research article rna sequencing reveals xyr1 as a...

Research ArticleRNA Sequencing Reveals Xyr1 as a Transcription FactorRegulating Gene Expression beyond Carbohydrate Metabolism

Liang Ma Ling Chen Lei Zhang Gen Zou Rui Liu Yanping Jiang and Zhihua Zhou

Key Laboratory of Synthetic Biology Institute of Plant Physiology and Ecology Shanghai Institutes for Biological SciencesChinese Academy of Sciences Shanghai 200032 China

Correspondence should be addressed to Zhihua Zhou zhouzhihuasippeaccn

Received 31 August 2016 Accepted 6 November 2016

Academic Editor Muhammad I Rajoka

Copyright copy 2016 Liang Ma et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Xyr1 has been demonstrated to be the main transcription activator of (hemi)cellulases in the well-known cellulase producerTrichoderma reesei This study comprehensively investigates the genes regulated by Xyr1 through RNA sequencing to producethe transcription profiles of T reesei Rut-C30 and its xyr1 deletion mutant (Δxyr1) cultured on lignocellulose or glucose xyr1deletion resulted in 467 differentially expressed genes on inducingmedium Almost all functional genes involved in (hemi)cellulosedegradation and many transporters belonging to the sugar porter family in the major facilitator superfamily (MFS) weredownregulated inΔxyr1 By contrast all differentially expressed protease lipase chitinase someATP-binding cassette transportersand heat shock protein-encoding genes were upregulated in Δxyr1 When cultured on glucose a total of 281 genes were expresseddifferentially in Δxyr1 most of which were involved in energy solute transport lipid amino acid and monosaccharide as wellas secondary metabolism Electrophoretic mobility shift assays confirmed that the intracellular 120573-glucosidase bgl2 the putativenonenzymatic cellulose-attacking gene cip1 theMFS lactose transporter lp the nmrA-like gene endo T the acid protease pepA andthe small heat shock protein hsp23were probable Xyr1-targetsThese results might help elucidate the regulation system for synthesisand secretion of (hemi)cellulases in T reesei Rut-C30

1 Introduction

Lignocelluloses have long been recognized as the mostabundant sustainable resources for the production of biofuelsand other biomaterials [1 2] Cellulosemust be hydrolyzed bycellulases to soluble carbohydrates to facilitate fermentationThe typical cellulase system consists of endoglucanases (EC3214) cellobiohydrolases (EC 32191) and 120573-glucosidase(BGL EC 32121) which act synergistically to hydrolyzecellulose to glucose [3] The ascomycete Trichoderma reesei(anamorph of Hypocrea jecorina) has been used widely as acellulose source since its discovery during World War II [4]However the cost of lignocellulolytic enzyme preparation isstill among the major limitations in the development of anacceptable technology to convert lignocellulose to biofuelsand other chemicals [5 6] Therefore T reesei should befurther genetically engineered to acquire an improved strainfor cellulase production

Several transcription factors (TFs) involved in the reg-ulation of cellulase gene expression have been identified in

T reesei including the activators Ace2 Xyr1 and Hap235as well as the repressor Ace1 and the carbon cataboliterepressor Cre1 [7] Xyr1 a homolog to XlnR in Aspergillusniger is a zinc binuclear cluster protein binding to a 51015840-GGCTAA-31015840 motif arranged as an inverted repeat [8] Itwas demonstrated to play an essential role in transcriptionalregulation of cellulolytic and xylanolytic genes such asxyn1 xyn2 bxl1 abf2 cbh1 cbh2 egl1 and bgl1 [9 10] Inaddition Xyr1 was reported to receive the lactose inductionsignal and regulate lactose metabolism by directly activatingxylose reductase 1 transcription and indirectly influencingtranscription of 120573-galactosidase 1 (bga1) [11] Unlike xyr1 in Treesei the deletion of xlnR (ortholog to xlnR inAspergillus) inFusarium oxysporum affects only xylanase activity [12]

Recently theTF xylan degradation regulator 1 (XLR-1) anortholog to XlnRXyr1 in A niger and T reesei was identifiedin Neurospora crassa Deletion of xlr-1 in N crassa preventedgrowth on xylan and xylose but its cellulolytic activity wasonly slightly affected indicating a different role from xyr1 inT reesei [13] Besides secretome analyses of wild type and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 4841756 20 pageshttpdxdoiorg10115520164841756

2 BioMed Research International

the xlnRxlr1xyr1 deletion mutants of five fungi showed thatT reeseiXyr1 has a different regulatory pattern compared to itsorthologs in other fungi [14] The above findings combinedwith the demonstration that Xyr1 in T reesei could bindnot only to the 51015840-GGCTAA-31015840 motif but also to the 51015840-GGC(AT)3-3

1015840 motif [15] suggest that Xyr1 behaves as apleiotropic regulator in T reesei

Recently transcription profiling of the T reesei Qm 9414and its Δxyr1 mutant grown on cellulose sophorose andglucose were performed and defined the role of the transcrip-tional factor Xyr1 during cellulose degradation [16] T reeseimutant Rut-C30 is a hyperproducer of cellulolytic enzymeswith its genome has been released [3 17 18] Rut-C30 wasobtained through several rounds of random mutagenesisfromwtQm6aThe rearrangement of chromosomes carryinggenes encoding cellulolytic enzymes [19] and the missinggt100 kb of genomic DNA [20] including the truncation ofcarbon catabolite repressor cre1 [17]may contribute to its highprotein secretory ability and cellulase production Portnoyet al reported challenging results indicating that the fulltranscription of xyr1 required Cre1 in T reesei Qm9414under induction conditions [21] Due to the special geneticbackground of Rut-C30 we assumed that its Xyr1 harboredrather special regulatory mechanisms compared to T reeseiQm9414

Wheat (Triticum aestivum L) bran which contains lig-nocelluloses as a major component is rich in hemicellulosescellulose and lignin [22 23] Therefore wheat bran behavesas an inducer for lignocellulolytic enzymes In this studyRNA sequencing (RNA-seq) was performed to investigate thefunctions of Xyr1 through comparison between a wild-typestrain Rut-C30 and an xyr1 disruptant under lignocelluloseand glucose conditions Our results shed new light on themechanism by which Xyr1 controls cellulose and hemicel-lulose utilization and determines the pleiotropic functionsof Xyr1 These new findings could offer strategies for strainimprovement of T reesei Rut-C30

2 Materials and Methods

21 Fungal Strains and Cultivation Conditions T reesei Rut-C30 (ATCC 56765) was purchased from ATCC and its xyr1deletion mutant strain Δxyr1 was constructed as followingPlasmids were propagated in Escherichia coli DH5120572 Thevector backbone used in constructing the plasmids wasbinary vector pCambia1300 (CAMBIA Canberra Australia)The E coli cultivations were performed overnight at 37∘C inLuria-Bertani (LB) medium plus kanamycin (100 120583gmlminus1) asselective agent

The xyr1 deletion vector was constructed using the binaryvector pCambia1300 as a recipient Primers used are given inTable S1 in Supplementary Material available online at httpdxdoiorg10115520164841756 First the plasmid pSilent-1[24] was used as template to amplify the ORF and terminatorof hygromycin B phosphotransferase gene hph (conferringhygromycin B resistance) with primers HygxhoI and OrfhygSecond the pyruvate kinase (pki GenBank accession num-ber L07060) promoter was amplified by primers Ppki andBampki with T reesei Rut-C30 genome as template Overlap

PCR was conducted to fuse the pki promoter to hphORFand the PCR product was digested with BamHI and XhoIand ligated into the BamHI and XhoI digested pCambia1300resulting in pPH Then the 13 kb fragment for 51015840 region ofxyr1 was amplified with primers xyr1xhf and xyr1xhr Afterdigestion by XhoI it was inserted to the XhoI digested pPHto generate pPH5X At last the 31015840 region of xyr1was amplifiedwith primers xyr1bam and xyr1sal and digested by BamHIand SalI before inserting into the corresponding restric-tion enzymes digested product of pPH5X to yield the xyr1knockout vector pPHXThe resultant pPHXwas transformedinto T reesei by Agrobacterium-mediated transformation asdescribed previously [25] Transformantswere then subjectedto verification of homologous recombination event PrimersX5 and TtrpC were used to amplify a fragment of 22 kb inthe xyr1 knockout strain and similarly primers X3 and Ppkiwere used to amplify a fragment of 23 kb in the xyr1 knockoutstrain and the results were further verified by sequencing thePCR products while random insertion could not yield anyspecific PCR products

The xyr1 recomplementation strain was constructed asthe phleomycin resistance gene (ble) [26] was used as aselection marker The binary vector pPB was constructed ina similar way except replacing the hph ORF with ble Thexyr1 genewas amplifiedwith primers X3BamHandX5BamHand the terminator of xyr1 was amplified with primersTxyr5Xho and Txyr3Xho (Table S1) The PCR products weredigested by BamHI and XhoI respectively and then insertedinto corresponding site of the pPB to yield pPBReX Thetransformation into Δxyr1 strain was performed as describedabove and transformants were selected on PDA containingphleomycin (4 120583gmlminus1) as selection agent The homologousintegration of pPBReX at the xyr1 locus of the Δxyr1 strainwas verified by PCR Primers B3homo and pki-phe wereused to amplify a fragment of 26 kb in the xyr1 homologousintegration retransformant and similarly primers X5 andPpki (Table S1) were used to amplify a fragment of 57 kband the results were further verified by sequencing the PCRproducts while random insertion could not yield any specificPCR products

All of the fungal strains were maintained on potato-dextrose agar (PDA) Then 107 conidia of both strainswere inoculated in 50-ml shake flasks containing 10ml ofSabouraudrsquos dextrose broth (SDB) at 28∘C for two daysand then the pregrown mycelia were collected by filtrationthrough miracloth and washed with 09 NaCl thoroughlyThen the mycelia of both strains were transferred intothe cellulase-inducing medium described by Ma et al [25]which contains 2 wheat bran 3microcrystalline cellulose(Avicel) and the cellulase-repressing medium containing2 glucose in place of wheat bran and Avicel respectivelyThe flasks were incubated on an orbital shaker at 200 rpm28∘C For RNA isolation the samples of mycelium werecollected after 15 hours of cultivation and then subjectedto RNA isolation For fungal growth and protein secretionanalysis 500 120583l samples were collected after being inducedfor 1 day 2 days 3 days and 7 days After centrifugationthe culture supernatant was subjected to electrophoresis

BioMed Research International 3

extracellular protein concentration assay enzymatic assaysand the mycelia which were used to quantify the biomass

22 RNA Isolation Sequencing and Data Analysis Fungalmycelia of four samples were harvested by filtration andcentrifugation frozen and ground under liquid nitrogenTotal RNA were isolated by TRIzol (Invitrogen) accordingto the instruction and RNA extracts were monitored byelectrophoresis and quantified using a spectrophotometer

Total RNA were provided to Chinese National HumanGenome Center at Shanghai for sequencing mRNA purifiedfrom total RNA using the MicroPoly(A)Purist kit (Ambion)was used for library preparation and latter sequencingusing the Illumina Hiseq2000 platform The quality controlwas performed with FASTX-Toolkit (Version 0013) andthe reads were filtrated when more than 20 of baseswith PHRED score were lower than 30 The cleaned RNA-seq reads were mapped to the T reesei Rut-C30 genomeusing Tophat V132 [27] with Bowtie V01270 [28] witha minimum intron size of 20 bp and a maximum intronsize of 100000 bp the mean inner distance between matepairs and the standard deviation for the distribution oninner distances between mate pairs were set to 150 and 75respectively Transcript abundance indicated as the numberof fragments per kilobase per million fragments mapped(FPKM) was estimated with the program Cuffdiff from thepackage Cufflinks v083 [29] Differential expression valueswere determined using DEseq [30 31] FDR (False DiscoveryRate) control (the cutoff was 001) was used to identifythe significance of differences for multiple tests [32] Thegenome sequence and gene structure information forT reeseiQM6a (httpgenomejgidoegovTrire2Trire2homehtml)and Rut-C30 (httpgenomejgidoegovTrireRUTC30 1TrireRUTC30 1homehtml) were obtained from the JGIgenome Portal site [18 33] The FunCat [34] annotationfor QM6a was downloaded from the PENDANT genomedatabase [35] The FunCat annotation for Rut-C30 wasperformed using a blastn [36] search between QM6a andRut-C30 The differentially expressed genes were furtherfine-sorted after being classified according to their FunCatannotations

23 Fungal Growth and Protein Secretion Analysis The cul-ture filtrate was collected by centrifugation at 4∘C 8000119892 for10min For SDS-PAGE analysis 10 120583l of culture supernatantsof T reesei Rut-C30 its xyr1 deletion strain and xyr1 recom-plementation transformant were subjected to SDS-PAGEProtein concentration of each culture supernatant was deter-mined using Modified Bradford Protein Assay Kit (SangonShanghai China) according to the manufacturerrsquos guidelinesThe absorbance at 595 nm was measured with a VarioskanFlash microplate reader (Thermo electron Finland)

For enzymatic activity measurement the culture super-natants from the parent strain and different transformantswere prepared for filter paper assay (FPA) which has beenwidely used to determine the total cellulase activity secretedby fungi [37] Filter paper activity was measured accordingto the absorbance at 540 nm [38] in a Varioskan Flashmicroplate reader Xylanase activities of different samples

were determined according to Turunen et al [39] withmodifications The diluted supernatants (50 120583l) were incu-bated with 50 120583l of 1 beechwood xylan (Sigma) dis-solved in acetate buffer (100mM pH 48) at 50∘C for10min Then 100 120583l of DNS was added and incubated at95∘C for 10min and the absorbance at 540 nm was mea-sured One unit of activity was defined as the amount ofenzyme required to release 1 120583mol of reducing sugars perminute

For fungal growth determination the mycelia of differentsamples were used And the biomass assays were performedas Zhao et al reported [40] with some modifications Brieflythe mycelia contained in 500120583l samples were collected andwashed by distilled water twice and transferred to a new15ml EP tube suspended by 100 120583l distilled water Then1ml of diphenylamine reagent was added into the tubevotex thoroughly and reaction in 60∘C for 1 hour Aftercentrifugation for 10min at 10000119892 200120583l supernatant wastransferred to the ELISA plate and the absorbance at 595 nmwas measured with a Varioskan Flash microplate reader Thebiomass was indicated by the amount of DNA (mg) per mlsample Three independent measurements were performedfor all quantification assays and the data are averages of thethree independent determinations Studentrsquos two-tailed 119905-testwas performed using Excel 2007 (Microsoft WA)

24 Quantitative Real-Time PCR (qRT-PCR) The RNA sam-ples obtained from T reesei Rut-C30 and Δxyr1 strain oneither cellulose or glucose were reverse transcribed intocDNA using PrimeScript RT reagent Kit with gDNA Eraser(TaKaRa Dalian China) according to the manufacturerrsquosprotocol All real-time PCR were carried out on a Mas-tercycler (Eppendorf Hamburg Germany) with twintechreal-time PCR plate 96 (Eppendorf) and Masterclear real-time PCR Film (Eppendorf) For the reaction the SYBRPremix Ex Taq (TaKaRa Dalian China) was used for 25 120583lassays Primers used were given in Additional File TableS1 Three replicates were performed per experiment Theamplification protocol consisted of an initial denature step(30 sec at 95∘C) followed by 40 cycles of denaturation (5 secat 95∘C) annealing (30 sec at 58∘C) and elongation (15 secat 72∘C) The data analysis was done using Realplex soft-ware (Eppendorf Germany) The gpda gene measurementwas performed for reference calculation Two independentexperiments were performed and the data are averages ofthe duplicate determinations For every experiment twobiological replicates were carried out with three technicalreplicates each Studentrsquos two-tailed 119905-test was performedusing Excel 2007 (Microsoft WA)

25 In Silico Analysis of Xyr1 Binding Sites of the 51015840-UpstreamRegion of Genes Gene models in the T reesei Rut-C30genomewere downloaded from theT reesei genome databaseat the Joint Gemome Institute website (httpgenomejgi-psforgTrireRUTC30 1TrireRUTC30 1homehtml) 51015840upstreamregions (1 kb) for each gene were extracted from the scaf-folds The occurrence of 51015840-GGC(AT)3-3

1015840 motifs and 51015840-GGC(AT)4-3

1015840 motifs in the 51015840-upstream regions was deter-mined for both strands


26 Overexpression and Purification of the DNA BindingDomain of Xyr1 TheDNAbinding domain (residues 55ndash195)of Xyr1 was expressed by the pGEX system according to themanufacturerrsquos guidelinesThe first-strand cDNAwas used asa template to amplify the fragment encompassing the ORF ofDNA binding domain of Xyr1 using the primers indicated inTable S1 The fragment was then ligated into plasmid pGEX-4T-1 via BamHI andXhoI double digestion to produce pGEX-4T-Xyr1-Binding and subsequently introduced into E coliBL21 (DE3) for protein production Purification and verifi-cation of the GST-fused proteins were performed accordingto the methods described previously [41]

27 Electrophoretic Mobility Shift Assays (EMSAs) A univer-sal primer (51015840-ACTAACTCGCGTACTG-31015840) was labeled at51015840-terminal with Cyanine 5 (Cy5) (Sangon Shanghai China)Cy5-labeled DNA probes were generated by two steps PCRamplification using the primers as shown in Table S1 firstdouble-stranded DNA fragments were amplified from thegenomic DNA of T reesei Rut-C30 using specific primerpairs with universal primer sequence in their 51015840-terminalssecond Cy5-tag was added to the above DNA fragments byPCR reaction using the universal primer labeled with Cy5The resulting Cy5-labeled probes were recovered by agarosegel electrophoresis EMSA was performed using a constantamount (10 ng) of labeled DNA probe The purified proteinof Xyr1 binding domain was preincubated with Cy5-labeledprobe and then subjected to electrophoresis according to Renet al [42] The gel was visualized using Starion FLA-9000Scanner (FujiFilm Japan)

3 Results and Discussion

31 Deletion of xyr1 in T reesei Abolished LignocellulolyticEnzyme Production We constructed an xyr1 deletion strain(Δxyr1) by replacing the xyr1 open reading frame (ORF)with the hygromycin B resistance gene in T reesei Rut-C30We also constructed an xyr1 recomplementation strain (xyr1-rec) by replacing the hygromycin B resistance gene with aphleomycin resistance gene and the xyr1 gene in Δxyr1 (TableS1 Fig S1) Rut-C30 and xyr1-rec displayed normal profilesof secreted proteins when cultured on cellulose-inducingmedia whereas Δxyr1 produced significantly less detectablesecreted protein under the same conditions as shown in thepolyacrylamide gel electrophoresis profile (Figure 1(a)) andthe assay of extracellular protein concentrations (Figure 1(b))

We measured the cellulase and xylanase activities of thesecreted proteins from Rut-C30 and Δxyr1 under induc-ing conditions The cellulase activity of Δxyr1 was almostcompletely abolished whereas the secreted proteins of theparent strain Rut-C30 had an FPA (filter paper activity) of144 IUmlminus1 (Figure 1(c)) Although the culture supernatantsof Rut-C30 had a xylanase activity of 5292 IUmlminus1Δxyr1 hadnearly no detectable xylanase activity (Figure 1(d)) A similarphenomenon was reported previously in T reesei Qm9414and its xyr1 deletionmutant [9]The role of Xyr1 in regulatingcellulase and xylanase gene expression is strain-independent

During 7 d of cultivation on lignocelluloses the biomassof Rut-C30 increased from 54 120583gmlminus1 on the first day to

87120583gmlminus1 on the seventh day whereas the biomass of Δxyr1did not increase detectably (Figure 1(e))Δxyr1 appeared to beunable to produce lignocellulolytic enzymes that hydrolyzedthe lignocellulolytic substrates into monosaccharaides forfurther utilization as a carbon source for mycelia growth Treesei Qm9414 and its Δxyr1 strain showed similar growthrates on plates containing xylan or cellulose [9]This discrep-ancy might be due to the different methods used to measuregrowth rate Similar to our findings XLR-1 was previouslyrecognized as a homolog of Xyr1xlnR and regulated somehemicellulase gene expression in N crassa Deletion of xlr1resulted in minimal growth on xylan whereas the parentalstrain grew well [13]

To exclude the growth difference in measuring proteinsecretion we normalized protein secretion by the biomass ofRut-C30 andΔxyr1 Extracellular protein concentration of theparent strain Rut-C30 was also significantly higher than thatof Δxyr1 (Figure 1(f)) By contrast no significant differencein growth secretive protein concentrations or activities ofFPase and xylanasewas observed betweenRut-C30 andΔxyr1cultured in medium with glucose as the sole carbon sourceAll valueswere similar to those ofΔxyr1 cultured on cellulose-inducing media (Figures 1(b)ndash1(f))

32 RNA-SeqData Processing and FunCat Analysis In prepa-ration for RNA-seq the T reesei Rut-C30 and Δxyr1 strainswere precultured in SDB (Sabouraudrsquos dextrose broth) for48 h and then the mycelium was collected washed andtransferred intomedium containing lignocellulose or glucosefor another 15 h and then samples were prepared for RNAisolation and further sequencing The 268ndash498M readsgenerated corresponded to different samples After sequencequality control and mapping the number of properly pairedreads per sample ranged from 1396 to 2656M (Table S2)The transcription levels of 22 genes which were expresseddifferently in the parent strain Rut-C30 and its xyr1 deletionstrain according to RNA-seq data were further analyzed byquantitative reverse-transcriptase polymerase chain reaction(Table S3) The results were consistent with the results fromtranscription profiling

When cultured on lignocellulosic medium 467 geneswere expressed differentially in the Δxyr1 strain com-pared with the parental strain Rut-C30 (Table S4) Amongthese genes 177 were found to be downregulated andthe other 290 were upregulated in Δxyr1 Polysaccharidemetabolism transport cell rescuedefense and virulencelipid metabolism interaction with the environment proteinfate energy secondary metabolism biogenesis of cellularcomponents transcription amino acid metabolism the sig-nal transduction mechanism cell fate protein synthesisnucleotide metabolism and aromate metabolism were themain functional categories (fine-sorted after being classifiedby FunCats) to which the 467 differentially expressed geneswere allocated (Figure 2)

We also examined the expression of genes affected byxyr1 deletion when strains were cultured on glucose Atotal of 281 genes were found to be significantly affected186 genes were downregulated in the Δxyr1 strain and95 genes displayed significantly higher expression levels in


M Rut-C30 Rut-C30MW

(kDa)Lignocellulose Glucose

CBH1

Δxyr1xyr1-recxyr1-recΔxyr1

143

201

29

443

664972

(a)

2 3 71Time (day)

000510152025303540455055

Prot

ein

conc

entr

atio

n (m

gmlminus

1)

lowast

lowast

lowast

Rut-C30 lignocelluloseΔxyr1 lignocellulose

Rut-C30 glucoseΔxyr1 glucose

(b)

Rut-C30Δxyr1 Δxyr1Rut-C30

Lignocellulose Glucose

000204060810101214161820

lowast

Fpas

e (U

mlminus

1)

(c)

Rut-C30 Δxyr1Δxyr1 Rut-C30


300

400

500

600

0005011520

Xyla

nase

(Umlminus

1)

lowast

(d)

2 3 71Time (day)

0102030405060708090

100

Biom

ass (

120583gm

lminus1)

lowastlowastlowast

lowast



(e)

lowast

lowast

lowast

2 3 71Time (day)

0

10

20

30

40

50

60

Prot

ein

biom

ass (

mgm

gminus1)



(f)

Figure 1 Analysis of fermentation liquor of strains Rut-C30 Δxyr1 and xyr1-rec in lignocellulosic and glucose medium (a) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of culture supernatants of Rut-C30 Δxyr1 and xyr1-rec For each sample 10 120583l supernatantwas loaded (b) Protein concentration (c) FPase activity (d) xylanase activity (e) biomass and (f) protein concentration (normalizedby biomass of samples) of Rut-C30 and Δxyr1 in lignocellulosic and glucose medium For (b) (e) and (f) samples were collected aftercultivation for 1 2 3 and 7 d For SDS-PAGE (a) and characterization of FPase activity (c) and xylanase activity (d) samples collected 7 dafter fermentation were used All values presented in (b)ndash(f) are means of three independent measurements error bars indicate standarddeviations lowast119901 lt 001


2112

7 105 6 10

4 5 4 2 3 1 0 1

27

15

4129

26 2120 17 11

16 11 11 11 86 6 10

10

20

30

40

50

60

Num

ber o

f gen

es

Poly

sacc

harid

e met

abol

ism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Ener

gy

Seco

ndar

y m

etab

olism

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Sign

al tr

ansd

uctio

n m

echa

nism

Cel

l fat

e

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism

Figure 2 Significant examples of functional categories of genes differentially expressed under the lignocellulose condition The blue barsindicate genes downregulated and the red bars indicate genes upregulated in the xyr1 deletion strain Δxyr1 compared with Rut-C30

Δxyr1 (Table S5) Among the 281 genes 47 also appearedwith the genes transcriptionally affected by xyr1 deletionunder induced conditions (Table S6) The main categoriesrepresented among the 186 downregulated genes were thoseinvolved in cell rescue defense virulence energy transportlipid metabolism secondary metabolism energy amino acidmetabolism interaction with the environment and polysac-charide metabolism (Figure 3) On the other hand the maincategories represented among the 95 upregulated genes wereinvolved in energy transport amino acid metabolism andprotein fate and synthesis (Figure 3 Table S5)

In the parental strain Rut-C30 338 genes were upreg-ulated and 741 genes were downregulated under induced(lignocellulose) compared with repressed (glucose) condi-tions (Table S7) Notably there were many more genes withchanged expression levels than the Δxyr1 strain under theinduced and repressed conditions which may be because(1) full activation of the function of Xyr1 required specialculture conditions and (2) changing the culture conditionsaffected not only Xyr1 but also other regulators Analysis ofgenes existing in the intersection of Rut-C30Δxyr1 underinduced conditions and the Rut-C30 (lignocellulose)Rut-C30 (glucose) would facilitate understanding of the regula-tion mechanisms of these genes (Table S8)

33 Functional Xyr1 Stimulated the Expression of Lignocel-lulose Degradation-Related Genes When cultured on ligno-cellulosic medium most of the 177 downregulated genes inthe Δxyr1 strain were involved in carbohydrate metabolismcompared with the parent strain Rut-C30 (Table S4) Inaddition to previously reported cbh1 cbh2 egl1 and bgl1 [9]expression of the other functional cellulase genes egl2 egl3

egl4 (cel61a) egl5 cel61b and bgl2 was significantly impairedin the Δxyr1 strain (Table 1)

Additional nonenzymatic cellulose-attacking proteinswere also regulated in a coordinated fashion with other cel-lulose-degrading enzymes (Table 1) These proteins includeswollenin SWO1 which is a protein carrying a cellulose-binding domain and an expansin-like domain that disruptsthe crystalline cellulose structure [43] and cip1 and cip2which encode a CE15 glucuronyl esterase [44] Both cip1 andcip2 contain cellulose-binding domains and signal sequences[45]

A previous report indicated that Xyr1 is not involvedin the activation of bgl2 (TrireRUTC30 127115) expression[9] Although the results from our study showed that it wassignificantly downregulated in the xyr1 deletion mutant thefunction of cip1 remains unknown To further determinewhether Xyr1 could directly regulate bgl2 and cip1 the DNAfragments from promoter regions of bgl2 and cip1 werechosen as the candidates in electrophoretic mobility shiftassays (EMSAs) Substantial gel shifts were observed for thelabeled probes corresponding to the promoter regions ofbgl2 and cip1 and typical protein concentration-dependentbinding trends appeared (Figures 4(a) and 4(b)) The resultssuggest that Xyr1 activated the expression of bgl2 and cip1directly by binding to their promoter regions just as itwas previously reported to bind to the promoter regionsof cellulase genes [15] As each of the two promoters hasmore than one 51015840-GGC(AT)3-3

1015840 motif various Xyr1-DNAcomplexes could be formed which was reflected in differentlow mobility bands (Figures 4(a) and 4(b))

Similarly deletion of xyr1 affected expression of hemicel-lulase genes on the inducing medium (Table 1) In T reesei


Table 1 Changes in CAZome gene expression between T reesei Rut-C30 and Δxyr1 under the lignocellulose condition

Protein ID log2 ratio Rut-C30Δxyr1 119876-valueCellulases

Endoglucanase 1 cel7b 5304 964332 0Endoglucanase 2 cel5a 72489 104187 0Endoglucanase 3 cel12a 124438 846247 107119864 minus 07

Endoglucanase 5 cel45a 25940 922161 218119864 minus 08

Exoglucanase 1 cel7a 125125 108384 0


Cellulase enhancing protein cel61a 139633 101565 0

Cellulase enhancing protein cel61b 122518 10063 0

120573-Glucosidase 1 cel3a 136547 589937 0

120573-Glucosidase 2 cel1a 127115 183786 343119864 minus 05

120573-Glucosidase cel1b 77989 252453 564119864 minus 11

120573-Glucosidase cel3d 122639 49912 0120573-Glucosidase cel3e 74305 124505 0002047120573-Glucosidase 109567 minus118554 0004262

HemicellulasesAcetylxylan esterase axe1 139631 104907 0Acetylxylan esterase 88887 8921338 399119864 minus 08

120572-Galactosidase 71638 496894 137119864 minus 08

120572-N-Arabinofuranosidase abf1 102517 319423 150119864 minus 07

120572-Xylosidase 134448 6166894 222119864 minus 08


Endo-14-120573-xylanase 1 xyn1 38418 3793969 0000173



Mannan endo-16-120572-mannosidase 126869 163426 0001508

120572-L-Arabinofuranosidase abf2 118070 6129569 681119864 minus 07


120573-Mannosidase 67432 582358 0

Exo-14-120573-xylosidase 140746 11148 0

Mannan endo-14-120573-mannosidase 122377 7256397 435119864 minus 05

Xyloglucanase 111943 634823 0

Glucuronoxylanase xynC 93498 105903 556119864 minus 11

120572-Glucuronidase 90302 8110081 593119864 minus 6

Nonenzymatic cellulose attacking enzymesCip1 121449 10417 0Cip2 125575 110829 0Swollenin 104220 873037 0

ChitinasesChitinase 94061 minus1583 0009666Chitinase 142298 minus160663 300119864 minus 05

Chitinase 7503 minus174855 000568

Endochitinase 33168 minus171966 0002243

Endochitinase 33886 minus20161 409119864 minus 07


Endochitinase 142123 215448 561119864 minus 08

Exochitinase 104242 minus213026 456119864 minus 09

Glucan 13-120573-glucosidaseGlucan endo-13-120573-glucosidase 125426 minus171506 000101449Glucan endo-13-120573-glucosidase 125427 minus213884 000104273Glucan 13-120573-glucosidase 25104 minus227709 396119864 minus 08

Glucan endo-13-120573-glucosidase 103726 minus234806 249119864 minus 05


Table 1 Continued

Protein ID log2 ratio Rut-C30Δxyr1 119876-valueOther glycoside hydrolases

Probable exopolygalacturonase X 91667 179769119890 + 308 937119864 minus 09

Glucuronoxylanase xynC 90847 104524 0

Endo-120573-16-galactanase 11580 468839 437119864 minus 09

13-120573-Glucanosyltransferase gel4 103899 338036 0

Probable endopolygalacturonase 133383 307815 693119864 minus 06

14-120572-Glucan-branching enzyme 114146 122399 000350323

Putative endo-13(4)-120573-glucanase 2 100837 minus152054 0000245344

120572-12-Mannosidase 122299 minus159948 301119864 minus 05

Glucan endo-13-120572-glucosidase agn1 94877 minus305265 176119864 minus 13

28 25 2317

2115

11 83 6

2 0 2 2

9 129

13 813

7

38 1

4 6 2 1Po

lysa

ccha

ride m

etab

olism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism

0

5

10

15

20

25

30

35

40

Num

ber o

f gen

es

Seco

ndar

y m

etab

olism

Ener

gy

Figure 3 Significant examples of functional categories of genes differentially expressed under the glucose condition The blue bars indicategenes downregulated and the red bars indicated genes upregulated in Δxyr1 compared with Rut-C30

hemicellulase genes xyn1 xyn2 and bxl1 were previouslyreported to be transcriptionally activated by Xyr1 whenthe inducer was supplied [9] According to the RNA-seqdata in this study the 120573-14-xylan main chain degradingenzyme gene xyn3 and the exo-14-120573-xylosidase gene whichhydrolyzesD-xylose from the nonreducing end of xylan werefound to be downregulated in a coordinated fashion withxyn1 xyn2 and bxl1 Other genes encoding hemicellulaseswhich hydrolyze the side groups linked to the 120573-14-xylanmain chain were also reported to be activated by Xyr1These genes included120572-arabinofuranosidases (abf1 and abf2)which remove the arabinose side chain 120573-galactosidaseswhich catalyze the hydrolysis of 120573-galactosidase from theside groups 120573-mannosidases which hydrolyze the 120573-14-manno-oligomers acetylxylan esterases which remove anacetyl group from the xylan backbone 120572-glucuronidaseswhich cleave the 120572-12-glycosidic bond of the 4-O-methy-D-glucuronic acid side chain of xylans and xyloglucanase

which releases glucose from xyloglucan [46ndash48] Thusalmost all functional cellulase and hemicellulase genes in Treesei were coregulated by the TF Xyr1 and the disruptionof xyr1 severely blocked their expression under inducingconditions

In addition we analyzed the transcription of genespotentially involved in the xylosemetabolism pathway whichmay be crucial for the expression of xylanases [49] Twogenes encoding D-xylose and L-xylulose reductase werealso found to be downregulated in Δxyr1 on lignocellulose-inducingmedia BothD-xylose andL-xylulose reductasemayparticipate in xylose metabolism This result was consistentwith results from previous studies [11 26] In T reesei D-xylose reductase was required to metabolize D-xylose toachieve full induction of xylanase expression [49] The L-xylulose reductase transcript was reported to be absent ina T reesei Δxyr1 strain which severely affects its growthon D-xylose as the sole carbon source [9] The expression


02 04 08 08

Competitor S NS

08

complex

Free DNA

10

bgl2 (TrireRUTC30 127115)

Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(a)

Competitor

- 02 04 08 08

S NS

08

complex

Free DNA

10

cip1 (TrireRUTC30 121449)

Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(b)

complex

Free DNA

Competitor

02 04 08 08

S NS

08

10

Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---

---

--- --- --- --- --- ---

(TrireRUTC30 127980)MFS lactose transporter gene

(c)

complex

Free DNA

05 10 10 10

Competitor S NS

10

nmrA-like gene (TrireRUTC30 121828)

GST (120583M)

Xyr1-DNA

GST-Xyr1 (120583M)

--- --- --- ---

------

--- --- --- --- ---

(d)

Figure 4 Xyr1 functionally binds to the upstream regions of four genes downregulated in Δxyr1 on inducing medium Binding of Xyr1 tothe promoter regions of the intracellular 120573-glucosidase-encoding gene bgl2 (a) the nonenzymatic cellulose-attacking enzyme-encoding genecip1 (b) the putative MFS lactose transporter-encoding gene lp (c) and the nmr-like gene (d) The amounts of purified Xyr1 (120583M) used wereas indicated and about 10 ng of Cy5-labeled probes was added to each reactionThe specificity of shifts was verified by adding 100-fold excessunlabeled specific (S) and nonspecific (NS) competitor DNA Purified GST (1 120583M) was used as the negative control to exclude nonspecificbinding by GST

of xylose reductase genes in A niger is also dependent onXlnR a homolog to Xyr1 [50] These results indicate thatXyr1 regulates xylan-decomposing and xylose-metabolizinggenes suggesting that these genes are subjected to concertedevolution (Fig S2)

Although lignin is another main component of lig-nocellulose T reesei is not considered to be a potentiallignin-degrading fungus [51 52] and no ligninolytic activityhas been reported However a recent study demonstratedthat some lignin depolymerizing oxidoreductases includ-ing laccase glyoxal oxidase peroxidasecatalase L-ascorbateperoxidase copperzinc superoxide dismutase and severalother oxidoreductases are expressed when T reesei Rut-C30is grown on natural lignocellulosic biomass [53]

In this study expression levels of two laccase genes(TrireRUTC30 104519 TrireRUTC30 36885) were higher on

lignocellulose medium than on glucose-repressing mediumin Rut-C30 (Table S7) In the Δxyr1 strain one of thelaccase genes (TrireRUTC30 104519) could also be inducedon lignocellulosic medium (Table S9) However other lignin-degrading genes such as glyoxal oxidase and CuZn superox-ide dismutase (TrireRUTC30 26844 TrireRUTC30 112797)were not induced on lignocellulose in either strain (Tables S7and S9) No gene encoding typical lignin-degrading enzymesincluding the six laccase-like multicopper oxidase-encodinggenes [51] was downregulated in Δxyr1 Although severallignin degradation-related enzymes could be induced by lig-nocelluloses their expressionwas possiblyXyr1-independentwhich inferred a specific regulatory pattern in controlling theexpression of the lignin-degrading enzyme genes

When cultured on glucose xyr1 deletion in Rut-C30resulted in the downregulated expression of six genes


encoding lignocellulolytic enzymes (Table S5) For exampletranscriptional levels of cbh1 and cbh2 were reduced by54- and 8-fold respectively in Δxyr1 compared with thetranscription levels in Rut-C30 (Table S5) At the proteinlevel the band of CBH1 could be detected in Rut-C30and the xyr1 recomplementation strain xyr1-rec cultured onglucose but not obvious in the xyr1 deletion strain Δxyr1(Figure 1(a)) Although other lignocellulolytic enzymes didnot show statistically significant differences their expressionlevels decreased similarly (data not shown) These resultssuggest that for the carbon catabolite derepression strainRut-C30 Xyr1 was partially functional in controlling theexpression of lignocellulolytic enzymes even under repressedconditions

Among the 1079 differently expressed genes (338 upreg-ulated and 741 downregulated genes) of the parent strainRut-C30 cultured under the induced (lignocellulose) andrepressed (glucose Table S7) conditions a total of 69genes (53 upregulated and 16 downregulated) were cate-gorized as involved in carbohydrate metabolism accord-ing to the FunCat classification (Table S10) Among the53 upregulated genes 35 were detected among the down-regulated genes in Δxyr1 under lignocellulose conditionscompared with those of the parental strain Rut-C30 (Tables1 S8 and S10) which included almost all functional cel-lulase and hemicellulase genes These 35 putative Xyr1-regulated target genes may have been activated underthe lignocellulose condition to participate in carbohydratemetabolism while the remaining 18 genes were potentiallycoregulated by other unidentified regulators By contrastonly one cellulase gene TrireRUTC30 109567 (BGL GH3family FPKM [the number of fragments per kilobase permillion fragments mapped] Δxyr1 lignocellulose 107295FPKM Δxyr1 glucose 53187) was detected to be expresseddifferently in Δxyr1 under the induced and repressed condi-tions

Further in vivo or in vitro assays would be neededto detect whether any of the 53 upregulated genes weredirectly regulated by Xyr1 For example bgl2 (TrireRUTC30127115) was not detected as upregulated in Rut-C30 underlignocellulose compared with glucose conditions Because ofthese results bgl2 was previously regarded as not regulatedby Xyr1 [9] bgl2 could also bind with Xyr1 in the gel-retardation assays (Figure 4(a)) In this case bgl2 might besubjected to the combined reactions of Xyr1 and several othercoregulators

Overall the above results supported the global roleof Xyr1 as an essential regulator in activating lignocel-lulose degradation-related genes including cellulose- andhemicellulose-encoding genes and genes participating inxylose metabolism but not lignin degradation-related genesNonenzymatic cellulose-attacking enzymes and two glyco-side hydrolase family AA9 protein (Cel61a and Cel61b)-encoding genes harbored coordinated transcription changeswith (hemi)cellulase genes which suggested that they wereunder the same regulation of Xyr1 and played key roles inlignocellulose degradation Even under glucose conditionsXyr1 plays a partial role in regulation of the expressionof cellulase genes in T reesei Rut-C30 which implies its

direct or indirect interactions with Cre1 in T reesei wild-typestrains

34 Transcription Levels of Transporter Genes Affected byxyr1 Deletion in T reesei Rut-C30 Transporter was thesecond largest category of genes downregulated in the Δxyr1strain when cultured on lignocellulose which is comprisedof 21 transporter-encoding genes (Table S11 Figure 2)among which 18 genes belonged to the major facilita-tor superfamily (MFS) The MFS transporters are single-polypeptide secondary carriers capable of transporting onlysmall solutes [54] and they are distributed ubiquitouslythroughout virtually all currently recognized organismalphyla [55] The 18 MFS genes were classified into sevenfamilies according to the Transporter ClassificationDatabase(httpwwwtcdborg) which included the sugar porter(SP) fructose H+ symporter nitratenitrite porter fami-lies the phosphate H+ symporter monocarboxylate porteranioncation symporter and vacuolar basic amino acid trans-porter families (Figure 5(a))

Among the 18 MFS genes 11 genes belong to the SPfamily (Table S11) which is the largest MFS family trans-porting sugars such as glucose fructose mannose galactosexylose maltose lactose 120572-glucoside and quinate [56] Inaddition the expression levels of 9 of the 11 SP familymembers were significantly reduced in the parental strainRut-C30 which was cultured from inducing (lignocellu-lose) to repressing (glucose) conditions (Tables S8 and S11Figure 5) Our EMSA experiments by testing the purifiedXyr1 and a putative lactose transporter gene TrireRUTC30127980 belonging to these SP family members (Figure 4(c))confirmed that the transcription of SP transporters might bedirectly regulated by Xyr1 in T reesei or its homolog in otherorganisms

A sophorose-inducible 120573-diglucoside permease was pre-viously reported to be involved in the induction of the cellu-lases in T reesei [57] indicating that some sugar transportershave an important role in induction of cellulase expressionThe induced expression of sugar transporters might be theprevious and necessary step in the induction of cellulose-encoding genes by promoting the transportation of inducibleglucose into the cell Galazka et al [58] introduced thecellodextrin transporter from N crassa into Saccharomycescerevisiae which has led to efficient growth of this yeast oncellodextrins However the functions of the Xyr1-dependenttransporters have not yet been characterized Recently oneof these Xyr1-dependent transporters (TrireRUTC30 109243Trire2 3405) was identified as essential for lactose uptake andcellulase induction by lactose [59] In another study the dele-tion strain of Trire2 3405 crt1 showed severe growth defectson Avicel [60] These results indicated that Xyr1rsquos role as amajor activator of cellulases was achieved by regulating thetranscription of some transporters Likewise 11 carbohydratetransporter genes were identified to depend on a functionalXLR-1 for increased expression levels when exposed to xylanin N crassa [13] indicating that the impact of Xyr1 or itshomolog on the expression of transporter genes is not aphenomenon exclusive to T reesei As the uptake of celluloseoligosaccharides could play an important role in cellulase


2A11 the sugar transporter family2A17 the fucoseH+ symporter (FHS) family2A18 the nitratenitrate porter (NNP) family2A19 the phosphateH+ symporter (PHS) family2A113 the monocarboxylate porter (MCP) family2A114 the anioncation symporter (ACS) family2A148 the vacuolar basic amino acid transporter(VBAAT) family

Major facilitator superfamily transportersMajor intrinsic proteinPredicted cation transporterUncharacterized transporter

111

111

2 1

18

11 1

(a)

Major facilitator superfamily transportersATP-binding cassette (ABC) transportersPredicted cation transportersPredicted ammonium transport outward proteinMajor intrinsic proteinPutative amino acid transporterUncharacterized transporter

2A11 the sugar transporter family2A12 the drugH+ antiporter-1 (12 spanners) (DHA1)family2A112 sialateH+ symporter (SHS) family2A116 the siderophore-iron transporter (SIT)family

9

7

5

12

2 1

4

3

1

1

(b)

Figure 5 Distribution of putative transporters differentially expressed in Δxyr1 compared with Rut-C30 when cultured on lignocelluloseDistribution of putative transporters downregulated (a) and upregulated (b) in Δxyr1 compared with Rut-C30 when cultured under thelignocellulose culture condition Details of major facilitator superfamily (MFS) transporter analysis are provided in the right panels of (a) and(b)

formation [57] identification of the characteristics of varioussugar transporters is important and would further assist inthe identification of the cellulase induction mechanism

ATP-binding cassettes (ABCs) containing ABC trans-porters are also reported to be important sugar transportersOur RNA-seq data demonstrated that no ABC transportergene was downregulated by deletion of xyr1 or by cultureunder the repressing condition (Tables S11 S12) Howeverseven genes encoding ABC transporters were upregulatedafter xyr1 deletion under lignocellulose-inducing conditions(Table S11 Figure 5(a)) These results implied that the ABCtransporters are inconsistent for the expression trends of(hemi)cellulase genes that might be coregulated by Xyr1By contrast two ABC transporters (Tr 2687 and Tr 58366)were reported downregulated under the cellulose or glu-cose condition in the Δxyr1 mutant strain compared to itsparental strain Qm9414 [16] Another nine MFS transportergenes also exhibited increased expression levels after deletion

of xyr1 under lignocellulose-inducing conditions (TableS11)

Whether the increased expression of these transporterscontributed to nutrient uptake under starvation due to aninability to utilize lignocelluloses in Δxyr1 is not knownThus two MFS transporter-encoding genesmdashthe putativeMFS glucose transporter rco3 (TrireRUTC30 136988) andthe predictedMFS siderophore iron transporter sit (Trire-RUTC30 115870)mdashwere selected for EMSAs However nogel retardation was observed for the probes correspondingto the promoters of these two genes (Fig S3A and B) Theseresults suggest that the upregulated transporter genes werenot regulated directly by Xyr1

35 Several Genes Relevant to Basal MetabolismWere Inclinedto Be Repressed by Xyr1 More genes encoding enzymesrelated to basalmetabolism such as lipidmetabolism proteinfate amino acid metabolism and nucleotide metabolism


Polysaccharidemetabolism Lipase Protease Amino acid

metabolismNucleotidemetabolism

41 0 0 4 0DownregulatedUpregulated 15 8 8 11 6

0

10

20

30

40

50

60

Num

ber o

f gen

es

Figure 6 Distribution of differentially expressed genes involved in basal metabolism after xyr1 deletion under lignocellulosic medium

were detected to be upregulated in Δxyr1 than in Rut-C30under inducing conditions (Figure 2) For example xyr1deletion resulted in increased expression of 11 genes encodingenzymes participating in amino acid metabolism and 6 genesparticipating in nucleotide metabolism when cultured onlignocelluloses (Figure 6) In addition eight lipase genesand eight protease genes were upregulated (five of themwere predicted to be secreted using SignalP V40 program[httpwwwcbsdtudkservicesSignalP] Figure 6)

Chitinases participate in fungal cell-wall morphogene-sis including spore germination hyphal elongation hyphalbranching and autolysis of mycelium [61ndash63] Membersof the fungal genus Trichoderma are known to producechitinase but very little is known about the regulationof expression of these chitinase genes According to ourRNA-seq data six genes (TrireRUTC30 94061 (chi18-12)142298 7503 (chi18-15) 33168 (chi18-6) 124526 (chi18-5) and104242) encoding secreted chitinases [64] were found to beupregulated on lignocellulose after xyr1 deletion (Table 1)By contrast the transcription levels of these genes did notdiffer between strains in themediumwith glucose as a carbonsource In accordance with this as Bischof et al reported oneof themajor differences in theT reesei transcriptome betweenwheat straw and lactose is the wheat straw specific chitinasesand mannosidase which were significantly higher expressedin an xyr1-deletion strain [65] Seidl et al found that chi18-12 belonged to Group B chi18-6 and chi18-5 belonged toGroup A while chi18-15 did not belong to any group In thiscase we could predicted that TrireRUTC30 94061 33168 and124526 were Chitinases possibly involved in mycoparasitism[64] Induction of chitinase genes could be influenced notonly by the presence of chitin but also by carbon cataboliterepression the N source and starvation [66] As the strainΔxyr1 might be subjected to a shortage of nutrients asreflected in retarded mycelia growth (Figure 1(e)) thesechitinase genes seemed to be induced by carbon starvation

One of the upregulated putative chitinase genes endoT (TrireRUTC30 142298) was shown to be not involvedin chitin degradation but it has mannosyl-glycoproteinendo-N-acetyl-120573-D-glucosaminidase activity [67] It wasreported to be responsible for N-deglycosylation of proteinsexpressed and secreted by T reesei [68] According to ourRNA-seq data the endo T in Rut-C30 was significantlyupregulated under lignocellulose conditions (FPKM= 4287)compared with glucose conditions (FPKM = 374) Howeverendo T further displayed significantly increased expressionlevels in the xyr1 deletion mutant Δxyr1 compared with Rut-C30 when cultured on lignocelluloses with FPKM valuesof 13057 and 4287 respectively These controversial resultssuggest that the regulatory mechanism of endo T is verycomplex

Foreman et al [45] reported that endoT is not coregulatedwith the expression of cellulase genes in T reesei Qm6a orRL-P37 Whether the increased expression of endo T was inresponse to carbon starvation remains unknown as does themechanism of regulation by Xyr1 As a result EMSAs wereperformed and strong gel retardation was observed for theprobe corresponding to the upstream region of the putativeendo T with Xyr1 (Figure 7(a)) In T reesei RutC-30 theexpression of endo T was potentially activated by Xyr1 as wellas lignocellulose degradation-related genes when culturedon lignocellulose Thus the resulted single GlcNAc on theheterologous expressed TrBglS benefitted from its enzymaticactivity and thermostability compared with PpBglS [69]After deletion of xyr1 the upregulation of endo T might bedue to starvation of carbon sources which might be an effortto deglycosylate the glycan coat of the glycoprotein composedof the cell wall and contribute to further protease degradation[67]

In filamentous fungi extracellular proteases are usuallyproduced in response to carbon or N derepression [70]These phenomena mean that genes related to nonpreferredN source utilization were stimulated in the Δxyr1 strain


05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---

(TrireRUTC30 142298)endo T

(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------

(TrireRUTC30 104564)pepA

(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---

hsp23 (TrireRUTC30 122251)

(c)

Figure 7 DNA binding of Xyr1 to the upstream regions of three genes upregulated after xyr1 deletion Xyr1 functionally binds to thepromoter regions from themannosyl-glycoprotein endo-N-acetyl-120573-D-glucosaminidase the putative acid aspergillopepsin I and the possibleheat shock protein Hsp23-encoding genes endo T (a) pepA (b) and hsp23 (c) which were upregulated in Δxyr1 when cultured on theinducing medium Strong gel shifts were observed after Xyr1 was added to the reactions The protein-DNA complex increased with proteinconcentrationThe amounts of purified Xyr1 (120583M) used were as indicated and about 10 ng Cy5-labeled probe was added to each reactionThespecificity of shifts was verified by adding 100-fold excess unlabeled specific (S) and nonspecific (NS) competitor DNA Purified GST (1 120583M)was used as the negative control to exclude nonspecific binding by GST

possibly due to the inability to utilize lignocelluloses Amongthe five upregulated secreted proteases the pepA (Trire-RUTC30 104564)-encoding putative acid aspergillopepsinI but not the tryp (TrireRUTC30 94189)-encoding putativetrypsin with an optimal operating pH of approximately75ndash85 was a putative Xyr1 target in Rut-C30 (Figures7(b) and S3C) These results imply that Xyr1 also tends torepress the acid peptidases directly to reduce the degradationof cellulases in medium with a pH value of about 5 Inaddition an 120572-12-mannosidase gene mds1 (TrireRUTC30122299) which participated in N-glycosylation modifica-tion of glycoprotein showed increased expression levels inΔxyr1 (FPKM values ranged from 3151 in Rut-C30 to 9551in Δxyr1) The 120572-12-mannosidase was reported to readily

convert Man8GlcNAc2 or a mixture of Man(6minus9)GlcNAc2oligosaccharides to the respective Man5 structures [71] andwas suggested to be localized in the Golgi apparatus [72]No interaction between Xyr1 and the promoter region ofthis 120572-12-mannosidase gene mds1 was detected in EMSAs(Fig S3D) In this case the upregulated 120572-12-mannosidaseexpression level in Δxyr1 might be attributed largely to itscarbon starvation status

36 Transcription of Genes Related to Energy MetabolismIn our study three genes predicted to be alcohol dehydro-genase genes (TrireRUTC30 128036 TrireRUTC30 26479and TrireRUTC30 133809) were also upregulated when xyr1was deleted in the lignocellulose condition (Table S4) In


the glucose condition however their expression exhibited nosignificant difference between Rut-C30 and Δxyr1 (Table S5)In Aspergillus nidulans an alcohol dehydrogenase (ADHII)was previously shown to be induced by carbon starvationstress [73] We speculated that the transcription of thesealcohol dehydrogenase genes could be sensitive to carbonstarvation but with no relationship to the existence ofXyr1 In agreement with this speculation the expression ofTrireRUTC30 133809 was upregulated significantly in Rut-C30 as well as the xyr1 deletion strain Δxyr1 under inducedconditions compared with repressed conditions (Tables S7S9)

The expression of TrireRUTC30 26479 was also stim-ulated in Δxyr1 when transferred from the glucose tothe lignocellulose condition (Table S9) In addition Trire-RUTC30 87029 the Podospora anserine PaATG1 orthologwas upregulated in Δxyr1 compared with Rut-C30 under alignocellulose condition (Table S4) TrireRUTC30 87029 is aserine-threonine kinase composed of an N-terminal kinasedomain and a C-terminal domain with unknown func-tion The PaATG1 mutant displayed developmental defectscharacteristic of abrogated autophagy in P anserine [74]Autophagy is a process of protein and organelle degradationby the vacuole (lysosome) This process is conserved inorganisms and functions as a cell survivalmechanism duringnutrient starvationTherefore elevatedATG1 gene expressionprobably contributed to accommodating the Δxyr1 strainduring carbon starvation reflecting an xyr1 deletion thattotally eliminated the capability to utilize lignocelluloses asa carbon source

The glycolysis and citric acid cycles are among the mostimportant energy-releasing pathways in glucose metabolismComparing the gene expression levels between Rut-C30 andΔxyr1 under a glucose condition deletion of xyr1 led tosignificant variation in a succinyl-CoA ligase gene (Trire-RUTC30 135123) and a glycerol-3-phosphate dehydrogenasegene (TrireRUTC30 116453 Table S5) The AD-lactate dehy-drogenase (TrireRUTC30 73249) andNAD-dependentmalicenzyme (TrireRUTC30 69465) genes (Table S5) were alsoinfluenced by xyr1 deletion and these two enzymes wereinvolved in yielding the important carbohydrate metabolicintermediate pyruvate The binding sites of Xyr1 weredetected in the promoter regions of the genes mentionedabove in xyr1 deletion mutants However more investigationis required to confirm whether Xyr1 also participates in theglucose metabolism and energy-releasing pathways

37 Transcription Changes of Putative Heat Shock ProteinsHeat shock proteins are often induced when organismsrespond to extreme temperature and other stresses such asstarvation [75 76] Based on transcription profiling of Rut-C30 and Δxyr1 under induced and repressed conditions weattempted to explain the roles that heat shock proteins playin lignocellulose degradation as well as their transcriptionalregulation mechanism

The transformation of hsp23 (small heat shock protein-encoding gene of Trichoderma virens) into Trichodermaharzianumwas reported to confer thermotolerance and resultin higher biomass accumulation under thermal stress [77]

Under lignocellulose-inducing conditions a gene homolo-gous to hsp23 TrireRUTC30 122251 was upregulated inΔxyr1 relative to its parent strain (Table S4) These resultsillustrate the process of lignocellulose degradation by Treesei Rut-C30 in which the heat shock protein-encodinggenes harbored expression profiles similar to profiles of thelignocellulose degradation-related genes

Due to the putative Xyr1-binding consensuses in theupstream region of this hsp23 homolog EMSAs were per-formed The results indicated that Xyr1 could bind to theprobe corresponding to the upstream region of this hsp23homolog (Figure 7(c)) In this case we speculated thatunder induced conditions Xyr1 was inclined to repress theexpression of heat shock proteins to maintain the balanceof the parent strain Rut-C30 and to ensure maximumlignocellulose degradation In agreement with these resultseven under the glucose condition deletion of xyr1 causedupregulation of a heat shock protein Hsp78-encoding gene(TrireRUTC30 73724 Table S5) However another heatshock protein Hsp70-coding gene TrireRUTC30 25176 wasdownregulated in Δxyr1 suggesting that the regulation ofheat shock proteins is much more complex in Rut-C30 underthe glucose condition Two putative heat shock protein-encoding genes Hsp70 (TrireRUTC30 97499) and DnaJ(Hsp40 TrireRUTC30 137482) were downregulated in Rut-C30 under the lignocellulose condition compared with theglucose condition (Table S7)

38 Expression Profiles of Transcription Factors With theexception of genes participating in lignocellulose degradationand transporters the expression profiles of characterized TFgenes were analyzed We found that the Ace3 (TrireRUTC3098455)-encoding gene (FPKM = 1373ndash683) and the geneencoding the NmrA-like family domain-containing protein(TrireRUTC30 121828 FPKM = 2784ndash588) were also down-regulated in Δxyr1 compared with the parent strain under alignocellulose-inducing condition (Table S4)

Ace3 is a recently identified transcription activator of cel-lulase genes [78] InΔxyr1 its transcription level decreased byabout 50 Hakkinen et al [78] reported that the expressionlevel of xyr1 was lower in the ace3 deletion strain than inthe parent strain Qm9414 In this case Xyr1 and Ace3 mightbe cross-regulated which requires further study by protein-DNA and protein-protein interaction assays In T reeseiKap8 was identified to be essential for the nuclear transportof the Xyr1 and deletion of kap8 completely abolished thetranscription of 42 CAzymes (including all the cellulases andhemicellulases) which resemble the phenomenon of a xyr1loss-of-function mutant [79] However the transcription ofxyr1 itself is not affected by kap8 deletion implying that itstranscription is hardly dependent onXyr1-autoregulationOnthe contrary Ace3 exhibited significant reduced expressionlevel after kap8 deletion under the induced condition indi-cated that its expression is partially Xyr1-dependent [79]

NmrA is a negative transcriptional regulator involved inN metabolite repression In A nidulans deletion of nmrAresulted in partial derepression of activities with utilizationof nonpreferred N sources in the case of N repression [80]This phenomenon was also observed in other filamentous


Table 2 The transcription of several identified TFs in Rut-C30 under the induced compared with repressed condition

Encoding genes of TFs Protein ID TrireRUTC30 FPKM (glucose) FPKM (lignocellulose) Significantxyr1 98788 4373 4293 Nocre1-1 23706 49807 80249 Noace1 122363 33792 20020 Noace2 32395 237 2107 Noace3 98455 8156 13733 NobglR 91236 22527 12807 Nohap2 93466 3957 2140 Nohap3 24298 7116 7536 Noclr-1 68701 1114 983 Noclr-2 76250 321 280 NonmrA 121828 215 2785 NoareA 140814 5295 3658 No

fungi such asN crassa [81]We speculated that the decreasedexpression of this nmrA-like gene might facilitate the uti-lization of an N source in response to carbon starvationin the xyr1-deleted strain or be directly regulated by Xyr1EMSA was employed to detect the potential interactionbetween Xyr1 and this nmrA-like gene and a strong gelshift of the probe was observed when Xyr1 was addedto the reaction (Figure 4(d)) The results suggested thatunder a lignocellulose-inducing condition Xyr1 activates theexpression of nmrA to repress utilization of nonpreferred Nsources possibly through repression of the activity of theA nidulans AreA [82] homolog in T reesei Rut-C30 andensure degradation of lignocellulose However the precisecharacteristic of this gene product remains to be determined

Similar to the effect of xyr1 deletion on the expression ofthe nmrA-like gene when cultured on lignocellulose Δxyr1showed a significantly lower level of the nmrA-like genewhenboth strains were cultured on glucose Obviously decreasedexpression of this nmrA-like gene was not a response tocarbon starvation As described in the gel-retardation assaythe nmr-like gene was a putative target of Xyr1 (Figure 4(d))The results further imply that the nmrA-like gene was aputative target downstream of Xyr1 and repressed by Xyr1regardless of the culture conditions

The transcription levels of genes encoding transcrip-tion regulators for cellulase or hemicellulase genes werealso compared in Rut-C30 under induced and repressedconditions These genes included cre1-1 ace1 ace2 ace3bglR hap2 hap3 clr-1 and clr-2 However none of thesegenes showed a significant response (Table 2) These resultsimply that all of these genes had constitutive expression andwere not induced when transferred to lignocellulose-inducedconditions The two transcription regulators involved in Nmetabolism nmrA-like and areA were also analyzed and thesame expressionmodewas detected (Table 2) suggesting thatcomplex posttranslational regulation such as phosphoryla-tion on Cre1 [83] acted on these transcription regulatorswhen T reesei Rut-C30 was transferred from repressed toinduced conditions Considering the lack of a significantdifference in xyr1 transcription levels of Rut-C30 underinduced (FPKM = 4293) and repressed (FPKM = 4373)

GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if

Figure 8 Frequencies of appearance of 51015840-GGC(AT)3-31015840 and 51015840-

GGC(AT)4-31015840 motifs in the upstream regions of Xyr1-upregulated

genes

conditions these results imply that the function of Xyr1 wasseverely repressed under repressive culture conditions

39 Putative Regulation Mechanism of Xyr1 in T reeseiAs the 51015840-GGC(AT)3-3

1015840 and 51015840-GGC(AT)4-31015840 motifs play

important roles as functional Xyr1-binding sites in T reesei[15] we searched for these motifs in the 1-kb 51015840-upstreamregions of all genes We identified an obvious tendency ofincreasing occurrence of Xyr1 binding sites in the upstreamregions of Xyr1-upregulated genes under the lignocellulolyticcondition (Table S7) An average of 23 51015840-GGC(AT)4-3

1015840

or 54 51015840-GGC(AT)3-31015840 motifs was detected in the 1-kb

51015840-upstream regions of 177 genes An average of 53 51015840-GGC(AT)4-3

1015840 motifs was detected in the top 50 Xyr1-upregulated genes and the occurrence increased to 77 inthe top 10 Xyr1-upregulated genes (Figure 8) Similarly the


AreAXyr1

Non-enzymatic cellulosedegradation genes

endo T

nmrA-like gene NmrA

Genes involved in non-preferrednitrogen sources degradation

Lignocellulose

Degradation of lignocellulose to the largest extent

Mainly genes involved in primary metabolism

hsp23

Acid peptidase-encoding genes

MFS sugar transporter-encoding genes

(hemi)cellulase genes (including bgl2)

ActivateRepress

Figure 9 The predicted model of Xyr1-mediated gene regulation in T reesei Rut-C30 under lignocellulose-inducing conditions Xyr1 playspleiotropic regulatory roles in the process of lignocellulose degradation and may be involved in primary metabolism It is also implicatedin interaction with the nonpreferred N source degradation repressor NmrArarr positive transcriptional control ⊣ negative transcriptionalcontrol[ indirect regulation

51015840-GGC(AT)3-31015840 motifs displayed an increasing frequency

of appearance from 86 to 109 corresponding to the top 50and top 10 Xyr1-upregulated genes respectively

In a previous study [15] the frequency of appearanceof the 51015840-GGC(AT)3-3

1015840 motifs in the 16 Xyr1-regulatedgenes was compared with those of all other annotated andpredicted ORFs in the T reesei genome database The 51015840-GGC(AT)3-3

1015840 motifs were present at a higher frequencyin the Xyr1-upregulated genes This finding combined withours suggests the involvement of motifs in Xyr1-mediatedgene expression However Xyr1-downregulated genes suchas pepA and hsp23 cultured on lignocellulose showed nosuch frequency ofmotif appearance in their promoter regions(data not shown) In this case the mechanism of Xyr1 in fine-tuning the expression of these genes requires further study

4 Conclusions

In T reesei all ever-identified (hemi)cellulase genes theintracellular bgl2 genes encoding nonenzymatic cellulose-attacking enzymes and some MFS transporter genes (most

belonging to the SP family) responsible for transporting glu-cose fructose mannose galactose xylose maltose lactose120572-glucosides and quinate were activated by Xyr1 underthe lignocellulose condition Transcription levels of most ofthese Xyr1 targets were significantly decreased in the parentstrain Rut-C30 cultured under the inducing (lignocellulose)condition compared with the repressing (glucose) condition

The genes encoding TFs NmrA and Ace3 Endo T(TrireRUTC30 142298) with mannosyl-glycoprotein endo-N-acetyl-120573-D-glucosaminidase activity and the acid pepti-dase pepA as well as a small heat shock protein-encodinggene hsp23 were putative Xyr1 targets All of these tran-scriptional regulations might contribute to the production of(hemi)cellulases and ensure the digestion of lignocelluloseto the largest extent (Figure 9) On the other hand thetranscription levels ofmost genes relevant to basal and energymetabolism were potentially affected by the starvation ofcarbon sources

Furthermore deletion of xyr1 also affected various geneseven when cultured on glucose indicating that Xyr1 haspleiotropic functions in biological processes In this study


lignocellulose-inducing conditions do not induce expressionlevel of xyr1 but activate its function possibly throughposttranslational modifications These findings not onlyimprove our understanding of the regulatory roles of Xyr1including lignocellulose degradation transport and interac-tion with N metabolism but also provide useful informationfor further exploration of genes involved in lignocellulosedegradation in T reesei

Abbreviations

SP Sugar porterMFS Major facilitator superfamilyEMSA Electrophoretic mobility shift assayTF Transcription factorRNA-seq RNA sequencingFPKM The number of fragments per kilobase per

million fragments mappedFPA Filter paper activityORF Open reading frameABC ATP-binding cassetteqRT-PCR Quantitative real-time PCRSDB Sabouraudrsquos dextrose brothPDA Potato-dextrose agarN Nitrogen

Disclosure

The RNA-seq data discussed in this study have beendeposited in the Sequence Read Archive of NCBI (httpwwwncbinlmnihgovTracessra) with the accession num-ber SRP028306 All the datasets supporting the results andconclusions of this article are included within the article andthe additional files

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Liang Ma constructed the Δxyr1 and xyr1-recmutant strainsprepared the RNA samples and drafted the manuscript LingChen performed electrophoretic mobility shift assays andprepared the figures Lei Zhang analyzed the transcriptionprofiling data and performed the annotations and statisticsGen Zou carried out real-time PCR analysis Rui Liu andYanping Jiang performed the FunCat analysis Zhihua Zhoudesigned the study and revised the manuscript All authorsread and approved the final manuscript Liang Ma and LingChen contributed equally to this work

Acknowledgments

This work was financially supported by the National NaturalScience Foundation of China (31300073 and 31470201) andHigh-Tech Research and Development Program of China(8632013AA102806)

References

[1] L R Lynd C E Wyman and T U Gerngross ldquoBiocommodityengineeringrdquo Biotechnology Progress vol 15 no 5 pp 777ndash7931999

[2] M E Himmel S-Y Ding D K Johnson et al ldquoBiomassrecalcitrance engineering plants and enzymes for biofuelsproductionrdquo Science vol 315 no 5813 pp 804ndash807 2007

[3] B S Montenecourt and D E Eveleigh ldquoSelective screeningmethods for the isolation of high yielding cellulase mutants ofTrichoderma reeseirdquo in Hydrolysis of Cellulose Mechanisms ofEnzymatic and Acid Catalysis vol 181 ofAdvances in Chemistrychapter 14 pp 289ndash301 1979

[4] E T Reese ldquoHistory of the cellulase program at the US armyNatick Development Centerrdquo Biotechnology and BioengineeringSymposium vol 6 no 6 pp 9ndash20 1976

[5] C E Wyman ldquoWhat is (and is not) vital to advancing cellulosicethanolrdquo Trends in Biotechnology vol 25 no 4 pp 153ndash1572007

[6] L R Lynd M S Laser D Bransby et al ldquoHow biotech cantransformbiofuelsrdquoNature Biotechnology vol 26 no 2 pp 169ndash172 2008

[7] C P Kubicek M Mikus A Schuster M Schmoll and BSeiboth ldquoMetabolic engineering strategies for the improvementof cellulase production byHypocrea jecorinardquo Biotechnology forBiofuels vol 2 article 19 2009

[8] R Rauscher E Wurleitner C Wacenovsky et al ldquoTranscrip-tional regulation of xyn1 encoding xylanase I in Hypocreajecorinardquo Eukaryotic Cell vol 5 no 3 pp 447ndash456 2006

[9] A R Stricker K Grosstessner-Hain E Wurleitner and R LMach ldquoXyr1 (Xylanase regulator 1) regulates both the hydrolyticenzyme system andD-xylosemetabolism inHypocrea jecorinardquoEukaryotic Cell vol 5 no 12 pp 2128ndash2137 2006

[10] E Akel B Metz B Seiboth and C P Kubicek ldquoMolecular reg-ulation of arabinan and L-Arabinose metabolism in hypocreajecorina (Trichoderma reesei)rdquo Eukaryotic Cell vol 8 no 12pp 1837ndash1844 2009

[11] A R Stricker M G Steiger and R L Mach ldquoXyr1 receivesthe lactose induction signal and regulates lactosemetabolism inHypocrea jecorinardquo FEBS Letters vol 581 no 21 pp 3915ndash39202007

[12] F Calero-Nieto A Di Pietro M I G Roncero and C HeraldquoRole of the transcriptional activator XlnR of Fusarium oxys-porum in regulation of xylanase genes and virulencerdquoMolecularPlant-Microbe Interactions vol 20 no 8 pp 977ndash985 2007

[13] J Sun C Tian S Diamond and N L Glassa ldquoDecipheringtranscriptional regulatory mechanisms associated with hemi-cellulose degradation in Neurospora crassardquo Eukaryotic Cellvol 11 no 4 pp 482ndash493 2012

[14] S Klaubauf H M Narang H Post et al ldquoSimilar is not thesame differences in the function of the (hemi-)cellulolytic reg-ulator XlnR (Xlr1Xyr1) in filamentous fungirdquo Fungal Geneticsand Biology vol 72 pp 73ndash81 2014

[15] T Furukawa Y Shida N Kitagami et al ldquoIdentification ofspecific binding sites for XYR1 a transcriptional activatorof cellulolytic and xylanolytic genes in Trichoderma reeseirdquoFungal Genetics and Biology vol 46 no 8 pp 564ndash574 2009

[16] L dos Santos Castro R G de Paula A C Antonieto G FPersinoti R Silva-Rocha and R N Silva ldquoUnderstanding therole of the master regulator XYR1 in Trichoderma reesei byglobal transcriptional analysisrdquo Frontiers in Microbiology vol7 article 175 2016


[17] M Ilmen C Thrane and M Penttila ldquoThe glucose repressorgene cre1 of Trichoderma isolation and expression of a full-length and a truncated mutant formrdquo Molecular and GeneralGenetics vol 251 no 4 pp 451ndash460 1996

[18] J A Serrato L A Palomares A Meneses-Acosta and O TRamırez ldquoHeterogeneous conditions in dissolved oxygen affectN-glycosylation but not productivity of a monoclonal antibodyin hybridoma culturesrdquo Biotechnology and Bioengineering vol88 no 2 pp 176ndash188 2004

[19] A L Mantyla K H Rossi S A Vanhanen M E Penttila PL Suominen and K M H Nevalainen ldquoElectrophoretic kary-otyping of wild-type andmutant Trichoderma longibrachiatum(reesei) strainsrdquo Current Genetics vol 21 no 6 pp 471ndash4771992

[20] S Le Crom W Schackwitz L Pennacchio et al ldquoTracking theroots of cellulase hyperproduction by the fungus Trichodermareesei using massively parallel DNA sequencingrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 38 pp 16151ndash16156 2009

[21] T Portnoy A Margeot V Seidl-Seiboth et al ldquoDifferentialregulation of the cellulase transcription factors XYR1 ACE2and ACE1 in trichoderma reesei strains producing high and lowlevels of cellulaserdquo Eukaryotic Cell vol 10 no 2 pp 262ndash2712011

[22] A S Sandberg H Andersson B Hallgren K HasselbladB Isaksson and L Hulten ldquoExperimental model for in vivodetermination of dietary fibre and its effect on the absorptionof nutrients in the small intestinerdquo British Journal of Nutritionvol 45 no 2 pp 283ndash294 1981

[23] F Woldesenbet A P Virk N Gupta and P Sharma ldquoEffect ofmicrowave irradiation on xylanase production fromwheat branand biobleaching of eucalyptus kraft pulprdquoApplied Biochemistryand Biotechnology vol 167 no 1 pp 100ndash108 2012

[24] H Nakayashiki S Hanada N B Quoc N Kadotani Y Tosaand S Mayama ldquoRNA silencing as a tool for exploring genefunction in ascomycete fungirdquo Fungal Genetics and Biology vol42 no 4 pp 275ndash283 2005

[25] L Ma J Zhang G Zou C Wang and Z Zhou ldquoImprovementof cellulase activity in Trichoderma reesei by heterologousexpression of a 120573-glucosidase gene from Penicillium decum-bensrdquo Enzyme andMicrobial Technology vol 49 no 4 pp 366ndash371 2011

[26] A Miettinen-Oinonen and P Suominen ldquoEnhanced produc-tion of Trichoderma reesei endoglucanases and use of the newcellulase preparations in producing the stonewashed effect ondenim fabricrdquoApplied and Environmental Microbiology vol 68no 8 pp 3956ndash3964 2002

[27] C Trapnell L Pachter and S L Salzberg ldquoTopHat discoveringsplice junctions with RNA-Seqrdquo Bioinformatics vol 25 no 9pp 1105ndash1111 2009

[28] B Langmead C Trapnell M Pop and S L Salzberg ldquoUltrafastandmemory-efficient alignment of short DNA sequences to thehuman genomerdquo Genome Biology vol 10 no 3 article no R252009

[29] C Trapnell B A Williams G Pertea et al ldquoTranscriptassembly and quantification by RNA-Seq reveals unannotatedtranscripts and isoform switching during cell differentiationrdquoNature Biotechnology vol 28 no 5 pp 511ndash515 2010

[30] S Anders and W Huber ldquoDifferential expression of RNA-Seqdata at the gene levelmdashthe DESeq packagerdquo 2012

[31] L Wang Z Feng X Wang X Wang and X Zhang ldquoDEGseqan R package for identifying differentially expressed genes fromRNA-seq datardquo Bioinformatics vol 26 no 1 pp 136ndash138 2010

[32] Y Benjamini and Y Hochberg ldquoControlling the false discoveryrate a practical and powerful approach to multiple testingrdquoJournal of the Royal Statistical Society Series B Methodologicalvol 57 no 1 pp 289ndash300 1995

[33] R A Ogert M K Lee W Ross A Buckler-White M AMartin and M W Cho ldquoN-linked glycosylation sites adjacentto and within the V1V2 and the V3 loops of dualtropichuman immunodeficiency virus type 1 isolateDH12 gp120 affectcoreceptor usage and cellular tropismrdquo Journal of Virology vol75 no 13 pp 5998ndash6006 2001

[34] D Surmeli O Ratmann H-WMewes and I V Tetko ldquoFunCatfunctional inference with belief propagation and feature inte-grationrdquo Computational Biology and Chemistry vol 32 no 5pp 375ndash377 2008

[35] M C Walter T Rattei R Arnold et al ldquoPEDANT covers allcomplete RefSeq genomesrdquo Nucleic Acids Research vol 37 no1 pp D408ndashD411 2009

[36] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[37] T M Wood and K M Bhat ldquoMethods for measuring cellulaseactivitiesrdquoMethods in Enzymology vol 160 pp 87ndash112 1988

[38] Z Xiao R Storms and A Tsang ldquoMicroplate-based filter paperassay to measure total cellulase activityrdquo Biotechnology andBioengineering vol 88 no 7 pp 832ndash837 2004

[39] O Turunen M Vuorio F Fenel and M Leisola ldquoEngineeringof multiple arginines into the SerThr surface of Trichodermareesei endo-14-120573-xylanase II increases the thermotoleranceand shifts the pH optimum towards alkaline pHrdquo ProteinEngineering vol 15 no 2 pp 141ndash145 2002

[40] Y Zhao S Xiang X Dai and K Yang ldquoA simplified dipheny-lamine colorimetric method for growth quantificationrdquoAppliedMicrobiology and Biotechnology vol 97 no 11 pp 5069ndash50772013

[41] L ChenG Zou L Zhang et al ldquoThedistinctive regulatory rolesof PrtT in the cell metabolism of Penicillium oxalicumrdquo FungalGenetics and Biology vol 63 pp 42ndash54 2014

[42] C Ren Y Gu Y Wu et al ldquoPleiotropic functions of catabolitecontrol protein CcpA in Butanol-producing Clostridium aceto-butylicumrdquo BMC Genomics vol 13 no 1 article no 349 2012

[43] M Saloheimo M Paloheimo S Hakola et al ldquoSwollenin aTrichoderma reesei protein with sequence similarity to the plantexpansins exhibits disruption activity on cellulosic materialsrdquoEuropean Journal of Biochemistry vol 269 no 17 pp 4202ndash42112002

[44] X-L Li S Spanikova R P de Vries and P Biely ldquoIdentifica-tion of genes encoding microbial glucuronoyl esterasesrdquo FEBSLetters vol 581 no 21 pp 4029ndash4035 2007

[45] P K Foreman D Brown L Dankmeyer et al ldquoTranscriptionalregulation of biomass-degrading enzymes in the filamentousfungus Trichoderma reeseirdquo Journal of Biological Chemistry vol278 no 34 pp 31988ndash31997 2003

[46] Y Noguchi M Sano K Kanamaru et al ldquoGenes regulatedby AoXlnR the xylanolytic and cellulolytic transcriptionalregulator in Aspergillus oryzaerdquo Applied Microbiology andBiotechnology vol 85 no 1 pp 141ndash154 2009

[47] D Shallom and Y Shoham ldquoMicrobial hemicellulasesrdquo CurrentOpinion in Microbiology vol 6 no 3 pp 219ndash228 2003


[48] S G Grishutin A V Gusakov A V Markov B B Ustinov MV Semenova and A P Sinitsyn ldquoSpecific xyloglucanases as anew class of polysaccharide-degrading enzymesrdquo Biochimica etBiophysica Acta vol 1674 no 3 pp 268ndash281 2004

[49] A R Mach-Aigner M E Pucher and R L Mach ldquoD-xyloseas a repressor or inducer of xylanase expression in Hypocreajecorina (Trichoderma reesei)rdquo Applied and EnvironmentalMicrobiology vol 76 no 6 pp 1770ndash1776 2010

[50] A A Hasper J Visser and L H de Graaff ldquoThe Aspergillusniger transcriptional activator XlnR which is involved inthe degradation of the polysaccharides xylan and cellulosealso regulates D-xylose reductase gene expressionrdquo MolecularMicrobiology vol 36 no 1 pp 193ndash200 2000

[51] A Levasseur M Saloheimo D Navarro et al ldquoExploringlaccase-like multicopper oxidase genes from the ascomyceteTrichoderma reesei a functional phylogenetic and evolution-ary studyrdquo BMC Biochemistry vol 11 article no 32 2010

[52] T Kirk ldquoDegradation of ligninrdquo in Microbial Degradation ofOrganic Compounds Marcel Dekker New York NY USA 1984

[53] S S Adav L T Chao and S K Sze ldquoQuantitative secretomicanalysis of Trichoderma reesei strains reveals enzymatic compo-sition for lignocellulosic biomass degradationrdquo Molecular andCellular Proteomics vol 11 no 7 2012

[54] S S Pao I T Paulsen and M H Saier Jr ldquoMajor facilitatorsuperfamilyrdquo Microbiology and Molecular Biology Reviews vol62 no 1 pp 1ndash34 1998

[55] V S ReddyM A Shlykov R Castillo E I Sun andM H SaierJr ldquoThe major facilitator superfamily (MFS) revisitedrdquo FEBSJournal vol 279 no 11 pp 2022ndash2035 2012

[56] M H Saier Jr ldquoFamilies of transmembrane sugar transportproteinsrdquo Molecular Microbiology vol 35 no 4 pp 699ndash7102000

[57] C P Kubicek R Messner F Gruber M Mandels and EM Kubicek-Pranz ldquoTriggering of cellulase biosynthesis bycellulose in Trichoderma reesei Involvement of a constitutivesophorose-inducible glucose-inhibited 120573- diglucoside perme-aserdquo Journal of Biological Chemistry vol 268 no 26 pp 19364ndash19368 1993

[58] J M Galazka C Tian W T Beeson B Martinez N L Glassand J H D Cate ldquoCellodextrin transport in yeast for improvedbiofuel productionrdquo Science vol 330 no 6000 pp 84ndash86 2010

[59] C Ivanova J A Baath B Seiboth and C P Kubicek ldquoSystemsanalysis of lactosemetabolism in trichoderma reesei identifies alactose permease that is essential for cellulase inductionrdquo PLOSONE vol 8 no 5 Article ID e62631 2013

[60] W Zhang Y Kou J Xu et al ldquoTwomajor facilitator superfamilysugar transporters from Trichoderma reesei and their roles ininduction of cellulase biosynthesisrdquo The Journal of BiologicalChemistry vol 288 no 46 pp 32861ndash32872 2013

[61] G W Gooday W-Y Zhu and R W OrsquoDonnell ldquoWhat are theroles of chitinases in the growing fungusrdquo FEMS MicrobiologyLetters vol 100 no 1ndash3 pp 387ndash391 1992

[62] N Takaya D Yamazaki H Horiuchi A Ohta and M Tak-agi ldquoCloning and Characterization of a Chitinase-encodingGene (chiA) from Aspergillus nidulans Disruption of WhichDecreases Germination Frequency and Hyphal Growthrdquo Bio-science Biotechnology and Biochemistry vol 62 no 1 pp 60ndash651998

[63] H Yamazaki D Yamazaki N Takaya M Takagi A Ohta andH Horiuchi ldquoA chitinase gene chiB involved in the autolyticprocess of Aspergillus nidulansrdquo Current Genetics vol 51 no 2pp 89ndash98 2007

[64] V Seidl B Huemer B Seiboth and C P Kubicek ldquoA com-plete survey of Trichoderma chitinases reveals three distinctsubgroups of family 18 chitinasesrdquo FEBS Journal vol 272 no22 pp 5923ndash5939 2005

[65] R Bischof L Fourtis A Limbeck C Gamauf B Seibothand C P Kubicek ldquoComparative analysis of the Trichodermareesei transcriptome during growth on the cellulase inducingsubstrates wheat straw and lactoserdquo Biotechnology for Biofuelsvol 6 no 1 article 127 2013

[66] V Seidl ldquoChitinases of filamentous fungi a large group ofdiverse proteins with multiple physiological functionsrdquo FungalBiology Reviews vol 22 no 1 pp 36ndash42 2008

[67] I Stals B Samyn K Sergeant et al ldquoIdentification of a genecoding for a deglycosylating enzyme in Hypocrea jecorinardquoFEMS Microbiology Letters vol 303 no 1 pp 9ndash17 2010

[68] I Stals S Karkehabadi S Kim et al ldquoHigh resolution crystalstructure of the Endo-N-Acetyl-120573-D-glucosaminidase respon-sible for the Deglycosylation of Hypocrea jecorina cellulasesrdquoPLoS ONE vol 7 no 7 Article ID e40854 2012

[69] W Wei L Chen G Zou et al ldquoN-glycosylation affects theproper folding enzymatic characteristics and production of afungal 120573-glucosidaserdquo Biotechnology and Bioengineering vol110 no 12 pp 3075ndash3084 2013

[70] M E Katz S M Bernardo and B F Cheetham ldquoThe interac-tion of induction repression and starvation in the regulationof extracellular proteases in Aspergillus nidulans Evidence fora role for CreA in the response to carbon starvationrdquo CurrentGenetics vol 54 no 1 pp 47ndash55 2008

[71] M Maras N Callewaert K Piens et al ldquoMolecular cloningand enzymatic characterization of a Trichoderma reesei 12-120572-D-mannosidaserdquo Journal of Biotechnology vol 77 no 2-3 pp255ndash263 2000

[72] F Van Petegem H Contreras R Contreras and J Van Beeu-men ldquoTrichoderma reesei 120572-12-mannosidase structural basisfor the cleavage of four consecutive mannose residuesrdquo Journalof Molecular Biology vol 312 no 1 pp 157ndash165 2001

[73] I G Jones V Fairhurst and H M Sealy-Lewis ldquoADHII inAspergillus nidulans is induced by carbon starvation stressrdquoFungal Genetics and Biology vol 32 no 1 pp 33ndash43 2001

[74] B Pinan-Lucarre A Balguerie and C Clave ldquoAccelerated celldeath in Podospora autophagy mutantsrdquo Eukaryotic Cell vol 4no 11 pp 1765ndash1774 2005

[75] M G Santoro ldquoHeat shock factors and the control of the stressresponserdquo Biochemical Pharmacology vol 59 no 1 pp 55ndash632000

[76] J J Cotto and R I Morimoto ldquoStress-induced activation of theheat-shock response cell and molecular biology of heat-shockfactorsrdquo Biochemical Society Symposium vol 64 pp 105ndash1181999

[77] M Montero-Barrientos R E Cardoza S Gutierrez E Monteand R Hermosa ldquoThe heterologous overexpression of hsp23 asmall heat-shock protein gene fromTrichoderma virens confersthermotolerance to T harzianumrdquo Current Genetics vol 52 no1 pp 45ndash53 2007

[78] M Hakkinen M J Valkonen A Westerholm-Parvinen et alldquoScreening of candidate regulators for cellulase and hemicel-lulase production in Trichoderma reesei and identification ofa factor essential for cellulase productionrdquo Biotechnology forBiofuels vol 7 no 1 article 14 2014

[79] S Ghassemi A Lichius F Bidard et al ldquoThe 120573-importin KAP8(Pse1Kap121) is required for nuclear import of the cellulase


transcriptional regulator XYR1 asexual sporulation and stressresistance in Trichoderma reeseirdquo Molecular Microbiology vol96 no 2 pp 405ndash418 2015

[80] A Andrianopoulos S Kourambas J A Sharp M A Davisand M J Hynes ldquoCharacterization of the Aspergillus nidulansnmrA gene involved in nitrogenmetabolite repressionrdquo Journalof Bacteriology vol 180 no 7 pp 1973ndash1977 1998

[81] N S Dunn-Coleman A B Tomsett and R H GarrettldquoThe regulation of nitrate assimilation in Neurospora crassabiochemical analysis of the nmr-1 mutantsrdquoMGGMolecular ampGeneral Genetics vol 182 no 2 pp 234ndash239 1981

[82] M Macios M X Caddick P Weglenski C Scazzocchio andA Dzikowska ldquoThe GATA factors AREA and AREB togetherwith the co-repressor NMRA negatively regulate argininecatabolism in Aspergillus nidulans in response to nitrogen andcarbon sourcerdquo Fungal Genetics and Biology vol 49 no 3 pp189ndash198 2012

[83] A Cziferszky R L Mach and C P Kubicek ldquoPhosphorylationpositively regulates DNA binding of the carbon cataboliterepressor Cre1 of Hypocrea jecorina (Trichoderma reesei)rdquoJournal of Biological Chemistry vol 277 no 17 pp 14688ndash146942002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the xlnRxlr1xyr1 deletion mutants of five fungi showed thatT reeseiXyr1 has a different regulatory pattern compared to itsorthologs in other fungi [14] The above findings combinedwith the demonstration that Xyr1 in T reesei could bindnot only to the 51015840-GGCTAA-31015840 motif but also to the 51015840-GGC(AT)3-3

1015840 motif [15] suggest that Xyr1 behaves as apleiotropic regulator in T reesei

Recently transcription profiling of the T reesei Qm 9414and its Δxyr1 mutant grown on cellulose sophorose andglucose were performed and defined the role of the transcrip-tional factor Xyr1 during cellulose degradation [16] T reeseimutant Rut-C30 is a hyperproducer of cellulolytic enzymeswith its genome has been released [3 17 18] Rut-C30 wasobtained through several rounds of random mutagenesisfromwtQm6aThe rearrangement of chromosomes carryinggenes encoding cellulolytic enzymes [19] and the missinggt100 kb of genomic DNA [20] including the truncation ofcarbon catabolite repressor cre1 [17]may contribute to its highprotein secretory ability and cellulase production Portnoyet al reported challenging results indicating that the fulltranscription of xyr1 required Cre1 in T reesei Qm9414under induction conditions [21] Due to the special geneticbackground of Rut-C30 we assumed that its Xyr1 harboredrather special regulatory mechanisms compared to T reeseiQm9414

Wheat (Triticum aestivum L) bran which contains lig-nocelluloses as a major component is rich in hemicellulosescellulose and lignin [22 23] Therefore wheat bran behavesas an inducer for lignocellulolytic enzymes In this studyRNA sequencing (RNA-seq) was performed to investigate thefunctions of Xyr1 through comparison between a wild-typestrain Rut-C30 and an xyr1 disruptant under lignocelluloseand glucose conditions Our results shed new light on themechanism by which Xyr1 controls cellulose and hemicel-lulose utilization and determines the pleiotropic functionsof Xyr1 These new findings could offer strategies for strainimprovement of T reesei Rut-C30

2 Materials and Methods

21 Fungal Strains and Cultivation Conditions T reesei Rut-C30 (ATCC 56765) was purchased from ATCC and its xyr1deletion mutant strain Δxyr1 was constructed as followingPlasmids were propagated in Escherichia coli DH5120572 Thevector backbone used in constructing the plasmids wasbinary vector pCambia1300 (CAMBIA Canberra Australia)The E coli cultivations were performed overnight at 37∘C inLuria-Bertani (LB) medium plus kanamycin (100 120583gmlminus1) asselective agent

The xyr1 deletion vector was constructed using the binaryvector pCambia1300 as a recipient Primers used are given inTable S1 in Supplementary Material available online at httpdxdoiorg10115520164841756 First the plasmid pSilent-1[24] was used as template to amplify the ORF and terminatorof hygromycin B phosphotransferase gene hph (conferringhygromycin B resistance) with primers HygxhoI and OrfhygSecond the pyruvate kinase (pki GenBank accession num-ber L07060) promoter was amplified by primers Ppki andBampki with T reesei Rut-C30 genome as template Overlap

PCR was conducted to fuse the pki promoter to hphORFand the PCR product was digested with BamHI and XhoIand ligated into the BamHI and XhoI digested pCambia1300resulting in pPH Then the 13 kb fragment for 51015840 region ofxyr1 was amplified with primers xyr1xhf and xyr1xhr Afterdigestion by XhoI it was inserted to the XhoI digested pPHto generate pPH5X At last the 31015840 region of xyr1was amplifiedwith primers xyr1bam and xyr1sal and digested by BamHIand SalI before inserting into the corresponding restric-tion enzymes digested product of pPH5X to yield the xyr1knockout vector pPHXThe resultant pPHXwas transformedinto T reesei by Agrobacterium-mediated transformation asdescribed previously [25] Transformantswere then subjectedto verification of homologous recombination event PrimersX5 and TtrpC were used to amplify a fragment of 22 kb inthe xyr1 knockout strain and similarly primers X3 and Ppkiwere used to amplify a fragment of 23 kb in the xyr1 knockoutstrain and the results were further verified by sequencing thePCR products while random insertion could not yield anyspecific PCR products

The xyr1 recomplementation strain was constructed asthe phleomycin resistance gene (ble) [26] was used as aselection marker The binary vector pPB was constructed ina similar way except replacing the hph ORF with ble Thexyr1 genewas amplifiedwith primers X3BamHandX5BamHand the terminator of xyr1 was amplified with primersTxyr5Xho and Txyr3Xho (Table S1) The PCR products weredigested by BamHI and XhoI respectively and then insertedinto corresponding site of the pPB to yield pPBReX Thetransformation into Δxyr1 strain was performed as describedabove and transformants were selected on PDA containingphleomycin (4 120583gmlminus1) as selection agent The homologousintegration of pPBReX at the xyr1 locus of the Δxyr1 strainwas verified by PCR Primers B3homo and pki-phe wereused to amplify a fragment of 26 kb in the xyr1 homologousintegration retransformant and similarly primers X5 andPpki (Table S1) were used to amplify a fragment of 57 kband the results were further verified by sequencing the PCRproducts while random insertion could not yield any specificPCR products

All of the fungal strains were maintained on potato-dextrose agar (PDA) Then 107 conidia of both strainswere inoculated in 50-ml shake flasks containing 10ml ofSabouraudrsquos dextrose broth (SDB) at 28∘C for two daysand then the pregrown mycelia were collected by filtrationthrough miracloth and washed with 09 NaCl thoroughlyThen the mycelia of both strains were transferred intothe cellulase-inducing medium described by Ma et al [25]which contains 2 wheat bran 3microcrystalline cellulose(Avicel) and the cellulase-repressing medium containing2 glucose in place of wheat bran and Avicel respectivelyThe flasks were incubated on an orbital shaker at 200 rpm28∘C For RNA isolation the samples of mycelium werecollected after 15 hours of cultivation and then subjectedto RNA isolation For fungal growth and protein secretionanalysis 500 120583l samples were collected after being inducedfor 1 day 2 days 3 days and 7 days After centrifugationthe culture supernatant was subjected to electrophoresis











1015840 motifs and 51015840-GGC(AT)4-3















M Rut-C30 Rut-C30MW


CBH1


143

201

29

443

664972

(a)

2 3 71Time (day)

000510152025303540455055

Prot

ein

conc

entr

atio

n (m

gmlminus

1)

lowast

lowast

lowast



(b)



000204060810101214161820

lowast

Fpas

e (U

mlminus

1)

(c)



300

400

500

600

0005011520

Xyla

nase

(Umlminus

1)

lowast

(d)

2 3 71Time (day)

0102030405060708090

100

Biom

ass (

120583gm

lminus1)

lowastlowastlowast

lowast



(e)

lowast

lowast

lowast

2 3 71Time (day)

0

10

20

30

40

50

60

Prot

ein

biom

ass (

mgm

gminus1)



(f)



2112

7 105 6 10

4 5 4 2 3 1 0 1

27

15

4129

26 2120 17 11

16 11 11 11 86 6 10

10

20

30

40

50

60

Num

ber o

f gen

es

Poly

sacc

harid

e met

abol

ism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Ener

gy

Seco

ndar

y m

etab

olism

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Sign

al tr

ansd

uctio

n m

echa

nism

Cel

l fat

e

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism


































120573-Mannosidase 67432 582358 0

Exo-14-120573-xylosidase 140746 11148 0
















Table 1 Continued











28 25 2317

2115

11 83 6

2 0 2 2

9 129

13 813

7

38 1

4 6 2 1Po

lysa

ccha

ride m

etab

olism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism

0

5

10

15

20

25

30

35

40

Num

ber o

f gen

es

Seco

ndar

y m

etab

olism

Ener

gy






02 04 08 08

Competitor S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(a)

Competitor

- 02 04 08 08

S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(b)

complex

Free DNA

Competitor

02 04 08 08

S NS

08

10

Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---

---

--- --- --- --- --- ---


(c)

complex

Free DNA

05 10 10 10

Competitor S NS

10


GST (120583M)

Xyr1-DNA

GST-Xyr1 (120583M)

--- --- --- ---

------

--- --- --- --- ---

(d)



















111

111

2 1

18

11 1

(a)



9

7

5

12

2 1

4

3

1

1

(b)











0

10

20

30

40

50

60

Num

ber o

f gen

es








05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---


(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------


(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---


(c)






















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











1015840 motifs and 51015840-GGC(AT)4-3















M Rut-C30 Rut-C30MW


CBH1


143

201

29

443

664972

(a)

2 3 71Time (day)

000510152025303540455055

Prot

ein

conc

entr

atio

n (m

gmlminus

1)

lowast

lowast

lowast



(b)



000204060810101214161820

lowast

Fpas

e (U

mlminus

1)

(c)



300

400

500

600

0005011520

Xyla

nase

(Umlminus

1)

lowast

(d)

2 3 71Time (day)

0102030405060708090

100

Biom

ass (

120583gm

lminus1)

lowastlowastlowast

lowast



(e)

lowast

lowast

lowast

2 3 71Time (day)

0

10

20

30

40

50

60

Prot

ein

biom

ass (

mgm

gminus1)



(f)



2112

7 105 6 10

4 5 4 2 3 1 0 1

27

15

4129

26 2120 17 11

16 11 11 11 86 6 10

10

20

30

40

50

60

Num

ber o

f gen

es

Poly

sacc

harid

e met

abol

ism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Ener

gy

Seco

ndar

y m

etab

olism

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Sign

al tr

ansd

uctio

n m

echa

nism

Cel

l fat

e

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism


































120573-Mannosidase 67432 582358 0

Exo-14-120573-xylosidase 140746 11148 0
















Table 1 Continued











28 25 2317

2115

11 83 6

2 0 2 2

9 129

13 813

7

38 1

4 6 2 1Po

lysa

ccha

ride m

etab

olism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism

0

5

10

15

20

25

30

35

40

Num

ber o

f gen

es

Seco

ndar

y m

etab

olism

Ener

gy






02 04 08 08

Competitor S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(a)

Competitor

- 02 04 08 08

S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(b)

complex

Free DNA

Competitor

02 04 08 08

S NS

08

10

Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---

---

--- --- --- --- --- ---


(c)

complex

Free DNA

05 10 10 10

Competitor S NS

10


GST (120583M)

Xyr1-DNA

GST-Xyr1 (120583M)

--- --- --- ---

------

--- --- --- --- ---

(d)



















111

111

2 1

18

11 1

(a)



9

7

5

12

2 1

4

3

1

1

(b)











0

10

20

30

40

50

60

Num

ber o

f gen

es








05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---


(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------


(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---


(c)






















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














M Rut-C30 Rut-C30MW


CBH1


143

201

29

443

664972

(a)

2 3 71Time (day)

000510152025303540455055

Prot

ein

conc

entr

atio

n (m

gmlminus

1)

lowast

lowast

lowast



(b)



000204060810101214161820

lowast

Fpas

e (U

mlminus

1)

(c)



300

400

500

600

0005011520

Xyla

nase

(Umlminus

1)

lowast

(d)

2 3 71Time (day)

0102030405060708090

100

Biom

ass (

120583gm

lminus1)

lowastlowastlowast

lowast



(e)

lowast

lowast

lowast

2 3 71Time (day)

0

10

20

30

40

50

60

Prot

ein

biom

ass (

mgm

gminus1)



(f)



2112

7 105 6 10

4 5 4 2 3 1 0 1

27

15

4129

26 2120 17 11

16 11 11 11 86 6 10

10

20

30

40

50

60

Num

ber o

f gen

es

Poly

sacc

harid

e met

abol

ism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Ener

gy

Seco

ndar

y m

etab

olism

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Sign

al tr

ansd

uctio

n m

echa

nism

Cel

l fat

e

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism


































120573-Mannosidase 67432 582358 0

Exo-14-120573-xylosidase 140746 11148 0
















Table 1 Continued











28 25 2317

2115

11 83 6

2 0 2 2

9 129

13 813

7

38 1

4 6 2 1Po

lysa

ccha

ride m

etab

olism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism

0

5

10

15

20

25

30

35

40

Num

ber o

f gen

es

Seco

ndar

y m

etab

olism

Ener

gy






02 04 08 08

Competitor S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(a)

Competitor

- 02 04 08 08

S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(b)

complex

Free DNA

Competitor

02 04 08 08

S NS

08

10

Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---

---

--- --- --- --- --- ---


(c)

complex

Free DNA

05 10 10 10

Competitor S NS

10


GST (120583M)

Xyr1-DNA

GST-Xyr1 (120583M)

--- --- --- ---

------

--- --- --- --- ---

(d)



















111

111

2 1

18

11 1

(a)



9

7

5

12

2 1

4

3

1

1

(b)











0

10

20

30

40

50

60

Num

ber o

f gen

es








05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---


(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------


(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---


(c)






















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


2112

7 105 6 10

4 5 4 2 3 1 0 1

27

15

4129

26 2120 17 11

16 11 11 11 86 6 10

10

20

30

40

50

60

Num

ber o

f gen

es

Poly

sacc

harid

e met

abol

ism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Ener

gy

Seco

ndar

y m

etab

olism

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Sign

al tr

ansd

uctio

n m

echa

nism

Cel

l fat

e

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism


































120573-Mannosidase 67432 582358 0

Exo-14-120573-xylosidase 140746 11148 0
















Table 1 Continued











28 25 2317

2115

11 83 6

2 0 2 2

9 129

13 813

7

38 1

4 6 2 1Po

lysa

ccha

ride m

etab

olism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism

0

5

10

15

20

25

30

35

40

Num

ber o

f gen

es

Seco

ndar

y m

etab

olism

Ener

gy






02 04 08 08

Competitor S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(a)

Competitor

- 02 04 08 08

S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(b)

complex

Free DNA

Competitor

02 04 08 08

S NS

08

10

Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---

---

--- --- --- --- --- ---


(c)

complex

Free DNA

05 10 10 10

Competitor S NS

10


GST (120583M)

Xyr1-DNA

GST-Xyr1 (120583M)

--- --- --- ---

------

--- --- --- --- ---

(d)



















111

111

2 1

18

11 1

(a)



9

7

5

12

2 1

4

3

1

1

(b)











0

10

20

30

40

50

60

Num

ber o

f gen

es








05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---


(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------


(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---


(c)






















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























120573-Mannosidase 67432 582358 0

Exo-14-120573-xylosidase 140746 11148 0
















Table 1 Continued











28 25 2317

2115

11 83 6

2 0 2 2

9 129

13 813

7

38 1

4 6 2 1Po

lysa

ccha

ride m

etab

olism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism

0

5

10

15

20

25

30

35

40

Num

ber o

f gen

es

Seco

ndar

y m

etab

olism

Ener

gy






02 04 08 08

Competitor S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(a)

Competitor

- 02 04 08 08

S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(b)

complex

Free DNA

Competitor

02 04 08 08

S NS

08

10

Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---

---

--- --- --- --- --- ---


(c)

complex

Free DNA

05 10 10 10

Competitor S NS

10


GST (120583M)

Xyr1-DNA

GST-Xyr1 (120583M)

--- --- --- ---

------

--- --- --- --- ---

(d)



















111

111

2 1

18

11 1

(a)



9

7

5

12

2 1

4

3

1

1

(b)











0

10

20

30

40

50

60

Num

ber o

f gen

es








05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---


(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------


(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---


(c)






















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued











28 25 2317

2115

11 83 6

2 0 2 2

9 129

13 813

7

38 1

4 6 2 1Po

lysa

ccha

ride m

etab

olism

Tran

spor

ter

Cell

resc

ue d

efen

se an

d vi

rule

nce

Lipi

d m

etab

olism

Inte

ract

ion

with

the e

nviro

nmen

t

Prot

ein

fate

Biog

enes

is of

cellu

lar c

ompo

nent

s

Tran

scrip

tion

Am

ino

acid

met

abol

ism

Prot

ein

synt

hesis

Nuc

leot

ide m

etab

olism

Aro

mat

e met

abol

ism

0

5

10

15

20

25

30

35

40

Num

ber o

f gen

es

Seco

ndar

y m

etab

olism

Ener

gy






02 04 08 08

Competitor S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(a)

Competitor

- 02 04 08 08

S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(b)

complex

Free DNA

Competitor

02 04 08 08

S NS

08

10

Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---

---

--- --- --- --- --- ---


(c)

complex

Free DNA

05 10 10 10

Competitor S NS

10


GST (120583M)

Xyr1-DNA

GST-Xyr1 (120583M)

--- --- --- ---

------

--- --- --- --- ---

(d)



















111

111

2 1

18

11 1

(a)



9

7

5

12

2 1

4

3

1

1

(b)











0

10

20

30

40

50

60

Num

ber o

f gen

es








05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---


(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------


(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---


(c)






















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


02 04 08 08

Competitor S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(a)

Competitor

- 02 04 08 08

S NS

08

complex

Free DNA

10


Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- --- ---

---

------------------

---

(b)

complex

Free DNA

Competitor

02 04 08 08

S NS

08

10

Xyr1-DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---

---

--- --- --- --- --- ---


(c)

complex

Free DNA

05 10 10 10

Competitor S NS

10


GST (120583M)

Xyr1-DNA

GST-Xyr1 (120583M)

--- --- --- ---

------

--- --- --- --- ---

(d)



















111

111

2 1

18

11 1

(a)



9

7

5

12

2 1

4

3

1

1

(b)











0

10

20

30

40

50

60

Num

ber o

f gen

es








05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---


(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------


(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---


(c)






















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













111

111

2 1

18

11 1

(a)



9

7

5

12

2 1

4

3

1

1

(b)











0

10

20

30

40

50

60

Num

ber o

f gen

es








05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---


(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------


(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---


(c)






















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





0

10

20

30

40

50

60

Num

ber o

f gen

es








05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---


(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------


(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---


(c)






















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


05 10 10 10

Competitor S NS

Xyr1-DNAcomplex

Free DNA

10

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

---

---------------

---


(a)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

---

---

---

--- --- ---

---

------------


(b)

05 10 10 10

S NS

10

Competitor

Xyr1-DNAcomplex

Free DNA

Xyr1 (120583M)

GST (120583M)

--- --- --- ---

------

--- --- --- --- ---


(c)






















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















GGC(AT)4 GGC(AT)3

Top 50Top 40Top 30

Top 20Top 10

00

20

40

60

80

100

120

Freq

uenc

ies o

f app

eara

nce o

f mot

if



genes





1015840





AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


AreAXyr1


endo T

nmrA-like gene NmrA


Lignocellulose



hsp23




ActivateRepress







4 Conclusions







Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Abbreviations



Disclosure


Competing Interests




Acknowledgments


References































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article rna sequencing reveals xyr1 as a...

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