Article Figures & Data
Figures
- FIG. 1.
Heat map showing hierarchical clustering of Phanerochaete chrysosporium genes exhibiting highly significant (P < 0.001) accumulation of transcripts in aspen-grown versus glucose-grown cultures. Numbers within boxes indicate the fold difference in transcripts for aspen versus glucose cultures. Only those P. chrysosporium genes exceeding a 4-fold difference are shown. Underlined P. chrysosporium models correspond to genes with >50 cDNA tags in medium containing powdered oak (43). On the right, a heat map shows transcript levels of the most closely related P. placenta sequences or, in the absence of a clear homolog, labeled as “None.” Protein IDs followed by asterisks correspond to peptides unambiguously identified by LC-MS/MS in filtrates from aspen-grown cultures. The log2-based scales below maps are calibrated to the data shown above. Boldface denotes models in need of editing: GH10 xylanase model Ppl113670 has been superceded by an annotated preferred version, Ppl134783, which shows less pronounced upregulation (1.10) with a log2 signal of 11.3. GH27 α-galactosidase model Ppl128150 has been superceded by annotated preferred version Ppl13470, which shows more pronounced upregulation (3.08) with a log2 signal of 14.4 (Table 3; see also Table S1 in the supplemental material for detailed data).
- FIG. 2.
Heat map showing hierarchical clustering of P. placenta genes exhibiting highly significant (P < 0.001) accumulation of transcripts in aspen-grown versus glucose-grown cultures. Numbers within boxes indicate the fold difference in transcripts in aspen versus glucose cultures. Only those P. placenta genes exceeding a 4-fold difference are shown. On right is a heat map showing transcript levels of the most closely related P. chrysosporium sequences or, in the absence of a clear homolog, labeled as “None.” Protein IDs followed by asterisks correspond to peptides unambiguously identified by MS in filtrates from aspen-grown cultures. Scales below maps show log2-based signals. Underlined P. chrysosporium models correspond to genes with >50 cDNA tags in medium containing powdered oak (43).
- FIG. 3.
Transcript levels of P. placenta genes encoding copper radical oxidases (Ppl130305, Ppl56703, and Ppl64380), GMC oxidoreductases with distant similarity to glucose oxidase (GOX; Ppl108489 and Ppl128830), an intact laccase (Ppl89382), a truncated laccase model (Ppl46931), and the highly expressed FAD-dependent oxidoreductase Ppl114192 (Table 4). Gray bars and the left axis represent log2 microarray signals. Blackened bars and the right axis represent cDNA as determined by competitive RT-PCR. Asterisks indicate protein models whose peptides were identified in concentrated filtrates of aspen-grown cultures.
Tables
- TABLE 1.
Gene expression summary for P. chrysosporium and P. placenta after 5 days of growth in medium containing ball-milled aspen as sole carbon source
Feature No. of genes identified P. chrysosporium P. placenta Gene modelsa 10,048 17,173 (12,227) Microarray target genes 10,004 12,438 Proteins by LC-MS/MS 73 67 Transcript and/or extracellular proteins accumulating in aspen culturesb Total 77 160 Glycoside hydrolases 33 33 Hypotheticals 15 49 Oxidoreductases 4 21 ↵ a Based on gene predictions previously described (36, 37, 53). The parenthetical number refers to the predicted number of unique alleles within the parental dikaryon strain of P. placenta.
↵ b Number of genes whose transcripts accumulated >2-fold (P < .001) and/or whose predicted peptides were identified with Mascot scores of >40.
- TABLE 2.
P. chrysosporium genes encoding putative oxidoreductases upregulated >2-fold in BMA medium relative to glucose medium or matched to MS/MS-derived peptide sequencesa
Phanerochaete chrysosporium Postia placenta Model Putative function Signal (log2) in: Ratio P value Model Allelle Pairwise alignment Signal (log2) in: Ratio P value Glu BMA % ID Score Glu BMA 11098 Cellobiose dehydrogenase 12.30 15.61 9.93 <0.001 None 138350 Acetoin dehydrogenase 8.82 10.83 4.01 <0.001 88413 110831 75 1,035 8.37 8.33 0.97 0.521 6771 Oxidoreductase 10.09 11.71 3.09 0.00160 49605 61601 76 1,544 10.07 11.41 2.53 <0.001 41616 Aldo/keto reductase 10.37 11.93 2.96 0.00406 None 147 Cytochrome b562 + CBM1 12.22 13.62 2.64 <0.001 None 10221 Alcohol dehydrogenase 10.73 12.11 2.60 0.01050 None 10957 Peroxidase LiPA 9.58 10.92 2.54 0.02240 None 121806 Peroxidase LiPH 10.95 12.20 2.38 0.00352 None 1139 FRE-like iron reductase 12.65 13.88 2.35 0.00331 130043 130030 36 635 9.90 10.17 1.20 0.0349 38849 P450 11.72 12.88 2.24 <0.001 None 126879 Alcohol oxidase* 13.77 14.74 1.97 0.020 118723 129841 88 3090 11.76 14.44 6.43 <0.001 124398 Catalase* 14.24 14.90 1.59 0.004 114720 123169 73 2005 14.27 14.33 1.04 0.526 140211 Formate dehydrogenase* 13.04 13.53 1.41 0.110 119730 None 85 1628 12.65 14.17 2.86 <0.001 6270 Oxidoreductase (GMC)* 9.80 10.16 1.29 0.184 None ↵ a To identify conserved sequences, the 12,438 P. placenta protein models represented on microarrays were aligned with all 10,048 v2 P. chrysosporium protein models using Timelogic harware (Active Motif, Carlsbad, CA) with accelerated double affine Smith-Waterman alignments. Scores, percent identities, and related alignment parameters for all genes are listed in Table S1 of the supplemental material. Normalized microarray data are presented as the average log2 signal strength of three fully replicated experiments. Array-wide averaged signals (± standard deviations) for P. chrysosporium and P. placenta were 11.6 (1.6) and 10.9 (1.7), respectively. Significant accumulation (ratio) of transcripts in BMA relative to glucose (Glu) cultures was determined using the moderated t test (48) and associated false detection rates (4). Data are ranked according to the ratio, from highest to lowest. Underlined P. chrysosporium models correspond to genes with >50 cDNA tags in medium containing powdered oak (43). *, gene model whose proteins were identified in BMA filtrates by MS/MS. Table S1 in the supplemental material provides a summary of these data, and the complete MS/MS results are provided in Table S2.
- TABLE 3.
P. placenta genes encoding putative GHs with the highest transcript levels and/or positive MS/MS identification in BMA mediuma
Postia placenta Phanerochaete chrysosporium Model Putative function Allele Signal (log2) in: Ratio P value Model Signal (log2) in: Ratio P value Glu BMA Glu BMA 115648 GH5 endo-1,4-β-glucanase* 108962 14.62 15.04 1.33 0.0012 6458 12.14 14.83 6.46 <0.001 119525 GH18 glycosidase 120960 14.81 14.83 1.02 0.7560 137237 9.00 9.71 1.64 0.0060 121831 GH5 endo-β-1,4-mannosidase* 134772 13.46 14.65 2.28 <0.001 140501 9.04 12.89 14.44 <0.001 112941 GH16 endo-1,3(4)-β-glucanase* 61809 12.66 14.58 3.79 <0.001 10833 11.77 13.65 3.68 0.0019 121713 GH5 glucan 1,3-β-glucosidase 46077 12.73 14.53 3.48 <0.001 121774 10.14 11.63 2.80 0.0092 117860 GH72 1,3-β-glucanosyltransferase* 118950 14.29 14.51 1.16 0.0206 6433 13.83 13.33 0.71 0.0232 134790 GH27 α-galactosidase* 98662 12.73 14.35 3.08 <0.001 134001 11.68 13.89 4.64 <0.001 94557 GH51 α-N-arabinofuranosidase* None 13.06 14.31 2.38 <0.001 3651 12.20 12.96 1.70 0.0017 127993 GH35 β-galactosidase* 128101 13.65 14.17 1.43 0.0033 9466 11.26 11.69 1.34 0.0709 116267 GH55 glucan 1,3-β-glucosidase* 108648 13.35 14.16 1.75 <0.001 8072 12.03 13.98 3.88 0.0832 100251 GH51 α-N-arabinofuranosidase* 127046 12.31 14.05 3.34 <0.001 3651 12.20 12.96 1.70 0.00169 134924 GH31 α-glucosidase* 93878 13.33 13.51 1.13 0.0316 125462 12.38 12.62 1.18 0.373 113670 GH10 endo-1,4-β-xylanase* 134787 12.62 13.36 1.67 <0.001 138715 11.16 13.54 5.20 <0.001 57564 GH2 β-mannosidase* 56576 12.32 13.35 2.05 <0.001 135385 13.37 13.57 1.15 0.118 126692 GH79 glycosidase* 111332 11.71 13.34 3.10 <0.001 1999 10.55 11.88 2.52 0.0269 97540 GH37 trehalase* 115929 13.11 13.14 1.02 0.6770 140627 13.96 12.80 0.45 0.0254 112047 GH92 α-1,2-mannosidase* 116992 13.19 12.98 0.86 0.0134 3431 12.77 13.25 1.39 0.0109 126595 GH18 chitinase* 134923 12.59 12.86 1.21 0.1630 39872 14.02 13.71 0.81 0.172 112369 GH20 β-hexosaminidase* 61331 12.92 12.79 0.91 0.1450 37522 9.05 9.08 1.02 0.781 117345 GH15 glucoamylase* 113112 12.34 12.66 1.25 0.0420 138813 12.43 13.02 1.51 0.0771 120395 GH27 α-galactosidase* None 11.50 12.54 2.06 <0.001 125033 12.22 12.91 1.61 0.0193 115593 GH47 Man(9)-α-mannosidase* 134925 13.48 12.51 0.51 <0.001 4550 11.62 13.44 3.52 <0.001 110809 GH43 galactan 1,3-β-galactosidase* None 10.75 12.50 3.38 <0.001 297 10.64 11.10 1.38 0.0625 116199 GH5 glucan 1,3- β-glucosidase* 128425 12.15 12.47 1.24 0.0066 2220 12.43 13.66 2.35 0.00274 111730 GH28 polygalacturonase* 43189 10.53 12.40 3.66 <0.001 3805 10.90 14.57 12.70 <0.001 54405 GH16 endo-1,3(4)-β-glucanase* 135020 11.30 12.19 1.86 <0.001 123909 12.33 13.27 1.93 0.0283 127469 GH3 possible β-xylosidase* 51213 11.58 12.08 1.42 0.0047 9257 9.24 12.67 10.79 <0.001 107557 GH3 β-glucosidase* None 11.95 12.02 1.06 0.5000 139063 14.70 14.55 0.90 0.599 46915 GH3 β-glucosidase* 95677 11.08 11.93 1.81 <0.001 129849 11.26 12.91 3.13 <0.001 105534 GH10 endo-1,4-β-xylanase* None 8.38 11.71 10.06 <0.001 138345 9.23 10.43 2.29 0.014 55086 GH31 α-glucosidase* 19564 10.96 11.63 1.59 0.0014 968 11.64 12.73 2.12 0.0374 52194 GH13 α-amylase* 92881 11.45 11.62 1.13 0.1360 38357 13.86 13.60 0.83 0.141 62385 GH92 α-1,2-mannosidase* 48716 11.30 10.90 0.76 0.0278 133585 12.04 13.75 3.28 <0.001 130398 GH20 β-hexosaminidase* 134907 10.15 9.88 0.83 0.0052 140587 13.29 13.49 1.15 0.233 127046 GH51 α-N-arabinofuranosidase* 100251 9.50 9.81 1.24 0.0053 3651 12.20 12.96 1.70 0.00169 95568 GH5 endo-1,4-β-mannosidase* None 8.91 9.52 1.52 <0.001 6579 12.43 13.67 2.36 0.00279 ↵ a Calculations are essentially the same as described for Table 2. The genes or alleles are listed in order of decreasing transcript levels. Detailed scores, e-values, and alignment parameters for the best P. chrysosporium hits are listed in Table S1 of the supplemental material. *, detection of peptides by LC-MS/MS in BMA medium filtrate (see Tables S1 and S2). Underlined P. chrysosporium models correspond to genes with >50 cDNA tags in medium containing powdered oak (43). Data are ranked according to P. placenta log2 signals in BMA. The first eight genes exceeded the genome-wide average signal by 2 standard deviations (>14.24). Questionable GH51 models Ppl9457 and Ppl127046 lie within a 10-kb region of scaffold 110, and alignments suggest possible duplication or assembly error. The 3′ termini of P. placenta models GH20 Ppl130398 and GH10 Ppl113670 are problematic and require additional data and manual annotation. However, MS/MS protein assignments are based on reliable (5′) regions of these models.
- TABLE 4.
P. placenta genes encoding putative oxidoreductases upregulated >2-fold in BMA medium relative to glucose medium or matched to MS/MS-derived peptide sequencesa
Postia placenta Phanerochete chrysosporium Model Putative function Allele Signal (log2) in: Ratio P value Model Pairwise alignment Signal (log2) in: Ratio P value Glu BMA % ID Score Glu BMA 109824 Ferroxidase Fet3 134624 10.01 12.72 6.55 <0.001 26890 68 2275 14.40 12.95 0.37 0.0124 107061 Oxidoreductase 51016 9.90 12.61 6.54 <0.001 138174 61 1126 10.07 10.14 1.05 0.698 114245 Polyphenol oxidase 109291 10.62 13.32 6.48 <0.001 None 118723 Alcohol oxidase 129841 11.76 14.44 6.43 <0.001 126879 88 3090 13.77 14.74 1.97 0.0199 124517 1,4-Benzoquinone reductase 64069 8.41 11.02 6.10 <0.001 10307 74 821 9.77 10.05 1.21 0.11 34850 Hydroquinol 1,2-dioxygenase 87645 9.86 12.21 5.09 <0.001 41330 71 943 11.20 12.02 1.77 0.0252 111395 Short chain dehydrogenase 64558 8.95 11.15 4.60 <0.001 5258 78 404 10.63 11.12 1.40 0.0162 98518 Formate dehydrogenase 89897 10.12 12.03 3.76 <0.001 140211 82 1578 13.04 13.53 1.41 0.106 61437 Aldehyde dehydrogenase 89252 8.75 10.57 3.51 <0.001 133924 33 483 12.25 11.03 0.43 0.0143 46931 Laccase (fragment) 111314 10.0 11.65 3.15 0.007 10581 40 206 8.6 8.67 1.05 0.45 108989 2-Nitropropane dioxygenase None 11.60 13.20 3.04 <0.001 218 75 1412 11.98 12.12 1.10 0.258 113908 Acireductone dioxygenase 63611 10.51 12.11 3.03 <0.001 123880 53 508 12.90 12.82 0.94 0.491 24981 Hydroquinol 1,2-dioxygenase 107881 11.60 13.13 2.88 <0.001 136660 79 984 11.34 11.89 1.46 0.0769 119730 Formate dehydrogenase None 12.65 14.17 2.86 <0.001 140211 85 1628 13.04 13.53 1.41 0.106 51235 P450* 20385 11.73 13.22 2.81 <0.001 1200 55 1355 11.40 11.51 1.08 0.38 43234 α-Aminoadipate reductase None 10.83 12.29 2.75 <0.001 6025 29 851 9.48 9.52 1.03 0.605 61079 Chloroperoxidase 25391 8.97 10.42 2.72 <0.001 3274 63 922 11.07 11.37 1.23 0.0271 116179 Short chain dehydrogenase None 11.22 12.57 2.55 <0.001 132770 69 956 9.71 10.05 1.27 0.0673 49605 Galacturonic acid reductase 61601 10.07 11.41 2.53 <0.001 6771 76 1544 10.09 11.71 3.09 0.0016 115028 Aryl alcohol dehydrogenase 89343 11.56 12.69 2.18 <0.001 128103 71 1459 10.45 10.91 1.38 0.00751 108358 2-Nitropropane dioxygenase 56793 10.01 11.08 2.11 <0.001 4432 87 1486 10.89 11.16 1.21 0.0874 126817 Flavin monooxygenase 104883 10.54 11.62 2.11 0.008 6291 57 1825 11.87 12.06 1.14 0.47 111823 Oxidoreductase 120225 12.67 13.71 2.05 <0.001 2284 68 1305 8.49 8.83 1.26 0.036 56703 CRO5* 99632 12.73 13.60 1.82 0.0022 8882 66 3651 12.72 12.55 0.89 0.277 122772 OR FAD linked* 114192 13.22 14.07 1.80 <0.001 138076 53 1151 8.93 8.85 0.95 0.527 128830 Glucose oxidase-like* None 13.25 13.11 0.91 0.3910 131961 31 734 12.32 12.20 0.93 0.349 ↵ a Calculations are essentially the same as described for Table 2. The P. chrysosporium homolog with the highest alignment score was reported for each P. placenta gene. Scores, percent identity, and related alignment parameters for all genes are listed in Table S1 of the supplemental material. With one exception (galacturonic acid reductase), transcripts of the corresponding P. chrysosporium gene exhibited relatively lower accumulation ratios and a concomitantly higher false discovery rate (4) probability (far right column). Underlined P. chrysosporium models correspond to genes with >50 cDNA tags in medium containing powdered oak (43). Data are ranked according to the P. placenta ratio, from highest to lowest. *, detection of corresponding peptides by MS/MS in BMA medium filtrate.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Postia placenta protein models matched to best-hit Phanerochaete chrysosporium models (Table S1).
Zipped Microsoft Excel file, 2.5 MB. - Supplemental file 2
-
Detailed mass spectrometry results for Phanerochaete chrysosporium and Postia placenta (Table S2).
Zipped Microsoft Excel file, 40K. - Supplemental file 3
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Primers and predicted lengths of Postia placenta genes analyzed by qRT-PCR (Table S3).
Zipped Microsoft Excel file, 5K. - Supplemental file 4
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Scatter plots showing average log2 transcript levels for Phanerochaete chrysosporium and Postia placenta genes during growth on media containing ball-milled aspen or glucose as the sole carbon source (Fig. S1).
Microsoft PowerPoint file, 1.3 MB.
- Supplemental file 1
-
Postia placenta protein models matched to best-hit Phanerochaete chrysosporium models (Table S1).
