basidiomycete 
 fungi are attracting increased interest because of 
 their plant pathogenic properties , along with an ability to produce 
 enzymes , in particular glycoside hydrolases , which can degrade recalcitrant 
 carbon sources , thereby increasing the range of substrates available 
 for bioethanol production .
although the 
 genomes of a number of basidiomycete species have been sequenced and 
 a number of proteomic studies have been carried out , one genera in 
 particular , armillaria , has received little attention 
 to date . yet , armillaria spp .
are among the 
 most serious plant root pathogens and cause major economic losses 
 following infection of tree species of both agronomic importance and 
 those grown for timber production purposes .
the genera consists of at least 40 species , including a. mellea , a. gallica , a. tabescens , and a. borealis , with the former species attracting the 
 most attention .
in particular , a. mellea has 
 been studied because 
 of its virulence , bioluminescent properties , ability to produce unusual 
 natural products and from a strain typing perspective , for definitive 
 identification of the armillaria spp .
recently , a 
 novel infection model system for a. mellea has been 
 established which deployed confocal microscopy and quantitative pcr 
 for the estimation of fungal biomass produced during grape rootstock 
 infection .
this approach has facilitated 
 a more quantitative assessment of fungal infection of plant roots , 
 in addition to distinguishing between susceptible and resistant plant 
 varieties .
armillaria spp . also produce uncommon 
 secondary metabolites , such as sesquiterpene aryl esters , diketopiperzines 
 and sphingolipids .
next generation sequencing has dramatically accelerated 
 research 
 into basidiomycete genomics , for example the genomes of over 50 basidiomycetes 
 are currently available , including the maize and teosinte pathogen ustilago maydis , the 
 ectomycorrhizal fungus , laccaria bicolor , white rots ( phanerochaete 
 chrysosporium and schizophyllum commune(11 ) ) and the multicellular species coprinopsis cinerea . in initial basidiomycete proteomics 
 studies ,
2-d page coupled with maldi - tof 
 or lc  ms / ms led 
 to identification of 25 extracellular proteins from p. chrysosporium , mainly those involved in carbohydrate polymer degradation , and 
 noted difficulties with peptide identification caused by protein glycosylation . 

soon after , these techniques were used to assess the effects of vanillin , 
 an intermediate in lignin degradation on the same fungus and revealed 
 up- and down - regulation of the citric acid and glyoxylate cycles , 
 respectively .
a proteomic approach was also 
 used to investigate the differential secretome composition of p. chrysosporium grown under ligninolytic conditions , where 
 peroxidases were mainly detected
. a greater diversity of fungal glycoside 
 hydrolases was identified during culture in biopulped material including 
 cellulases , hemicellulases , putative glucan 1,3--glucosidases 
 and -glycosidases .
this work , which was the first differential 
 secretome characterization of p. chrysosporium , highlighted 
 the power of high - throughput proteomic analysis to reveal culture - dependent 
 proteome remodelling due to substrates used for fungal culture .
although 
 no genome sequences are available , proteomic 
 analyses were also carried out on two economically important edible 
 basidiomycetes , sparassis crispa and hericium 
 erinaceum , yielding identification of 77 and 121 proteins 
 from both species , respectively , 21 of which were found to be common 
 in both species .
l. bicolor is an ectomycorrhizal 
 basidiomycete , which exists in either a saprotrophic phase in soil 
 or an extended mutualistic interaction with tree roots .
a recent proteomic study of the l. bicolor secretome in liquid culture identified 224 proteins by a combined 
 electrophoretic fractionation and mass spectrometry - based shotgun 
 approach .
this work significantly extended 
 knowledge of the secretome of this saprophytic organism and identified 
 multiple glycoside hydrolases and proteases , hypothesized to be involved 
 in either plant colonisation or basidiomycete defense . of interest , 
 among proteins of unknown function ,
an induced small secreted protein , 
 probably a putative effector , has been detected .
functional assignment of unknown proteins produced by basidomycetes 
 is set to become one of the major challenges in fungal biology in 
 the coming years . however , despite such advances in other basidiomycete 
 species , 
 to date , only 16 distinct a. mellea proteins are 
 curated in genbank including an atp synthase subunit 6 , gapdh isoform , 
 putative zinc finger / binuclear cluster transcriptional regulator , 
 rna polymerase ( two subunits ) , a laccase - like multicopper oxidase 
 lcc2 and a metalloendopeptidase .
this is especially problematic since 
 so little is known at the molecular level about this plant pathogen . here , we reveal the first extensive genome sequence 
 analysis of a. mellea , combined with a detailed proteomic 
 characterization .
in addition , we propose a novel infection model 
 system that utilizes candida albicans as a target 
 to investigate a. mellea virulence .
a. mellea ( vahl : fr ) 
 kummer dsm 3731 was purchased from dsmz ( braunschweig , germany ) and 
 maintained on malt extract agar ( 50
liquid cultures 
 ( 200 ml ) were undertaken in 500 ml erlenmeyer flasks using potato 
 dextrose broth ( pdb ) ( 2.4% ( w / v ) ) or minimal media ( 5 mm ammonium 
 tartarate as nitrogen - source , trace elements according to schrettl 
 and colleagues and containing 1% ( w / v ) 
 of one of the following carbon - sources : lignin , xylan , cellulose , 
 rutin , yeast extract ) .
ethanol ( 0.15% ( v / v ) ) was added to liquid cultures 
 before inoculation with mycelia and incubation at 25 c in darkness 
 for 26 weeks .
mycelia from liquid cultures were harvested 
 by filtration through miracloth , washed with distilled water , dried 
 thoroughly with absorbent paper , and put immediately into liquid nitrogen 
 and stored at 70 c .
malt extract agar ( mea ) plates were inoculated 
 with a. mellea mycelia and incubated at 25 c 
 in darkness for 26 weeks .
coculture experiments involving a. 
 mellea and candida albicans were carried 
 out on pdb agar . c. albicans ( 5 l ; 10 cells / ml ) was applied in quadruplicate around central mycelial 
 cultures of a. mellea .
after 52 days coculture , c. albicans plugs were collected for subsequent growth ( via 
 reculture on pdb agar ) and viability assessment ( via fluorescein diacetate 
 ( fda ) ) , in the absence of a. 
 mellea . briefly
, fda ( 2 g/100 l ) and propidium 
 iodide ( pi : 0.6 g/30 l ) were added to 2  10c .
after sequential washing with phosphate buffered saline ( pbs ) and 
 20% ( w / v ) bovine serum albumin ( bsa ) in pbs , fluorescence was determined 
 using an olympus fluoview 1000 confocal microscope .
viability was 
 also assessed by replating serial dilutions of monoculture and coculture - derived c. albicans on mea . proteins of a. mellea produced during coculture were extracted by gently grinding mycelia 
 and agar ( 25 ml ) to avoid mycelial lysis with 50 mm kh2po4 ph 7.0 .
after overnight agitation and filtration , 
 proteins were tca precipitated , resolubilized in 50 l of 10 
 mm ammonium hydrogen carbonate containing 10% ( v / v ) acetonitrile and 
 trypsin ( 65 ng / ml ) and digested ( 37 c ) overnight .
resultant 
 peptides were diluted 1/4 in 0.1% ( v / v ) formic acid ( 20 l ) 
 and analyzed directly by lc 
a. mellea dna was extracted 
 using a zr fungal / bacterial dna kit ( zymo research group ) using a 
 modified protocol .
mycelia were ground into a fine powder in liquid 
 n2 , and 300 mg were added to tubes containing glass beads 
 ( 200 mg , 0.4 mm ) . lysis solution ( 750 l ) was added , specimens 
 were bead - beaten for 2 min at 20 hz followed by centrifugation at 
 10000 g for 1 min .
supernatants ( 400 l ) 
 were transferred to zymo - spin iv spin filters and the manufacturer s 
 instructions were then followed .
dna was precipitated by adding 3 
 m sodium acetate ( 1/10 vol . ) and 3 vol .
ice - cold ethanol ( 100% ( v / v ) ) . 
 following incubation at 20 c for 3 h
, specimens were 
 centrifuged at 13000 g for 10 min and supernatants 
 removed .
pellets were washed again with ice - cold ethanol ( 75% ( v / v ) ; 
 180 l ) followed by centrifugation at 13000 g for 10 min .
after supernatant removal , pellets were air - dried to 
 remove traces of ethanol before resuspending in milli - q h2o ( 20 l ) .
pcr to confirm a. mellea dna presence 
 was carried out using two different primer pairs ; regf1 and regr1 
 primers and amel3 and its4 primers .
a single 
 no - pcr paired - end library was generated from a. mellea genomic dna according to the method of kozareva and turner with an estimated fragment size of 168  
 92 bp ( inferred by remapping the reads onto the assembly with maq 
 software ) .
the library was sequenced 
 on three lanes of an illumina gaii at 76 bp read length producing a total of 6.72 gb q20 bases .
the raw sequence data have been submitted 
 to the european nucleotide archive ( ena : http://www.ebi.ac.uk/ena/ ) under accession : erp000894 .
gene models were predicted using the ab initio gene finder augustus . based on a close phylogenetic relationship
blast2go 
 was also used to assign gene ontology ( go ) terms to all gene models .
proteins that may 
 be secreted via the nonclassical secretion pathway were located using 
 the secretomep 2.0 server .
a. mellea multigene families 
 were located by performing an  all against all  blastp 
 database search and the resulting hits 
 were clustered into families using the mcl algorithm with an inflation value of 2 .
numbers along branches represent 
 bootstrap support values . two ascomycete species ( candida 
 albicans and saccharomyces cerevisiae ) were chosen as outgroups .
the initial genome assembly and predicted protein coding 
 genes 
 are available at http://www.armillariamellea.com .
our representative 
 genome species tree consisted of 23 basidiomycete species and 2 ascomycete 
 outgroups ( supplementary table 1 , supporting information ) .
genome data was obtained from the relevant sequencing centers 
 ( supplementary table 1 , supporting information ) . in total , 8860 homologous gene families ( containing 4 or more 
 genes ) were identified using a previously described randomized blastp 
 approach .
gene families were aligned using the multiple 
 sequence alignment software muscle v3.7 with the default settings . misaligned or fast evolving regions of 
 alignments were removed with gblocks .
permutation tail probability ( ptp ) tests were performed on each alignment to ensure that the presence of 
 evolutionary signal was better than random ( p < 
 0.05 ) .
we found that 2924 gene families failed the ptp test , these 
 were removed , leaving a data set of 5936 gene families .
optimum models 
 of protein evolution were selected using modelgenerator and these were used to reconstruct maximum likelihood 
 phylogenies in phyml v3.0 .
bootstrap 
 resampling was performed 100 times on each alignment and majority 
 rule consensus ( threshold of 50% ) trees were reconstructed .
gene families 
 were used to reconstruct supertrees using gene tree parsimony implemented in the software duptree version 1.48 .
mycelial protein 
 extraction was 
 carried out at 4 c using two different methods . for 2-d page , 
 mycelia were ground in liquid n2 and 1 g resuspended in 
 6 ml 10% ( w / v ) trichloroacetic acid
( tca ) ( 4 c ) and sonicated 
 six times ( bandelin senoplus hd2200 sonicator , cycle 6 , 10 s , power 
 10% ) .
samples were then centrifuged at 20200 g for 10 min ( 4 c ) , followed by washing in ice cold acetone , 
 twice .
pellets were finally resuspended in ief buffer ( 10 mm tris , 
 8 m urea , 2 m thiourea , 4% ( w / v ) chaps , 1% ( v / v )
triton x-100 , 65 
 mm dithiothreitol ( dtt ) and 0.8% ( w / v ) ipg buffer ) , centrifuged at 
 14000 g for 5 min ( 4 c ) and supernatants 
 used for further analysis .
proteins were quantified using the bradford 
 assay and 300 g were used for ief using 13 cm strips ( ph range 
 310 ; ipgphorii ; ge healthcare ) ( step : 50 v  12 h ; step : 
 250 v  0.25 h ; gradient to 5000 v  2 h ; step : 5000 v  
 5 h ; gradient to 8000 v  2 h ; step : 8000 v  1 h ) followed 
 by sds - page separation ( 60 v , 30 min ; 120 v , 1.5 h ) on protean plus 
 dodeca cell ( bio - rad ) and colloidal coomassie blue staining .
for shotgun 
 analysis , 1 g of mycelial powder was resuspended in 6 ml of 25 mm 
 tris - hcl , 6 m guanidine - hcl , 10 mm dtt , ph 8.6 and sonicated five 
 times to generate whole cell lysates .
dtt ( 1 m ; 10 
 l / ml lysate ) was added to supernatants and incubated at 56 
 c for 30 min .
samples were cooled to room temperature and 55 
 l of 1 m iodoacetamide per milliliter of lysate was added followed 
 by incubation ( in the dark ) for 20 min .
samples were then dialyzed 
 twice against cold 100 mm ammonium bicarbonate , ( 2  50 vol . ) , 
 for at least 3 h with stirring .
one - hundred microliters of denatured 
 protein solution was transferred to an eppendorf tube and 5 l 
 trypsin ( promega v5111 ) ( 0.4 g/l in 10% ( v / v ) acetonitrile 
 in 10 mm ammonium bicarbonate ) was added .
after overnight incubation 
 at 37 c , the protein precipitates disappeared and protein digests 
 were spin filtered ( agilent technologies , 0.22 m cellulose 
 acetate ) prior to lc  ms / ms analysis . for extracellular 
 protein isolation , a modified version of that described by fragner 
 et al . was used .
culture supernatants 
 ( 40 ml ) were frozen 20 c , thawed , centrifuged at 48000 g for 40 min , transferred to clean tubes and frozen in liquid 
 n2 .
samples were lyophilized to 4 ml , brought to 10% ( w / v ) 
 tca and centrifuged 10000 g for 5 min before 
 washing with ice cold acetone ( 3 times ) with centrifugation steps 
 of 10000 g for 5 min .
protein pellets were 
 solubilized with 100 l of 10% ( w / v ) sodium dodecyl sulfate 
 ( sds ) , 0.1 m dtt and 5 l of sds - page solubilization buffer 
 and boiled for 10 min prior to sds - page . for protein extraction of 
 fungal cultures grown on solid phase , mea plates ( 25 ml ) were briefly 
 ground with a pestle , transferred to containers and brought to 50 
 ml with 50 mm kh2po4 ph 7.0 .
samples were agitated overnight at 4 c , after which 
 liquid was removed by gravity filtration through miracloth .
polysaccharides 
 were removed by centrifugation at 48000 g for 
 30 min , and supernatants were precipitated with 10% ( w / v ) tca under 
 agitation overnight at 4 c .
samples were centrifuged at 1700 g for 40 min , pellets were washed ( 3 times ) with 20% ( v / v ) 
 50 mm tris - hcl in ice cold acetone followed by washing in pure ice - cold 
 acetone before air drying and storage 20 c .
proteins 
 were digested overnight at 37 c with 50 l of trypsin 
 ( 65 ng/l in 10% ( v / v ) acetonitrile in 10 mm ammonium bicarbonate ) . 

gel digestions ( sds - page bands and 2-d page spots ) were carried 
 out according shevchenko and colleagues with little modification .
spots were excised and destained with 10% ( v / v ) 
 acetonitrile in 100 mm ammonium bicarbonate .
gel pieces were shrunk 
 with 500 l of acetonitrile and digested with 50 l of 
 trypsin ( 13 ng/l ) at 37 c overnight before filtration 
 and lc  ms / ms analysis . an agilent 6340 ion trap 
 mass spectrometer
running chemstation 
 0.01.03-sr2 ( 204 ) and a protiid - chip150 c18 150 mm ( g424062006 ) 
 chip was used for peptide separation from in - gel protein digests , 
 while a 160 nl 150 mm c18 chip ( g424062010 ) was used for tryptic 
 peptides prepared by total protein digestion .
solvent a : 0.1% ( v / v ) 
 formic acid in water and solvent b : 0.1% ( v / v ) formic acid in 90% 
 ( v / v ) acetonitrile / 10% ( v / v ) water . for protein identification following 
 sds- or 2-d page , a flow rate of 0.6 l / min with a linear gradient 
 of 5 to 70% solvent b over 7 min followed by 1 min increase to 100 
 % solvent b for 1 min and decrease to 5% solvent b for
proteomic studies , a flow rate of 0.4 l / min with 
 non - linear acetonitrile / water + 0.1% ( v / v ) formic acid gradients , 
 optimised on a sample - specific basis from initial chromatographic 
 analyses , were used . for shotgun proteomic studies , a flow rate of 
 0.4 l / min with a nonlinear acetonitrile / water + 0.1% ( v / v ) formic 
 acid gradient was optimized on a sample - specific basis from initial 
 chromatographic analyses .
gradients ranging from 20 to 180 min were 
 optimized for identification of the maximum number of proteins per 
 sample : peptide identification from protein spots or bands required 
 20 min gradients , while shotgun ( complex mixtures ) gradients were 
 generally up to 180 min duration .
all lc  ms spectra were 
 analyzed using spectrum mill software ( rev a.03.03.084 sr4 ; agilent 
 technologies ) to identify proteins against the translated a. mellea cdna database .
ms / ms searches were carried out 
 with the following settings : enzymatic trypsin digestion , carbamidomethylation 
 as fixed modification and two variable modifications were set : deamination 
 and oxidation of methionine .
the minimum scored peak intensity was 
 set to 70 , with precursor mass tolerance 2.5 da and product 
 mass tolerance 0.7 .
the maximum ambiguous charge was set to 
 3 with a sequence tag length > 3 and minimum detected peaks set 
 to 
 4 .
the genome of a. mellea ( vahl : fr ) kummer dsm 3731 was sequenced using 
 an illumina genome analyzer ii .
a total of 6.72 gb of sequence data 
 was obtained from 3 single read lanes . on the basis of nuclear dna 
 measurements by rflp analysis , this represents 
 57.25-fold coverage of the a. mellea genome .
assembly 
 of the illumina reads with the velvet assembler resulted in 58.35 
 mb of sequence data in 4377 scaffolds with an n50 size of 36.7 kb 
 ( table 1 ) .
the overall gc content of the genome 
 is 49.1% , while in the coding regions it is 53.2% .
a total of 
 14473 putative a. mellea open reading 
 frames ( orfs ) were predicted ( table 2 ) which 
 was subsequently used as the reference database for proteomic analyses . 

the average gene length was found to be 1575 bp , similar to what is 
 observed in close relatives of a. mellea , such as l. bicolor and coprinopsis cinerea ( figure 1 and table 2 ) .
the average 
 exon / intron lengths were also comparable among these species , as were 
 the average number of introns per gene ( 4.72 , 4.44 and 4.66 , respectively ) . 

blast2go successfully assigned a go term to 9514 ( 65.7% ) of the predicted 
 genes ( data not shown ) .
the signalp webserver identified 1903 proteins 
 with secretion signal peptides , while phobius predicted 4035 proteins 
 ( 27.8% ) with membrane regions , 2746 ( 19% ) with transmembrane regions 
 and 246 with transmembrane regions plus signal peptide regions .
homology searches followed 
 by markovian clustering inferred that 
 9939 ( 68.7% ) of the a. mellea orfs belonged to multigene 
 families . using the same approach we calculated that 68.5% of l. bicolor genes belong to multigene families .
approximately 
 18.8% ( 2721 ) of the predicted a. mellea genes have 
 no significant homologues in the ncbi database , a figure consistent 
 with other newly sequenced basidiomycete genomes .
we also successfully 
 located 240 trna genes which compares favorably to l. bicolor which has 297 trna genes .
given the paucity of a. mellea proteins curated 
 and present in genbank ( n = 16 ) , it was necessary 
 to interrogate the translated 
 version of the a. mellea cdna database with peptide 
 sequence data for complete and accurate de novo protein 
 identification . as a consequence
, a total of 921 proteins were identified 
 in both mycelia and culture supernatants from a. mellea grown under a range of different culture conditions .
these identifications 
 were based on the detection of 3641 unique peptides , 2839 of which 
 came from mycelia and 802 from the supernatant , respectively , with 
 197 common to both ( supplementary tables 2 and 3 , supporting information ) .
mycelial proteins ( n = 739 ) were identified by using 
 the a. mellea database 
 obtained from the translation of the protein - coding genes .
six - hundred 
 twenty - nine proteins were uniquely identified in mycelia ( supplementary 
 table 2 , supporting information ) , while 
 others were also identified in the a. mellea secretome 
 ( supplementary tables 3 and 4 , supporting information ) . of the mycelia - specific proteins ,
509 were uniquely identified 
 by a shotgun approach , with 98 and 9 proteins identified following 
 prior 2-d page and sds - page fractionation , respectively ( supplementary 
 figure 1 , supporting information ) .
supplementary 
 table 2 , supporting information , shows 
 the actual peptide sequence with corresponding spectrum mill scores 
 where values > 11 were deemed significant according to manufacturer s 
 guidelines .
sequence coverage ranged from 1 to 5% , 1 to 81% and 1 
 to 72% , respectively , using sds - page , 2-d page fractionation and shotgun 
 analysis . functional analysis undertaken with blast2go and database 
 searches classified proteins according to the biological process ( bp ) 
 in which they are involved , the molecular function ( mf ) and the cellular 
 component ( cc ) .
the largest categories were catabolic processes ( n = 114 ) followed by translation ( n = 88 ) , 
 carbohydrate metabolic processes ( n = 81 ) , precursor 
 metabolites and energy ( n = 56 ) , response to stress 
 ( n = 22 ) and secondary metabolic processes ( n = 19 ) ( figure 2a ) .
mf of mycelial 
 proteins revealed nucleotide binding ( n = 150 ) and 
 structural molecule activity ( n = 70 ) as the largest 
 categories , accounting for 53% of mycelial proteins identified . go 
 identified 38 proteins in mf as having peptidase activity ( figure 2b ) .
mycelial proteins cc mainly comprised ribosomal 
 proteins ( n = 78 ) and protein complexes ( n = 77 ) .
there were also 23 proteins classified as mitochondrial 
 ( figure 2c ) and 14 proteins that have not been 
 previously annotated and appear to be unique to a. mellea .
one - hundred two proteins were detected that were previously reported 
 as hypothetical or predicted ( table 3 ) .
as 
 expression of these proteins is now demonstrated , they can be reclassified 
 as unknown function proteins .
overall , 114 known domains were assigned to 55 proteins 
 ( table 3 ) .
eight proteins ( am15909 , am6889 , 
 am18190 , am14843 , am14848 , am14568 , am13925 and am17640 ) were found 
 to contain a nad(p)-binding domain , however no other domain 
 was over - represented in our analysis .
moreover , no go function was 
 identifiable for 73 proteins , while 26 showed no blast description . 
 among these are proteins with conserved domains which indicate a putative 
 process , function or cellular location such as am12246 containing 
 a start domain typical of proteins which transport ubiquinone during 
 respiration or am14390 containing a 60 residue conserved bsd domain 
 of unknown function usually located in btf2-like transcription factors , 
 synapse - associated proteins and dos2-like .
blast2go 
 annotation of mycelial protein classification : ( a ) biological 
 process ; ( b ) molecular function ; ( c ) cellular component .
the total 
 number of proteins in each category , as well as the overall % , are 
 given for each subdivision ( no . , % ) .
gravy score , grand 
 average of hydropathy 
 ( negative score indicates hydrophilicity while positive score indcates 
 hydrophobicity ) .
ms / ms 
 analysis , 2-d proteins separated in two dimensions prior to lc 
source : m , mycelia ; 
 sn , culture 
 supernatant / secretome . regarding 
 2-d page analysis ,
a reference 2-d page map is shown in figure 3 and information ( pi and mr ) on proteins identified 
 can be found in supplementary table 2 ( as  2d  under 
 column  method  , supporting information ) .
possible protein isoforms were identified in different spots ( e.g. , 
 spots 89 , 92 , 107 ( am10779 ) and 47 , 122 , 124 , 128 , 130 ( am16991 ) . 

one protein , am17600 , was identified in 17 spots ( nos . 1 , 3 , 11 , 58 , 
 88 , 89 , 90 , 97 , 103 , 104 , 107 , 117 , 118 , 119 , 131 , 132 , 133 ) probably 
 due to a protease activity or degrading events .
a total of 
 293 proteins were identified in culture supernatants derived from a. mellea , 183 of which were uniquely detected in the secretome , 
 while 111 were also identified in mycelia ( supplementary table 3 , supporting information ) . to overcome the considerable 
 difficulty of precipitating low abundance proteins from liquid culture , a. mellea was grown on agar , which was subsequently gently 
 macerated and the liquid phase subject to tca precipitation followed 
 by sds - page fractionation .
a nonredundant protein search of the translated a. mellea gene set , identified 226 proteins .
analysis of 
 lc  ms / ms data identified 157 unique proteins , while 37 were 
 uniquely identified by a shotgun strategy without sds - page fractionation . 

sequence coverage ranged from 1 to 55% and 1 to 46% following sds - page 
 and the shotgun approach , respectively , in the case of supernatant 
 protein analyses ( supplementary table 3 , supporting 
 information ) .
eight proteins were identified in culture supernatants 
 which were a. mellea - specific and included : am1874 , 
 am2320 , am5632 , am9253 , am11032 , am11414 , am13545 and am13611 .
ips 
 revealed low - complexity segments in am11414 , am2320 , am5632 and am9253 . 

analysis revealed two sigp hits ( am11032 and am13611 ) , three secp 
 signals ( am11414 , am13545 and am1874 ) and a tmhmm search predicted 
 am11032 to be a hydrophobic membrane protein .
the proportions 
 of proteins involved in catabolic processes ( n = 
 29 ) and carbohydrate metabolism ( n = 38 ) was greater 
 ( 55% ) than in mycelia ( 33% ) ( figure 4a ) .
go analysis related to the molecular function 
 of supernatant proteins showed peptidase activity as the largest functional 
 category ( n = 15 ) followed by nucleotide binding 
 proteins ( n = 14 ) .
the class including carbohydrate 
 binding proteins represented 14% in the secretome , yet in mycelia 
 it accounted for only 1% . proteins classified as having antioxidant 
 activity represent 11% of the secretome , while only 3% of mycelial 
 proteins were similarly classified ( figures 2b , 4b ) .
concerning go annotation of cc , the 
 largest categories in the secretome comprised protein complexes ( n = 11 ) , and extracellular proteins ( figure 4c ) .
( a ) 
 biological process ; ( b ) molecular function ; ( c ) cellular component . 

the total number of proteins in each category , as well as the overall 
 % , are given for each subdivision ( no . , % ) .
signalp analysis predicted signal peptide presence for 12% 
 ( n = 90 ) and 50.2% ( n = 147 ) of 
 mycelial 
 and secretome proteins , respectively .
furthermore secretomep indicated 
 that 99 secreted proteins ( 34% ) contained a nonclassical secretion 
 signal meaning that less than 20% ( 55 ) of those proteins do not contain 
 a secretion signal .
gravy and phobius evaluation of hydrophobicity 
 and transmembrane domain presence indicated that 12.7% ( n = 93 ) of mycelial proteins were hydrophobic in nature , 10.5% ( n = 77 ) contained transmembrane regions , and 1.4% ( n = 10 ) contained both a transmembrane region and signal 
 peptide .
six proteins ( am9118 , 13744 , 6597 , 5593 , 17215 and 19909 ) 
 contained greater than 10 transmembrane regions , while 71 proteins 
 contained between 1 and 8 transmembrane domains .
with respect to supernatant 
 proteins , 26.3% ( n = 77 ) were hydrophobic in nature , 
 29 contained predicted transmembrane domains , and 9 of these were 
 also predicted to possess signal sequences . although basidiomycetes have been 
 considered as sources of glycoside hydrolases ( gh ) , with potential 
 applications in recalcitrant carbohydrate digestion , no data whatsoever 
 exists on gh complement in a. mellea . here , we reveal 
 the presence of 226 gh genes in a. mellea ( supplementary 
 table 5 , supporting information ) , and further 
 demonstrate the expression of 52 of these at the protein level , in 
 culture supernatants , under a range of culture conditions ( table 4 )
. specifically , gh genes belonging to 43 families 
 are present ( supplementary table 5 , supporting 
 information ) .
the number of a. mellea gh genes 
 is greater than that identified in either p. chrysosporium ( n = 177 ) or ceriporiopsis subvermispora ( n = 171 ) , both of which are white rot fungi , a finding that may be related to the pathogenic 
 nature of a. mellea .
gravy score , grand 
 average of hydropathy 
 ( negative score indicates hydrophilicity while positive score indcates 
 hydrophobicity ) .
ms / ms 
 analysis , 2-d proteins separated in two dimensions prior to lc 
the a. mellea genome encodes 16 xyloglucan : xyloglucosyltransferases 
 ( gh16 family , supplementary table 5 , supporting 
 information ) .
xyloglucan , a hemicellulose , is a major component 
 of plant cell walls whereby its cellulose binding capacity provides 
 structural support for the cell wall .
gh 16 family enzymes cleave the 14 glycosidic backbone of 
 xyloglucan and transfer the segment either to another xyloglucan or 
 to a xyloglucan oligosaccharide ( xgo ) , thus restructuring and modifying 
 the primary cell wall .
xgos have been 
 shown to activate plant cellulases and promote plant growth , but plant 
 xgos do not activate fungal cellulase .
two enzymes ( am17743 and am19909 ) in this class were also implicated 
 in gtpase activity , which may indicate a signaling role . other gh families with increased or decreased 
 representation in the a. mellea genome compared to 
 lignolytic and cellulolytic fungi are reported in table 5 .
contrary to expectation that lignolytic fungi would have 
 similar frequencies of particular gh family proteins , a. mellea appears to contain more gh28 , gh61 , and gh76 family members than 
 the other white or brown rot fungi ( table 5 ) .
for example , 17 gh28 genes ( endo / exo-(rhamno)galacturonases , essential 
 for pectin degradation ) are present in the a. mellea genome ( supplementary table 5 , supporting information ) in comparison to the 6 identified in l. bicolor and the 4 present in p. chrysosporium .
moreover , gh109 , an -n - acetylgalactosaminidase present 
 in a. mellea and the mycorrhizial l. bicolor ( supplementary table 5 , supporting information ) , has previously been also identified in prokaryotes and its crystal 
 structure defined .
chitinases , 
 gh18 ( endo--n - acetylglucosaminidases ) 
 ( n = 11 ) , are also encoded within the genome , and 
 the expression of two of these was detectable in a. mellea culture supernatants ( am17468 and am17470 ) ( supplementary table 
 3 , supporting information ) .
in addition 
 to glycoside hydrolases , 72 glycosyltransferase and 19 polysaccharide 
 lyases have been identified ( supplementary table 5 , supporting information ) . besides glycoside 
 hydrolases , other cbm enzymes were discovered in the a. mellea genome .
cbm proteins exhibit three functions encompassing ( i ) carbohydrate 
 binding and concentration of enzymes on the substrate , ( ii ) binding 
 to the polysaccharides that are the substrate targets of the catalytic 
 moiety of the enzyme and ( iii ) substrate degradation .
three cbm proteins were identified ( am18408 , am9123 and 
 am11736 ) by lc 
ms / ms analysis , belonging to families 1 , 12 
 and 13 , respectively ( table 4 ) .
cbm12 previously only described 
 in bacteria , comprises 4060 residues , binds chitin , which 
 is unusual among cbm proteins which have broad polysaccharide specificity . 

the c - terminus of the chitin binding domain ( chbd ) binds very specifically 
 to insoluble chitin but not to chitin oligosaccharides or glucose .
cbm13 is a 150 residue module that has been 
 identified in galactose - binding plant lectins .
predominantly found in bacteria , they have also been identified 
 in eukaryotes , including some fungi ; cbm13 modules occur in glycoside 
 hydrolases and glycosyltransferases and also bind xylan .
compared to some basidiomycete 
 and ascomycete species , a. mellea has increased representation 
 of carbohydrate esterase ( ce ) families ( supplementary table 5 , supporting information ) , specifically ce4 , ce8 , 
 ce9 and ce14 comprised 94 genes .
ce1 is an acetyl xylan esterase ( axe ) 
 which depolymerises xylan microfibrils and solubilizes lignocellulosic 
 material and releases fermentable sugars . a ce1 enzyme ( am19807 )
was identified at the protein level in the 
 growth condition we tested ( supplementary table 2 , supporting information ) .
the ce4 family exhibits acetyl xylan , 
 chitin , chitooligosaccharide and peptidoglycan deacetylase activity . 

four ce4 enzymes ( am18938 , am3987 , am3988 , and am6064 ) have been identified 
 in a. mellea culture in both mycelia and secretome 
 ( supplementary tables 2 and 3 , supporting information ) .
these enzymes act on n - acetylglucosamine ( glcnac ) which is present 
 in bacterial as well as fungal cell walls ; have very broad substrate 
 specificity and catalyze the hydrolysis of acetyl groups from xylan 
 and carbon 
chitin deacetylases ( cda ) are secreted during 
 hyphal penetration of plants by colletotrichum lindemuthianum .
tsigos and bouriotis hypothesized that chitin of invading pathogens 
 triggers recognition by plant defenses inducing plant chitinase activity 
 which gives rise to formation of lignin and callus .
deacetylases modify plant chitinases thereby neutralizing 
 plant defenses , and modify chitin to chitosan which plant deacetylases 
 are unable to modify , thus enabling the fungus to invade the plant . 

pectin methylesterase ( pme ) are members of the ce8 family and catalyze 
 the demethyloxidation of pectin , a major plant cell wall polysaccharide . 

pmes are important in vegetative and reproductive 
 plant development , but pathogenic fungi can also degrade pectin in 
 the cell wall during plant invasion .
ce9 ( n - acetylglucosamine 6-phosphate 
 deacetylase ) is another gene family detected in a. mellea which is involved in carbohydrate , specifically n - acetyglucosamine , 
 catabolism .
surprisingly , a glcnac-6-phosphate 
 deacetylase ( dac1 ) has been linked to c. albicans virulence .
the ce14 deacetylase family 
 has a conserved lmbe domain which catalyzes the n - deacetylation of 
 appropriate substrates .
tanaka et al . 
 identified a deacetylase from thermococcus kodakarensis kod1 which degrades diacetylchitobiose ( glcnac2 ) to glcn 
the a. mellea genome encodes 
 6 laccases ( am380 , am12382 , am16732 , am16804 , am17018 and am19478 ) 
 ( figure 5 ) , all of which contain the canonical 
 l1-l4 signature sequences , characteristic of this multicopper oxidase 
 ( mco ) family of enzymes .
overall , 15 
 laccase - like enzymes are encoded within the a. mellea genome .
ms / ms analysis ( supplementary tables 2 and 3 , supporting information ) , while expression of 
 any other laccase was undetectable under the condition tested .
expression 
 of two laccase - like enzymes ( lcc5 : am2859 and am4666 ) was also evident 
 in the a. mellea secretome ( supplementary table 3 , supporting information ) .
notably , the genome 
 of the white rot fungus p. chrysosporium does not 
 encode any laccase , while those of c. subvermispora(51 ) and l. bicolor(69 ) encode 7 and 9 laccase 
 gene models , respectively .
one - hundred bootstrap replicates were applied 
 with the appropriate protein model using the software program phyml 
 ( v3.0 ) and summarized using the majority - rule 
 consensus method .
monophyletic laccases , canonical ferroxidases ( fet3 ) 
 and other multicopper oxidases ( mco ) are highlighted .
basidiomycete peroxidases 
 are a class of 
 oxidoreductase involved in lignin biodegradation in white rot fungi , ligninolytic haem peroxidases have been shown 
 to cleave the main linkage types in lignin due to their high redox - potential 
 and specialized catalytic mechanisms .
to date , three ligninolytic haem peroxidases have been isolated 
 and characterized in white - rot fungal species .
these include lignin 
 peroxidase ( lip ) , characterized by its ability to oxidize high redox - potential 
 compounds such as alcohol and methoxybenzenes and veratryl , manganese peroxidase ( mnp ) which exhibits an 
 ability to oxidize mn to mn and versatile peroxidase ( vp ) which can oxidize both lip 
 and mnp substrates .
our genome analysis 
 and subsequent homology modeling infers 
 that a. mellea contains nine putative ligninolytic 
 peroxidases . in an attempt to elucidate the function of the a. mellea peroxidases we reconstructed a phylogenetic tree 
 using representative mnp , lip and vp sequences from c. subvermispora and p. chrysosporium .
our phylogeny infers that the a. mellea peroxidases 
 are the result of a species - specific expansion as they are clustered 
 together in a monophyletic clade ( figure 6 ) . 

interestingly the a. mellea - specific clade is clustered 
 beside c. subvermispora mnp13 , and this gene has 
 previously been described as a short mnp .
the length of the a. mellea genes is comparable 
 to typical mnps however ( not shown ) .
the phylogeny also infers that 
 the a. mellea genes are more closely related to p. chrysosporium lip genes than the  typical  c. subvermispora mnp genes ( figure 6 ) .
however , close sequence inspection shows that the a. mellea peroxidases do not contain the conserved aromatic substrate oxidation 
 sites required for lip enzymatic activity .
furthermore , five of the nine a. mellea peroxidases 
 contain the three acidic residues ( two glutamates and an aspartate ) 
 required for the mn binding site of mnps .
based on this 
 observation we hypothesize that at least five of the peroxidases found 
 in a. mellea have mnp activity .
one hundred bootstrap replicates were used with 
 the appropriate protein model using the software program phyml ( v3.0 ) and summarized using the majority - rule consensus 
 method .
comparative growth studies of c. albicans recovered 
 from either mono- or coculture conditions 
 demonstrates that significant killing ( p = 0.0004 ) 
 of c. albicans by a. mellea occurs 
 in cocultures ( figure 7 ) .
this observation 
 can be associated with the detection of 30 a. mellea proteins , not identified under any other culture condition ( table 6 ) including redox - active , degradative and attack - type 
 proteins .
interestingly , cys-2 peroxiredoxin ( am10593 ) and thioredoxin 
 ( am7929 ) were both uniquely detected in fungal cocultures .
cys-2 peroxiredoxin 
 converts peroxides to water , with concomitant formation of intramolecular 
 disulfide linkages .
these disulfide linkages are in turn reduced by 
 thioredoxin to regenerate the active form of cys-2 peroxiredoxin .
we speculate that this system may be induced in a. mellea as an antifungal response to c. albicans presence . 

alternatively , peroxide dismutation may be required to attenuate products 
 originating from c. albicans cell wall degradation . 
 in this regard ,
it is notable that a class ii chitinase ( am13890 ) 
 was also uniquely detected in fungal cocultures which may be deployed 
 by a. mellea to degrade candida cell 
 wall material .
two proteins classified as prohibitins ( termed am14001 
 and am20304 ) , which have been proposed to be involved in the negative 
 regulation of cell proliferation , have also 
 been identified elsewhere .
these serine - threonine - rich 
 membrane proteins are anchored by glycosylphosphatidylinositol ligands . 

several gpi - anchored proteins have been characterized in fungi as 
 having multiple functions including cell wall organization in a. fumigatus , fruiting body formation in lentinus 
 edodes , and interaction with mapk , where they may have a 
 signaling role .
am3423 belongs to the 
 same protein family of drmip , hesp-379 , which is a haustorially expressed 
 secreted protein involved in host tissue penetration in l. 
 edodes .
in addition , a number 
 of oxidoreductases , monooxygenases , reductases and dehydrogenases 
 of basidiomycete origin were detected in the fungal cocultures ( table 6 ) .
similarly , metabolite extracts from a. 
 tabescens have also been observed to exhibit antifungal activity .
apart from demonstrating the power of proteomic 
 investigations to dissect fungal  fungal interactions , the coculture 
 system we have developed for a. mellea and c. albicans may represent a model system for the further 
 investigations of a. mellea pathogenicity .
( a ) culture plates : ( i ) c. albicans only ; ( ii ) coculture of c. albicans and a. mellea for up to 52 days .
reculture of c. albicans was carried out from multiple plugs taken from 
 inoculation points ( arrows ) .
( b ) comparative viable cell count of 
 recultured c. albicans from single and coculture 
 plates shows that coculture with a. mellea results 
 in significant ( p = 0.0004 ) killing of c. 
 albicans .
simultaneous fda ( live ) and pi ( dead ) cell staining of c. albicans . 1 . c. albicans live culture 
 ( 24 h ; positive control ) ; 2 .
monoculture of c. albicans and 4 . c. albicans following coculture with a. mellea ( a. ) .
ms / ms 
 analysis , 2-d proteins separated in two dimensions prior to lc 
ms / ms 
 has , for the first time , revealed new insights into the proteome of 
 the important fungal plant pathogen armillaria mellea , including the identification of multiple glycodegradative enzyme 
 systems which can be useful for recalcitrant carbon compound degradation 
 in biotechnology applications .
moreover , proteomics tools shed light 
 on potent antifungal properties of a. mellea , which 
 in part may be mediated by basidiomycete enzymes .
 armillaria mellea is a major plant 
 pathogen . yet , no large - scale  -omics  data are available 
 to enable new studies , and limited experimental models are available 
 to investigate basidiomycete pathogenicity . here 
 we reveal that the a. mellea genome comprises 58.35 mb , contains 14473 gene 
 models , of average length 1575 bp ( 4.72 introns / gene ) . 
 tandem mass 
 spectrometry identified 921 mycelial ( n = 629 unique ) 
 and secreted ( n = 183 unique ) proteins . 
 almost 100 
 mycelial proteins were either species - specific or previously unidentified 
 at the protein level . a number of proteins ( n = 111 ) 
 was detected in both mycelia and culture supernatant extracts . 
 signal 
 sequence occurrence was 4-fold greater for secreted ( 50.2% ) compared 
 to mycelial ( 12% ) proteins . 
 analyses revealed a rich reservoir of 
 carbohydrate degrading enzymes , laccases , and lignin peroxidases in 
 the a. mellea proteome , reminiscent of both basidiomycete 
 and ascomycete glycodegradative arsenals . 
 we discovered that a. mellea exhibits a specific killing effect against candida albicans during coculture . 
 proteomic investigation 
 of this interaction revealed the unique expression of defensive and 
 potentially offensive a. mellea proteins ( n = 30 ) . 
 overall , our data reveal new insights into the 
 origin of basidiomycete virulence and we present a new model system 
 for further studies aimed at deciphering fungal pathogenic mechanisms . 
Introduction
Experimental
Procedures
Results and Discussion
Conclusions