soil, indicating
that decomposition of ENB by M. importuna led to assimilation of N from soil towards ENB, during days 0-
15. Significant difference between day 0 and day 15 was judged by t test. Samples with a significantly
increased level of 15N relative abundance are labeled with asterisks. The p-values are provided in Table S8.
6. Expression of CAZymes
Table S6. Proteins identified in the metaproteomes, with relative abundance of the three replicates at day 15,
45 and 75. The table is of big size, and is therefore provided as multiple working-sheets in an individual
Excel file available online: TableS6.xls.
0.0
0.5
1.0
1.5
2.0
day0 day15
ENB+ ENB- SP+ SP-
15N relative abundance
in total N element (%)
*
*
ENB (M. importuna inoculated into the soil)
ENB (M. importuna not inoculated into the soil)
soluble proteins in ENB (M. importuna inoculated into the soil)
soluble proteins in ENB (M. importuna not inoculated into the soil)
9
Fig. S5. All the 88 CAZy-proteins of M. importuna SCYDJ1-A1 identified in ENB. Expression levels of the
Color coding of expression patterns
(> : significantly up-regulated defined by fold-change > 2 plus p-value < 0.05; < : significantly down-regulated defined by fold-change < 0.5 plus p-value < 0.05; ≈ : no significant up- or down- regulation)
10
transcripts and proteins were estimated by RNA-Seq and nanoLC-MS/MS, respectively. Steady-state
transcript level (in RPKM) and protein relative abundance are the mean of three biological replicates. ND:
not detected. Functions of the CAZymes were predicted according to their nearest analogs whose activities
had been characterized in previous studies, as provided by the CAZy database. Significant upregulation and
downregulation were judged by fold-change > 2 or fold-change < 0.5, respectively, while FDR-corrected p-
value < 0.05. Fold-change-values and p-values are provided in Table S8.
Fig. S6. Counts of proteins showing significant upregulation (fold-change > 2 and p-value < 0.05),
significant downregulation (fold-change > 0.5 and p-value < 0.05) or no significant shift, during days 15-45
or during days 45-75, respectively. Proteins of the metaproteomes of the major fungal taxa in ENB (M.
importuna SCYDJ1-A1, Mortierella, Trichoderma, Monodictys, Peziza, Cladosporium, Neonectria,
Penicillium, Fusarium, Oliveonia, Plectosphaerella), or those belonging to M. importuna SCYDJ1-A1 only,
were counted respectively. Subset panels show expressing regulation in the CAZymes of M. importuna
SCYDJ1-A1. Areas of the circular sectors are all proportional to gene counts.
570
804
6
1162
96
day15 → day45 day45 → day75
Proteins of the major fungal taxa
Proteins belonging to M. importuna SCYDJ1-A1
day15 → day45 day45 → day75
168
5
5
88
54
33
1
1
86
1
(CAZymes) (CAZymes)
122
660
740
11
Fig. S7. A. Distribution of occurrence of genes coding for CAZymes involved in decomposition of plant polysaccharides, lignin and lipids, compared between the genomes
of the commercially cultivated mushrooms M. importuna SCYDJ1-A1, A. bisporus, P. ostreatus and L. edodes. B. Presence of CAZymes in the proteomic profiles of M.
010 20 30
GH13 GH15
GH31 GH63
050 100 150 200 250
GH1 GH2 GH3 GH5 GH6 GH7
GH9 GH10 GH11 GH12 GH16 GH17
GH26 GH27 GH29 GH30 GH35 GH36
GH37 GH38 GH39 GH43 GH44 GH45
GH47 GH51 GH53 GH55 GH62 GH63
GH65 GH71 GH72 GH74 GH76 GH78
GH79 GH81 GH88 GH89 GH92 GH93
GH95 GH105 GH106 GH115 GH125 GH127
GH128 GH131 GH132 GH134 GH135 GH145
GH152 GH154 CE1 CE3 CE12 CE15
CE16 AA8 AA9
010 20 30
GH28 CE8
PL1 PL3
010 20 30
GH18 GH20 GH75
GH85 CE4
020 40 60 80
AA1 AA2
AA3 AA5
0 5 10
CE5
Morchella
importuna SCYDJ1-A1
Agaricus bisporus
var bisporus H97
Pleurotus
ostreatus PC15
Lentinus
edodes B17
A
B
starch cellulose, β-glucans and hemicellulose pectins chitins lignin lipids
Morchella
importuna SCYDJ1-A1
Agaricus bisporus
var bisporus H97
Pleurotus
ostreatus PC15
0 5 10
GH13
GH15
GH31
020 40 60
GH1 GH2 GH3 GH5 GH6 GH7
GH10 GH11 GH12 GH16 GH17 GH27
GH29 GH35 GH38 GH43 GH45 GH47
GH51 GH53 GH55 GH71 GH72 GH74
GH76 GH78 GH79 GH88 GH92 GH93
GH95 GH105 GH115 CE1 CE15 CE16
AA8 AA9
0 5 10
GH28 CE8
PL1 PL3
0 5 10
GH18
GH20
CE4
010 20 30
AA1 AA2
AA3 AA5
0 2 4
CE5
starch cellulose, β-glucans and hemicellulose pectins chitins lignin lipids
12
importuna SCYDJ1-A1, P. ostreatus and A. bisporus. The CAZy-genes are sorted in categories according to their targeted substrates. Significant over-representation and
under-representation were judged by Fisher’s exact test shown in Table S7. The number of expressed CAZy-proteins of P. ostreatus and A. bisporus are calculated from
available previous studies (Patyshakuliyeva et al., 2015; Fernández-Fueyo et al., 2016), while M. importuna SCYDJ1-A1 is from this study.
Table S7. Crosstabs showing all the results of Fisher’s exact test conducted in this study. Significant over-representation is judged by the adjusted residual-value > 1.96
(upper limit of 95% confidence of +1), and significant under-representation by the adjusted residual-value < -1.96 (lower limit of 95% confidence of -1), as the criteria
proposed by MacDonald and Gardner (2000). The table is of big size, and is therefore provided as multiple working-sheets in an individual Excel file available online:
TableS7.xls.
7. Significance of difference judged by statistical analyses
Table S8. p-values of all the statistical comparisons (except for Fisher’s exact test) in this study. The table is of big size, and is therefore provided as multiple working-
sheets in an individual Excel file available online: TableS8.xls.
13
II. Details in experimental procedures
1. Morel spawn and sowing
Commercial mushroom spawn of M. importuna SCYDJ1-A1 was purchased from Jindi-Tianlingjian Co.Ltd.,
Sichuan, China. The corporation is authorized and quality-controlled by the Soil and Fertilizer Institute,
Sichuan Academy of Agricultural Sciences, to commercially produce the mushroom spawn. Culture of M.
importuna SCYDJ1-A1 was amplified to produce the mushroom spawn for cultivation via three stages:
Stage A, the so-called maternal spawn: morel mycelium inoculated on potato dextrose agar (PDA) slant
in glass tube. Incubate at 15°C for 15 days.
Stage B, the so-called proto-spawn: 500 ml jar with ventilating but septic-proof lid containing 400 cm3 of
solid substrate, a mixture of 360 cm3 of sandy loam soil and 40 cm3 of logging debris. The bottled substrate
was autoclaved at 121°C for 2 h. Approximate 2 cm3 of the inoculated agar plug from stage A was
transferred to the top of the solid substrate in stage B, and incubate at 15°C for 20 days.
Stage C, the so-called cultivating spawn: 500 ml jar with ventilating but septic-proof lid containing 400
cm3 of solid substrate, a mixture of 200 cm3 of soaked wheat grains, 180 cm3 of sandy loam soil and 20 cm3
of logging debris. The bottled substrate was autoclaved at 121°C for 2 h. Approximate 20 cm3 of the
inoculated substrate (pre-homogenized under sterile condition) from stage B was transferred to the top of the
solid substrate in stage C, and incubate at 15°C for 20 days.
Before sowing, the cultivating spawn for all grids were combined and mixed to homogeneity before used.
Even weight of the mushroom spawn was used for each grid. 600 g of the pre-homogenized morel spawn
was fractioned for each grid (equal to 400 g m-2), evenly spread on the soil surface of the grid and covered
with a thin layer of the soil (about 1-2 cm). In this study, the morel was sown on 26th November 2017.
2. Making and placing of ENB
ENB was made of commercial wheat grains (Triticum aestivum L. subsp. aestivum) and rice husks (Oryza
sativa L. subsp. japonica), purchased from COFCO Corporation, China. Measured by dry weight, 85
aliquots of wheat grains and 15 aliquots of rice husks were soaked respectively until soft and then mixed
thoroughly. 350 g fresh weight of the mixture was filled in polypropylene casing and autoclaved at 121°C for
14
3 h. ENB was placed on 11th December 2017, which is 15 days after the sowing. Temperature inside ENB
was recorded throughout the whole course of morel cultivation as shown in Fig. S1.
3. Field management
Field managements for temperature and humidity of soil and air in the greenhouse were operated as
instructed in a published technical regulation (Tang et al., 2015), which were also summarized by Liu et al.
(2018). The morel began to form primordia in middle March of 2018.
4. Determination of chemical composition in ENB
Total C, N, phosphorus (P) and potassium (K) were determined with the procedures described by Nishanth
and Biswas (2008). Specifically, total C was determined by combustion method, total N was determined
from H2SO4-digested sample by micro-Kjeldahl method, total P was determined by the classical Vanado-
molybdophosphate spectrophotometric method (Tabatabai and Bremner, 1969) after the sample was digested
and oxidized with HNO3-HClO4 to convert all P into phosphate form, and total K was determined by flame
atomic absorption spectrophotometry. Organic N was determined with the alkaline persulfate oxidation
method as described by Griffiths et al. (2012). Ammonium N, which is N in NH3 or NH4+ form, was
determined using the micro-Kjeldahl method procedures but without digesting the sample. N in inorganic
nitrate form was determined with the salicylic acid colorimetric method as described by Tian et al. (2008).
Mineral P (inorganic phosphate) was determined from undigested sample by Vanado-molybdophosphate
spectrophotometric method.
Total carbohydrates were determined using the Total Carbohydrate Assay Kit (Sigma-Aldrich, U.S.)
based on phenol-sulfuric acid method. Total reducing sugars were determined with the 3,5-dinitrosalicylic
acid (DNS) method as described by Zhu et al. (2013). For determination of different carbohydrates, readily-
soluble monosaccharides, disaccharides and oligosaccharides were extracted in the first step by soaking four
times (2 h for each time) with a soaking buffer (20 mM Tris-HCl, 10 mM EDTA, pH 7.5) at 4°C. For each
time, the soaking liquid was replaced to new soaking buffer to ensure complete extraction as possible. The
extracts from the four soaking batches were combined and then determined by HPLC to estimate the amount
of free maltose, cellobiose, trehalose, glucose, fructose, galactose, mannose, xylose and arabinose. In the
15
second step, amylose and amylopectin were extracted by soaking four times (10 min each time) with the
soaking buffer in boiling-water bath and combined. Amylose and amylopectin in the extracts were
distinguished and estimated according to the Province Standard Method DB32/T 2265-2012 of Jiangsu
Province, China, which is based on dual-wavelength spectrophotometry. After removal of readily-soluble
small molecular saccharides and starch, macromolecular structural carbohydrates (cellulose and
hemicellulose) were determined with the HPLC analyses described in the National Renewable Energy
Laboratory (NREL) standard method (Sluiter et al., 2008). Protopectins and water-soluble pectins were
determined with the carbazole-colorimetric method as described by Lei et al. (2012).
Lignin was extracted and quantified with the procedures described in the NREL standard method.
Monomeric composition of p-hydroxyphenyl (H), syringyl (S) and guaiacyl (G) units in lignin was
determined with the method described by Finger-Teixeira et al. (2010), in which the H, S and G units
(despite cross-linked or free state) in lignin were oxidized by alkaline nitrobenzene into monomeric aldehyde
forms and then determined by HPLC. To determine the amount of free-state H, S and G monomers, 50%
(w/v) methanol was used to extract the free-state monomers from the ENB samples directly, followed by
HPLC determination of the free-state monomers. Ferulic acid was also determined by HPLC. Crude fats
were determined with the Soxhlet extraction method as described by Zhang et al. (2017). From the extracted
crude fats, triglycerides were determined using the Triglyceride Quantification Kit (Sigma-Aldrich, U.S.)
based on colorimetric quantification of glycerol released from triglycerides, and free fatty acids were
determined using the Free Fatty Acid Quantitation Kit (Sigma-Aldrich, U.S.) based on enzymatic oxidation
of free fatty acids. Total proteins and total soluble proteins were determined with Pierce BCA Protein Assay
Kit (Thermo Fisher Scientific, U.S.). Free amino acids were determined using the L-Amino Acid
Quantitation Kit (Sigma-Aldrich, U.S.) based on oxidative deamination by L-amino acid oxidase.
5. Determination of chemical composition in fruiting body and surface soil
For the fruiting body samples, total C, total N, total P, total K, total carbohydrates, reducing sugars, proteins,
free amino acids, and crude fats, were determined respectively. For the surface soil samples, total organic C,
total N, and ammonium N, were determined respectively. In order to determine total organic C in the soil, the
samples were pre-treated with H2SO3 to remove inorganic carbonate, as described by Aye et al. (2016).
16
6. Extraction of soluble proteins from ENB
Non-dried ENB sample was ground with liquid-nitrogen. 1000 g of wet ENB sample was used for each
replicate, ground to particle size around 120-180 μm diameter, as estimated with sieve, which could facilitate
exposure and soaking out of extracellular soluble proteins (including proteins released from cytosol of dead
cells into extracellular space) buried inside the ENB substrate, meanwhile minimize potential release of
fungal intracellular proteins as possible. Potential extracellular proteins were extracted by a soaking method
as previously used by Zhu et al. (2016), with a modification of using the soaking buffer instead of Milli-Q
water. The ground sample was soaked four times in 3 l of the soaking buffer, with ice bath and gentle
shaking. The total 12 l of soaking liquid was combined and concentrated to 100 ml, using an ultrafiltration
system (Merck-Millipore, U.S.) with 8 kDa molecular weight cut-off. Concentration of the extracted proteins
was determined with BCA Protein Assay Kit (Thermo Fisher Scientific, U.S.).
7. Enzymatic activity estimation
To estimate the enzymatic activity levels of the decomposition enzymes (CAZymes mainly) in the protein
extracts from ENB, detailed methods are included in Table S9. Since the protein extracts are a mixture of
different enzymes with various activities, the adopted methods are aiming for minimize potential interference
from unspecific detection of other activities.
17
Table S9. Methods for enzymatic activity estimation.
Activity
Quantification principle and method
Substrate for assay
α-amylase
(EC 3.2.1.1)
Ceralpha Method α-Amylase Assay Kit (Megazyme, Ireland), which can exclude potential interference from β-amylase, γ-
amylase and α-glycosidase, as the principle described by Sheehan and McCleary (1988)
benzylidene-(glucose)7-α-pNP (Sheehan
and McCleary, 1988)
β-amylase
(EC 3.2.1.2)
Betamyl-3 Method β-Amylase Assay Kit (Megazyme, Ireland), which can exclude potential interference from α-amylase, as
the principle described by McCleary and Codd (1989). The activity measured is a sum-up of β-amylase plus γ-amylase, thus
the γ-amylase activity should be deducted from the measurement
(glucose)3-β-pNP (McCleary and Codd,
1989)
γ-amylase
(EC 3.2.1.3)
Free glucose is cleaved successively from non-reducing end of amylopectin by this enzyme. Measure the release of free
glucose by HPLC. Reaction time is short (2 min) to avoid potential interference from the free glucose generated by a-
amylase + β-amylase + α-glycosidase working together to shred amylopectin chain completely
amylopectin from maize
(Sigma-Aldrich, U.S.)
α-glycosidase
(EC 3.2.1.20)
Maltose is hydrolyzed to two glucose by this enzyme. Measure the release of free glucose by HPLC
maltose
endo-β-1,4-glucanase
(EC 3.2.1.4)
CellG3 Method Cellulase Assay Kit (Megazyme, Ireland), which can exclude potential interference from exo-
cellobiohydrolase and β-glycosidase, as the principle described by McCleary et al. (2014)
4,6-O-benzylidene-2-chloro-4-nitrophenyl
β-D-cellotrioside (McCleary et al., 2014)
endo-β-1,3-glucanase
(EC 3.2.1.39)
β-1,3-glucan is randomly cleaved by the enzyme to expose an increasing number of reducing end. Measure the increasing of
reducing end generated from the enzymatic reaction (reducing sugars) by the classic DNS colorimetric method
β-1,3-glucan from Euglena gracilis
exo-cellobiohydrolase
(EC 3.2.1.91 + EC 3.2.1.176)
The assay measures the summed-up activity of exo-cellobiohydrolase type I (reducing end) and II (non-reducing end). Free
cellobiose is cleaved successively from both non-reducing and reducing ends of cellulose chains. Measure the release of free
cellobiose by HPLC
Avicel cellulose
(Sigma-Aldrich, U.S.)
β-glycosidase
(EC 3.2.1.21)
β-Glucosidase Activity Assay Kit (Sigma-Aldrich, U.S.). 4-nitrophenyl β-D-glucopyranoside is hydrolyzed by β-glycosidase
thereby releasing 4-nitrophenol. Monitor the release of 4-nitrophenol by colorimetry
4-nitrophenyl β-D-glucopyranoside
endo-β-1,4-xylanase
(EC 3.2.1.8)
XylX6 Method Endo-xylanase Assay Kit (Megazyme, Ireland), which can exclude potential interference from exo-β-1,4-
xylosidase, as the principle described by Mangan et al. (2017)
4,6-O-(3-ketobutylidene)-4-nitrophenyl β-
45-O-glucosyl xylopentaoside (XylX6)
(Mangan et al., 2017)
exo-β-1,4-xylosidase
(EC 3.2.1.37)
4-nitrophenyl β-D-xylopyranoside is hydrolyzed by exo-β-1,4-xylosidase thereby releasing 4-nitrophenol. Monitor the
release of 4-nitrophenol by colorimetry
4-nitrophenyl β-D-xylopyranoside (Sigma-
Aldrich, U.S.)
β-mannosidase
(EC 3.2.1.25)
4-nitrophenyl β-D-mannopyranoside is hydrolyzed by β-mannosidase thereby releasing 4-nitrophenol. Monitor the release
of 4-nitrophenol by colorimetry
4-nitrophenyl β-D-mannopyranoside
(Sigma-Aldrich, U.S.)
α-L-arabinofuranosidase
(EC 3.2.1.55)
4-nitrophenyl α-L-arabinofuranoside is hydrolyzed by α-L-arabinofuranosidase thereby releasing 4-nitrophenol. Monitor the
release of 4-nitrophenol by colorimetry
4-nitrophenyl α-L-arabinofuranoside
(Sigma-Aldrich, U.S.)
endo-chitinase
Chitinase Assay Kit (Sigma-Aldrich, U.S.). 4-nitrophenyl β-D-N,N′,N′′-triacetylchitotriose is hydrolyzed by endo-chitinase
4-nitrophenyl β-D-N,N′,N′′-
18
(EC 3.2.1.14)
thereby releasing 4-nitrophenol. Monitor the release of 4-nitrophenol by colorimetry
triacetylchitotriose
exo-chitinase
(EC 3.2.1.52)
Chitinase Assay Kit (Sigma-Aldrich, U.S.). 4-nitrophenyl N-acetyl-β-D-glucosaminide is hydrolyzed by exo-chitinase
thereby releasing 4-nitrophenol. Monitor the release of 4-nitrophenol by colorimetry
4-nitrophenyl N-acetyl-β-D-glucosaminide
pectin lyase
(EC 4.2.2.10)
Pectin Lyase Assay Kit (Comin Biotechnology, Suzhou, China) based on the principle describe by Collmer et al. (1988),
which measures the generation of 4,5-unsaturated residue
pectin from apple (high methylesterification
degree)
polygalacturonase
(EC 3.2.1.15)
Polygalacturonase Assay Kit (Comin Biotechnology, Suzhou, China) based on the principle describe by Collmer et al.
(1988), which measures the increasing of reducing end exposed by random endo-cleavage of polygalacturonic acid by
polygalacturonase
polygalacturonic acid
pectin esterase
(EC 3.1.1.11)
Monitor the release of methanol using gas-liquid chromatography, as described by Salas-Tovar et al. (2017)
pectin from apple (high methylesterification
degree)
pectate lyase
(EC 4.2.2.2)
Pectate Lyase Assay Kit (Comin Biotechnology, Suzhou, China) based on the principle describe by Collmer et al. (1988)
polygalacturonic acid
laccase
(EC 1.10.3.2)
Classic colorimetric method monitoring the oxidation of 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by
optical absorbance at 420 nm (Touahar et al., 2014)
ABTS
Mn peroxidase
(EC 1.11.1.13)
Classic colorimetric method monitoring the oxidation of Mn2+ to Mn3+ by optical absorbance at 240 nm (Touahar et al.,
2014)
Mn2+
versatile peroxidase
(EC 1.11.1.16)
Classic colorimetric method monitoring the oxidation of veratryl alcohol to veratraldehyde by optical absorbance at 310 nm
(Touahar et al., 2014)
veratryl alcohol
glyoxal oxidase
(EC 1.2.3.15)
Methylglyoxal is oxidized by this enzyme thereby generating H2O2. The H2O2 is estimated by a coupled Classic colorimetric
method monitoring the H2O2-dependent oxidation of ABTS (Yin et al., 2015), which is coupled with the H2O2-generating
reaction of oxidation. Unspecific oxidation of ABTS by laccase should be deducted from the total oxidized ABTS measured
methylglyoxal
(Sigma-Aldrich, U.S.)
lipase
(EC 3.1.1.3)
Measure the hydrolysis of triglycerides using the Triglyceride Quantification Kit (Sigma-Aldrich, U.S.) based on
colorimetric quantification of glycerol released from triglycerides
triglycerides
total protease
Protease Fluorescent Detection Kit (Sigma-Aldrich, U.S.). Use casein labeled with fluorescein isothiocyanate (FITC) as the
substrate to measure the total activity of protein-cleavage
FITC-casein
phosphatase
Monitor release of inorganic phosphate with the classical Vanado-molybdophosphate colorimetric method (Tabatabai and
Bremner, 1969). Citrate buffer was used instead of phosphate buffer to avoid background P interference
para-nitrophenyl phosphate (pNPP)
19
8. 15N-isotopic experiment
A fraction of the pre-homogenized sandy loam soil (described in Table S4) was used for the 15N-isotopic
experiment. 10 g of (15NH4)2SO4, with a 15N relative abundance (percentage of 15N in total N) of 98%, was
mixed thoroughly with 1 m3 of the soil, resulting in a 15N-labeled soil with a 15N relative abundance of 1.371
± 0.046%. The 15N-labeled soil was loaded evenly into six ventilated plastic containers (40 × 40 × 20 cm3
size) as mushroom beds. Two treatments, with three replicates for each, were set up: A. M. importuna
SCYDJ1-A1 inoculated into the mushroom beds; B. M. importuna SCYDJ1-A1 not inoculated (by using
autoclaved morel spawn) into the mushroom beds.
The morel spawn of M. importuna SCYDJ1-A1 was sown to the soil in the plastic containers as
described in the “Morel spawn and sowing”. Two ENBs was placed on the soil surface of each plastic
container. One ENB was sampled at day 0 and the other at day 15. Half of the ENB was kept as an ENB
substrate sample while the other half was used for extracting soluble proteins, using the method described
above. The samples were sent to the Isotope Analysis Center, Chinese Academy of Agricultural Sciences,
Beijing, China, for determination of 15N isotope. Relative abundance of 15N was determined with an
Isoprime-100 automatic elemental analyzer isotope ratio mass spectrometry (EA-IRMS) system (Elementar
Analysensysteme GmbH, Germany).
9. User-defined metagenome
According to the microbial community profiles determined by ITS metabarcoding survey, fifteen most
abundant OTUs represented 96.5% of the fungal community in ENB. The fifteen most abundant OTUs were
identified as belonging to M. importuna plus ten other fungal genera (Mortierella, Trichoderma, Monodictys,
Peziza, Cladosporium, Neonectria, Penicillium, Fusarium, Oliveonia, and Plectosphaerella (from high to
low abundance). They together were defined as the major fungal taxa in ENB. The genome of M. importuna
SCYDJ1-A1 plus available genomic data of the other ten fungal taxa were collected to form a metagenome,
as reference genomes for following fungal protein identification in metaproteome.
20
Table S10. User-defined reference metagenome.
Reference genome name
Accession number or download link
Target
Metagenome
M. importuna SCYDJ1-A1 v1.0
NCBI BioProject PRJNA334370
M. importuna SCYDJ1-A1
Major fungal taxa
in ENB,
composed of the
morel plus ten
other most
abundant fungal
genera
Mortierella elongata AG-77 v2.0
NCBI BioProject PRJNA196039
Mortierella genus
Trichoderma gamsii TGAM01v2
NCBI BioProject PRJNA252048
Trichoderma genus
Monodictys
N.A.
N.A
Peziza echinispora DOB1120
v1.0
https://genome.jgi.doe.gov/portal/pages
/dynamicOrganismDownload.jsf?organi
sm=Pezech1
Peziza genus
Cladosporium cladosporioides
ASM290114v1
NCBI BioProject PRJNA396076
Cladosporium genus
Neonectria ditissima Nd324p
v1.0
NCBI BioProject PRJNA285413
Neonectria genus
Penicillium freii ASM151392v1
NCBI BioProject PRJNA277835
Penicillium genus
Fusarium equisetii
ASM331317v1
NCBI BioProject PRJNA473205
Fusarium genus
Oliveonia pauxilla KC1149
Standard Draft v1.0
https://genome.jgi.doe.gov/portal/Olipa
1/Olipa1.download.html
Oliveonia genus
Plectosphaerella cucumerina
RP01 Standard Draft v1.0
https://genome.jgi.doe.gov/portal/Plecu
c1/Plecuc1.download.html
Plectosphaerella genus
Triticum aestivum cv. Chinese
spring
Uniprot UP000019116
wheat grain from ENB
substrate
Protein hits of the
wheat and rice
substrate
background, to be
manually
removed
Oryza sativa subsp. japonica
Uniprot UP000059680
rice husk from ENB
substrate
21
10. Metatranscriptomics
Mapping rate of metatranscript reads toward M. importuna SCYDJ1-A1 is decreasing significantly from day
15 to day 75, in line with the decreasing proportion of M. importuna hyphae in ENB as estimated with
metabarcoding.
Table S11. Mapping rate of RNA-Seq reads.
Sample
Total
clean reads
Reads mapping to M. importuna
SCYDJ1-A1 genome
Unmapped
reads
Mapping rate
(%)
day15-replicate1
80549674
61430005
19119669
76.26
day15-replicate2
81832728
62383364
19449364
76.23
day15-replicate3
80940974
61922381
19018593
76.50
day45-replicate1
96949240
24617206
72332034
25.39
day45-replicate2
99300520
25198851
74101669
25.38
day45-replicate3
94287492
24062437
70225055
25.52
day75-replicate1
81170760
2512770
78657990
3.10
day75-replicate2
81028848
2518992
78509856
3.11
day75-replicate3
82059410
2573088
79486322
3.14
22
11. Metabarcoding estimation of microbial diversity
Fig. S8. Rarefaction curves of bacterial 16S (a) and fungal ITS (b) sequences. ENB at day 15, 45 and 75 are
shown by solid lines, dash lines and dot lines, respectively. The three replicates of each time-point are
coloured in red, green and blue.
23
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