Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures

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Phosphoproteome profiles of the

phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during...

Article in Proteomics · July 2014

DOI: 10.1002/pmic.201300541 · Source: PubMed

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D

ATASET

B

RIEF

Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during

exponential growth in axenic cultures

Marl `ene Davanture

^1,2

, J ´er ˆome Dumur

³

, Nelly Bataill ´e-Simoneau

³

, Claire Campion

³

, Benoˆıt Valot

^1,2

, Michel Zivy

^1,2

, Philippe Simoneau

³

and Sabine Fillinger

⁴

1CNRS, Plateforme d’Analyse Prot ´eomique de Paris Sud Ouest, PAPPSO, Gif-sur-Yvette, France

2INRA/University Paris-Sud/CNRS/AgroParisTech, UMR 0320/UMR 8120 G én étique V ég étale, Gif-sur-Yvette, France

3INRA/Universit ´e d’Angers/ /Agrocampus-Ouest, UMR 1345 IRHS, Angers, France

4INRA UR1290 BIOGER, Thiverval-Grignon, France

Received: December 6, 2013 Revised: March 21, 2014 Accepted: May 8, 2014 This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungiAl-

ternaria brassicicolaandBotrytis cinereagrown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% inA. brassicicolaand at a slightly higher ratio inB. cinerea(1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast.

The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817).

Keywords:

Black spot / Gray mold / Microbiology / Protein phosphorylation / PTMs / Signal transduction

Additional supporting information may be found in the online version of this article at the publisher’s web-site

The phytopathogenic fungi Botrytis cinerea (Bc) and Alternaria brassicicola (Ab) belong to two distantly related taxa (i.e. Leotiomycetes and Dothideomycetes) within the As- comycetes. They provoke, respectively, gray mold on over 200 dicotyledonous plant hosts and black spot on majorBras- sicacrop species. These fungi are ubiquitous and use similar necrotrophic strategies to interact with their host plants.

Correspondence: Dr. Sabine Fillinger, INRA UR1290 BIOGER, BP01, F-78850 Thiverval-Grignon, France

E-mail:sabine.fillinger@versailles.inra.fr Fax:+33-1-3081-5306

Abbreviations: HK, histidine kinase;MAPK, mitogen-activated protein kinase

The genome sequences of both fungal species are available [1, 2] and genomic tools to investigate infectious strategies are being developed and used including tran- scriptomics, proteomics, yeast-one-hybrid, and others [3–5].

One bottleneck of proteomic studies is the identification of PTMs that are involved in the regulation of protein activity. Especially, phosphorylation and dephosphorylation are commonly used regulatory events involved in signal transduction (ST) particularly important for pathogenicity, development, and adaptation to changing environmental conditions including fungicides (reviewed in [6]). Only few papers report on global identification of phosphoproteins in nonmodel fungi [7, 8], whereas numerous studies describe the development of phosphoproteomics in several yeast species (reviewed in [9, 10]), but also in plants [11]. In

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2 M. Davanture et al. Proteomics2014,00, 1–7

this work, we established a gel-free phosphoproteomics protocol for the above cited fungi grown under nonlimiting in vitro conditions leading to a first set of roughly 1350 phosphopeptides per fungus representing over 800 phosphoproteins each.

Characterization of the Ab and Bc phosphoproteomes: Spores of theB. cinereareference strain B05.10 were inoculated at 2×10⁵spores/mL in liquid YSS (medium composition) (2 g/L KH2PO4, 1.5 g/L K2HPO4, 1 g/L (NH4)2SO4×7H2O, 2 g/L yeast extract, 10 g/LD-glucose). Spores ofA. brassicicola strain Abra43 were inoculated at 2×10⁵spores/mL in potato dextrose broth medium. Four independent cultures of 150 mL were grown for 22 h at 23⬚C in the dark on a rotary shaker at 150 rpm. The mycelium was harvested by filtration and immediately frozen in liquid nitrogen prior to freeze-drying.

To simplify the lecture of this paper, data derived fromB.

cinereawill be referred to as Bc and those fromA. brassicicola as Ab in the forthcoming sections.

Total extraction of proteins was performed on 100 mg of mycelium using phenol according to a protocol adapted from [12]. Pellets of proteins were suspended in 6 M urea, 2 M thiourea, 2% w/v CHAPS, and 30 mM Tris-HCl pH 8.8. Protein content was determined using the 2D Quant- kit (GE Healthcare, Velizy-Villacoublay, France) with BSA as standard.

For each biological replicate, 2 mg of proteins were reduced with 10 mM DTT and alkylated with 40 mM iodoacetamide.

Protein digestion was performed with modified trypsin (Promega, Madison, WI, USA) at an enzyme/substrate ratio of 1:33 w/w by overnight incubation at 37⬚C, and stopped by adding 1% formic acid v/v.

Prior to the enrichment of phosphopeptides using IMAC as described in [13], peptides were labeled using stable isotope as described by Boersema et al. [14] and separated in ten fractions using strong cation exchange chromatography (Zorbax 300, 5␮m, 2.1 mm id, 150 mm length).

Each enriched phosphopeptide fraction was separated on a Nano 2DLC-Ultra system (Eksigent) using a C18 column and analyzed online with an LTQ XL ion trap (Thermo Elec- tron, Courtaboeuf, France) using a nano-electrospray inter- face. Peptide ions were analyzed using Xcalibur 2.07 with the following data-dependent acquisition steps: (1) full MS scan in enhanced scan rate mode (m/z=350–1400, profile mode), (2) MS/MS on the two highest ions detected in full scan (qz

=0.25, activation time=30 ms, and collision energy=35%, centroid mode), (3) MS3 was performed when neutral loss of H3PO4was detected in the MS2 spectra. Dynamic exclusion was set to 45 s.

Identification of proteins and phosphorylation sites was performed using X!Tandem Cyclone (http://www.

thegpm.org/TANDEM/2010.12.01). Enzymatic cleavage was described to be due to trypsin digestion with one possible miss cleavage. Cys carboxyamidomethylation was set as static mod- ification while Met oxidation and phosphorylation of tyrosine, serine, and threonine residues were set as variable modifica-

tions. Precursor mass and fragment mass tolerance were 2.0 and 0.5 Da, respectively. Identifications were performed using theB. cinereagenome databases (Broad Institute of Har- vard at MIT, http://www.broadinstitute.org, 16 448 entries;

INRA URGI, http://urgi.versailles.inra.fr/Species/Botrytis, 16 360 entries) and theA. brassicicolagenome database (2010, DOE Joint Genome Institute, http://genome.jgi-psf.org/

Altbr1/Altbr1.home.html, 10 685 entries) completed with a homemade contaminant database. Identified proteins were filtered and grouped using the X!Tandem pipeline v3.3.0 (http://pappso.inra.fr/bioinfo/xtandempipeline/). Data fil- tering was achieved according to a peptide E value <0.01.

The false discovery rate was estimated<1% for both analyses (reverse database strategy). The assignment of phosphorylation sites in MS² and MS³ spectra was scored (Supporting Information Document 2) using the Olsen–Mann algorithm [15] implemented in PhosCalc [16]. Eighty-two percent of the phosphosites proposed by X!Tandem matched with the first Phoscalc hit or second Phoscalc hit when the difference between the first and second scores was<10. Phosphorylation site assignment was confirmed by manual inspection of the raw data for all phosphopeptides from proteins involved in signal transduction (Tables 1 and 2).

Identified proteins, phosphopeptides, and their corresponding spectra were deposited online using PROTICdb database [17] at the following URL: http://moulon.inra.fr/

protic/necrophos using the following login: “necrophos.”

The data have also been deposited to the ProteomeXchange Consortium database (http://www.proteomexchange.org) via the PRIDE partner repository [18] with the dataset identifier PXD000817 and DOI 10.6019/PXD000817 at the following URL: http://tinyurl.com/oorc5n9.

This experimental procedure leads to the identification of 1361 (Bc) and 1379 (Ab) phosphopeptides representing 818 (Bc) and 820 (Ab) phosphoproteins, respectively (Supporting Information Document 1). Among the 1439 detected phosphosites (p-sites) in the Bc extracts, 82.7% were phospho- serine, 15.8% phosphothreonine, and 1.5% phosphotyrosine.

The ratio found among the 1442 Ab phosphosites was slightly but significantly different (chi-square test,p-value=0.0037, df=2) with 80.4% serine, 18.7% threonine, and only 0.9% tyrosine residues phosphorylated. The experimental procedure did not allow the identification of the acid-labile phosphorylation of histidine or aspartate residues, because the buffers used for IMAC and strong cation exchange chromatography were at pH 3. Specific experimental conditions would have been necessary to identify these phosphorylation events. Most proteins contained a single phosphosite.

GOs in the phosphoproteomes: We then applied the GO annotations developed for both fungi in their respective genome projects to the phosphoproteomic data in order to identify the biological processes affected by protein phosphorylation. Statistical analyses conducted on the GOEAST web-server (http://omicslab.genetics.ac.cn/GOEAST/, [19]) highlighted those GO categories overrepresented among the

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Table1.SignaltransductionproteinsidentifiedamongtheA.brassicicolaphosphoproteins IDNameFunctionPhosphositepositionPhosphopeptides AB03810.1PQ-loop130,152/153:RD(pS)SNIGLPGSHR,A(s)(s)LPTIPDVGDGASEWVR AB03884.1RegulatorofGprotein(RGS)373FIGTTANETQSDSD(pS)IHEFSK AB08967.1Phosducin37HGVIPEKPP(pS)PTPMIQEALEQAR AB06955.1PKAregulatorysubunit52/53,71TEYLLSQQH(s)(s)PQGR,AENTFPGSNPFST(pS)PSSGSGFTR AB06955.1PKAregulatorysubunit99,163,164,166,175LEEEEENDTMTSP(pT)ASTFAAVDFGR,(p)T(p)SV(p)SAESLNPAS(p)SAADNFTPPFHQK AB08717.1PKAcatalyticsubunit25,311DLDSP(pT)SPIKDSNSPITPTTSR,EVPDITW(pT)LCGTPDYLAPEVVASK AB09880.1PKAcatalyticsubunit245/246/247E(t)(y)(t)LCGTPEYLAPEVIR AB01474.1AbSch9Sch9-likeproteinkinase179/180/181/182,289,291K(s)(s)(s)(t)VNPSPAPVSTCQVSDPR,(p)TE(p)SQMGTPMAIPMK AB01474.1AbSch9Sch9-likeproteinkinase572/574ANLTENAT(t)N(t)FCGTTEYLAPEVLLDEQGYTK AB08261.1CLK1-likeproteinkinase229,1069RN(pT)LPVLAAEDHTSK,RA(pS)NAPIDPSEELNLTR AB00575.1Hrk1-likeproteinkinase113,184SSIPGSPASAMH(pS)PPLTPAGTSSR,IAPQ(pS)PRPSSDLSGIVQPHPK AB00575.1Hrk1-likeproteinkinase378SGIM(pT)PPITNGAMSVPFADDHGLQSK AB01755.1Yak1-likeproteinkinase36,476,665/666,668QNSVPLQ(pS)PSGTASR,QTVYT(pY)IQSR,SG(s)(s)R(pS)PAPGVQEQQR AB04026.1Psk1/2-likeproteinkinase110,328AA(pS)LNIATPPVSR,TL(pT)DVAPLTIQSQLHDALAQPYR AB07904.1RhoGAPprotein175LVEDLEEEDKLAFQDPQSEAAQ(pS)PK AB07854.1RhoGAPprotein108,182,580SGAS(pT)PDLGRPVR,SK(pS)IKEGESSGSR,GNN(pS)PQVQSSK AB07854.1RhoGAPprotein616,657/658,699NGRA(pT)PPNPSLVLSEPSSNPVLR,GGGGGGGGGGGGGGGYGERE(t)(s)PER,APPSVSTEASSIDLDENA(pT)PTQAQHSR Ab03936.1RhoGAPprotein16SS(pS)LSAVPPPQHSSNYSPDLAK AB04861.1RhoGEFprotein843,920/927/931SNSN(pS)PNNPPPR,NM(s)PPLGAD(s)NIP)t)QLK AB01313.1RhoGEFprotein86/88/89,283,490QNDELFIGANSSPQLSQGPM(s)P(t)(t)GGYGGSYGYQR,YTSMSGNALPPTPEVALLHG(pS)PQR,VD(pT)FGTGGLEEPSAR Ab08544.1AbHhk5Histidinekinase–groupV115,121,127,207,219,393(pT)APSNA(pS)VAALG(pS)R,WA(pS)AFHMPFTSLSP(pT)VAAGQPR,QKSN(pT)SPDTPTPAPLGRPR AB05093.1AbHhk8Histidinekinase–groupVIII150,265,1263SAISVDPTP(pT)PK,ADRAP(pS)IGGASAR,(pS)LEHISSQSVVPPHKR AB01584.1Histidinekinase–groupXI742RTM(pT)GEEAMQTIK AB09877/8.1AbSsk1Responseregulator301/302/306(s)(s)SSL(s)PTTAANPEPNGVKR AB03493.1Responseregulator622/625/626,667RAD(s)PA(t)(s)DVGLSR,SMVPPPS(pS)PLR AB08549.1AbSkn7Responseregulator273MNGTLT(pT)PPPDFTR AB09970.1AbSte11MAPKKK208,212,219,442QIMNSDLGSYAYGAMPP(p)SRPG(p)SPLVDQE(p)TR,LTGESLAAVSYPL(pS)PTSAR AB05686.1AbHog1MAPK171,173IQDPQM(pT)G(pY)VSTR AB01438.1AbPbs2MAPKK24,29,125,137SP(pS)PAPS(pS)PTENASSPDLLSAERPPPR,GPAPQ(pS)PAPSPDNRNPM(pS)PGGNPGGIPGNFR AB01438.1AbPbs2MAPKK194,197,331,334FQGALPGGL(pS)GS(pS)PTAGGPAPPAGRK,NFDEEA(p)SPT(p)SAKPAAGTGIVMAMK AB07135.1AbSlt2MAPK98GFSMDPEENAGYM(pT)EYVATR AB04139.1AbMkk1MAPKK64,106LSVV(pT)PMGSNNAPHEGR,SG(pS)FGEVQANGSASSYHAALGFPGLQK AB04139.1AbMkk1MAPKK132,136,139,143,154/155A(p)TDPI(p)SAI(p)SQGG(p)SEGAPSMER,AN(s)(s)QPLPDLDAMAVEK AB02044.1AbBck1MAPKKK853LNADSQLD(pS)PGGR AB01652.1Ypk1/2-likeproteinkinase440DEDR(pT)NTFCGTPEYLAPELLTGAGYTK AB04922.1Ypk1/2-likeproteinkinase414(pS)QDGELHPTR Ab04691.1Pkh1/2-likeproteinkinase360,364,541AQ(pS)FVG(pT)AEYVSPELLTDK,LITELPPPSQLDIDW(pS)PVLTK AB07725.1Fpk1-likeproteinkinase157,433/435/439VA(pS)APNAQGLFSSGK,(t)N(s)FVG(t)EEYIAPEVIK AB00828.1Pom1-likeproteinkinase526AYE(pS)PKDPER AB01801.1Cdc28-likeproteinkinase57/58RL(s)(t)GDEDEETAK Ab03742.1Cdc15-likeproteinkinase654TQ(pS)QIEIQK AB07167.1Dbf20-likeproteinkinase451/453,503,532/534DNAMDRPG(t)P(t)EAFMSPQQTPQGSPSK,TKPG(pS)PTLPNK,LQLGSSGGADYSAQDSPSFAPG(s)P(t)RK AB07167.1Dbf20-likeproteinkinase555,874,1061ES(pS)YLTHAAQSR,(pS)IVGSPDYMAPEVLK,TADENGKPA(pS)PR AB09330.1Bur1-likeproteinkinase203EY(pT)TLVVTR Ab03799.1Yck1/2-likeproteinkinase419,449AAPA(pS)IPNPQQPR,LANVLCCGA(pS)K AB07752.1Cbk1-likeproteinkinase146GGYGYG(pS)QGGSSGSR AB09047.1p21-activatedserine/ threonineproteinkinase 65,295/297SV(pS)SASTVK,KD(s)Y(s)QGYGSQPSASSQQDR AB09315.1Proteinkinase353KR(pS)EPFLSTYSAAK AB09163.1Rim11-likeproteinkinase198ILVENEPNVS(pY)ICSR AB07449.1ProteinkinaseC601,603,606,608,659,684, 685,689,693/694

GQ(p)SS(p)SGP(p)SM(p)SQR,SPPPGPPRQQ(pS)YPTSAASIDAAR,ASFSTSGSSAAP(pT)(pS)PTA(pS)SRP(s)(s)GPR

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Table1.Continued IDNameFunctionPhosphositepositionPhosphopeptides AB07449.1ProteinkinaseC698/700/701,723,1003/1004(t)Q(s)(s)VAAAAAAMNK,SN(pT)DYQPQSGR,EEMWYGST(t)(s)TFCGTPEFMAPEILLDKK AB07962.1Stt4-likeproteinkinase296,383,472EASE(pS)DDEATPVKK,LAPLQTPG(pS)PDPVR,EIAADMNNAEI(pT)PQQR AB00947.1CalcineurinA425,550EELEEETPSSLASGPS(pS)PPLSSLDPDSTEFKR,RI(pS)MSSGSGR AB03945.1AbCrz1CN-regulatedTF158/159,255H(t)(s)LDPSAAYGQITEWGQMAGGFGQHR,FNLNGSNAHISAGN(pS)PHISPR AB02788.1Mechanosensitiveion channel

76,368GIQ(pS)PSQTR,(pT)PGQILTEAQK AB09176.1Ca2+/CaM-dependentkinase77SEYSN(pS)EDDGSAQHR AB00893.1Clk1-likeproteinkinase326,443VIWDTQTM(pT)PCGTVGYTAPEIVKDER,EADEPTYSAYDAPPLA(pT)PAAKK AB03068.1Kin4-likeproteinkinase105,151/153,437,438,441TS(pS)HRTPNTASASSQPSR,(s)A(t)AIGQPPVGTSLPR,GDLMQ(pT)(pS)CG(pS)PCYAAPELVVSDSLYTGR AB03068.1Kin4-likeproteinkinase642,648,832,898GEA(p)SPPVET(p)SPK,ESFTSTG(pS)YGGYPEEK,RF(pS)LLPSSLSK AB07973.1Dun1-likeproteinkinase32,65/67,637HSRL(pS)PPPNGR,ESTGDASEGYKEG(t)Q(s)PPPGK,GDQTLY(pS)EEEDGSR AB01426.1Ca2+/H+antiporter21,24,353R(p)SST(p)YGTGSDTPTQSASGTDSAR,LFFGPTTGTS(pS)LIGR AB00595.1Ca2+/H+antiporter49/51/54TR(s)E(t)HA(t)PAIEEQR AB00486/7.1Ca2+translocatingATPase80,81ESRP(pT)(pS)PHNISSPVTAWNNNAGLLNVPGAR AB08416/17.1Ca2+translocatingATPase40SS(pT)PSSTTSAHSLVSAQDTAQK AB07326.1Proteinphosphatase6172SIG(pS)ASDVDSAK AB00432.1Proteinphosphatase4401RL(pS)SVLNDSFQK AB00277/8.1Proteinphosphatase2C88/90QSLHLGGS(s)G(s)PTSSTSTMSEK AB03980.1Proteinphosphatase2C332,404,418IDD(pS)PDDIDMDLDSR,ES(pT)PGPQHESK,VQVTE(pS)PSSVQTEK AB05290.1Proteinphosphatase2C392FAASFVTD(pS)FGEER (pS):phosphorylatedsite;(p)S:peptidefoundmonophosphorylatedatdifferentsites,(s),(y),or(t)undistinguishablesite. Theannotationswereselectedfromthegenomedata(http://genome.jgi-psf.org/Altbr1/Altbr1.home.html,[2])andnamesofgenesindicatedifknown.

phosphoproteome in comparison to the genome content (Supporting Information Document 3). Figure 1A–B shows the categories of biological processes and molecular functions enriched in both phosphoproteomes according to the GOEAST analysis. As expected, regulation of signaling, of cellular processes, response to stimuli, and cell communica- tion figure among these, but also metabolic processes and inB. cinerea, the category of developmental processes seems enriched in phosphoproteins.

Regarding the molecular functions identified in our screen, those dealing with nucleoside or nucleotide binding build common categories for both fungi. The comparison of the enriched GO-categories between both fungi (Multi-GOEAST) pinpoints to a specific enrichment in the categories of oxidoreductase and protein kinase regulatory activity inA. brassicicola.

Signal transduction proteins: We then selectively screened for signal transduction proteins. Tables 1 and 2 summa- rize the hits for both fungi. Briefly, the principal protein kinases were found among the phosphoproteins in both fungi, such as the cAMP-dependent protein kinases PKA (catalytic and regulatory subunits, respectively), the Ca²⁺/calmoduline- dependent protein kinases, the mitogen-activated protein kinases (MAPKs) Hog1-like and Slt2-like as well as the corresponding MAPK-kinases. The third MAPK (Fus-like) and its kinase were identified only in the B. cinerea dataset.

As additional ST proteins, we identified regulators of G- proteins and small GTPases, but also protein phosphatases.

As mentioned above, we did not expect histidine kinases (HKs) in our datasets with the exception of potentially phosphorylated serine, threonine, or tyrosine residues in conserved signaling domains (e.g. PAS, GAF) typical for some classes of HKs [20]. Intriguingly, we found three and two phosphorylated HKs inA. brassicicolaandB. cinerea, respectively, of the classes V, VIII, XI (Ab), and I and III (Bc), respectively. AB08544.1 (class VIII) had five phosphorylated residues (serine and tyrosine), AB05093.1 (class V) had two, and the other HKs had one phosphosite, respectively. None of these residues affected conserved domains, and we were un- able to identify common sequence motifs between the phosphorylation sites (data not shown). It will therefore be chal- lenging to characterize the role of these phosphorylations in HK functions. A good candidate would be the class III HK Bos1 ofB. cinereawhose involvement in adaptation to hyper- osmotic conditions and to some fungicides is known. The same issue with the other HKs seems more difficult as their functions are still unknown.

This work is, to our knowledge, the first description of a phosphoproteomic dataset of two important necrotrophic ascomycetes based on a gel-free analysis protocol that was proven to work in different organisms [8, 11, 21]. With 1638 phosphoproteins identified in both fungi, these data cover 7.7% of the 10.688 predicted Ab proteins and 7.8% of the predicted 10.427 Bc B05.10 proteins [22], a starting point for the functional analysis of protein phosphorylation sites. With

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Table2.SignaltransductionproteinsidentifiedamongtheB.cinereaphosphoproteins IDNameFunctionPositionPeptide BC1G07268.1RegulatorofGprotein(RGS)426/428TSTTTADSD(s)L(s)EYKDGIAGVR BC1G12682.1Phosducin66SN(pS)GGLGGLSEPLR BofuT4P084160.1BcPKA2PKAcatalyticsubunit211/212/213E(t)(y)(t)LCGTPEYLAPEVIQSK BC1G10590.1BcPKARPKAregulatorysubunit57,60,171/172/174SV(p)SPG(p)TANRPR,(t)(s)V(s)AESLNPTASSNDNWSPPFHQK BC1G14873.1BPK2Sch9-likeproteinkinase368,369,425,427,908, 913,914

TD(p)S(p)SMGGTVVPPK,GPQSEEDDEDDGN(p)SH(p)SPGGIPIGGIPIGGIPITR,GF(p)TFVDE(p)S(p)SIEENMKDR BC1G05432.1BPK3CLK1-likeproteinkinase571TGS(pS)PSTASAIAK BC1G13633.1RhoGAPprotein597,599(p)SK(p)SFGGPVDENPR BC1G09755.1RhoGAPprotein545,636SISGPLLQ(pS)PIDDMVPPNR,NT(pS)SEASTSAR BC1G09755.1RhoGAPprotein651,680SV(pS)ETTAR,DIS(pS)PISMSSPNPGDDPALLR BC1G06319.1RhoGAPprotein128,671,685LVEDIEEENKLAFQDPET(pS)PK,AT(pS)PAPSPR,DRS(pS)GPPAAETR BC1G15133.1RhoGAPprotein392,396/398SF(pS)HGP(s)L(s)QHIVAPVVSPTNPISTSPDFNTWSPR BC1G01690.1RhoGAPprotein145SS(pS)NASAGADSVR BC1G09394.1BcCDC24RhoGEFprotein753,852,853LGGDSYF(pS)PIGESPAPSAR,SY(p)S(p)TPDINAQQGVR BC1G07524.1RhoGEFprotein112,113,268,270Y(p)Y(p)SGNAPALASDPYR,(p)SI(p)SSTAGGSSR BofuT4P012000.1M3M1Histidinekinase—groupI1136AIE(pS)PTPNALVEK BC1G00374.1BOS1Histidinekinase—groupIII1290/1293/1295AYTTTGPINHG(s)AE(s)P(s)LVTADAEDPLAR BC1G07872.1BRRG1Responseregulator506/507/508NSMGSLGALGGQGG(s)(s)(s)K BC1G03001.1BcSAK1MAPK171,173IQDPQM(pT)G(pY)VSTR BC1G07144.1BMP3MAPK186,188GFSVDPEENAGYM(pT)E(pY)VATR BC1G13966.1BMP1MAPK183SAASQEDNSGFM(p)TEYVATR BC1G07633.1BOS5MAPKK399,597,601ICDFGVSGNLVA(pS)IAK,APLDTV(pS)PLP(pS)PRE BC1G03809.1BcSTE7MAPKK358,365,377,381/383(pS)HLAPQL(pS)PASR,GDD(p)SPNA(s)Q(t)PTSGDIPIK BC1G11713.1BcMKK1MAPKK106,167SG(pS)FGPMDGK,DG(pS)MNGLEAFDK BC1G04606.1BOS4MAPKKK1138,1149TLVTATTA(pS)IANK,SMTG(pT)PMYMSPEVIK BC1G06557.1BcSTE11MAPKKK108YGIM(pS)PAEQMK BC1G07478.1BcPKC1ProteinkinaseC675,686,687,688,690, 691,719

AESYGAPSSSEATQAAQAMYQQSTT(pS)PQQRPAGPDR,(p)T(p)S(p)TS(p)S(p)SATAAAAAATAAMGGR,A(p)STDYTNQVGAQR BC1G00093.1BcCRZ1CN-regulatedTF403AV(pS)DPYNSPR BC1G07228.1BcCMK1Ca2+/CaM-dependentkinase90,116/117L(pT)QDPHSDLVLADFGIAK,DEVL(t)(t)MAGSFGYAAPEVMLK BC1G07228.1BcCMK1Ca2+/CaM-dependentkinase288,290,294,298,282N(p)TG(p)SSSA(p)SGST(p)SPGEK,MQEDDGEESDVPGNAAAAASDAIPG(pS)PERK BC1G06577.1BcCMK3Ca2+/CaM-dependentkinase144SA(pS)DIHINEQR BC1G13713.1Ca2+/H+antiporter37,38,39(p)S(p)S(p)TFPAQVPDEEALR BC1G01673.1Ca2+/H+antiporter566TG(pS)FTNYVMTK BofuT4P084940.1Proteinphosphatase2C335,337,339GPGVHHNFDD(pS)D(p)SG(pY)DVDMDQK BC1G01244.1ProteinphosphatasePP-Z35,84GSDNLSSYP(pS)FSK,NSIAGIGSLVDSNDKSDVA(pS)LK BC1G14507.1AC-associatedprotein222SD(pS)QSSINSNR BC1G00082.1Calreticulin/calnexin570ASEAVA(pS)GTDAVK BofuT4P154620.1cNMP-bindingprotein270/271EATIHA(s)(s)VR BC1G10054.1BcGBL1G␤-likeprotein288,292,297EPECV(pS)LAW(pS)ADGQ(pT)LFAGYTDNIIR BC1G06083.1Myosin1571MEYTQDDIAEQ(pS)DNEEAK BC1G10821.1Myosin1145/1146(s)(t)PTPSLAGGLAEALR BC1G05708.1Penta-EF-handprotein71,91RL(pS)PQMHPPGNYGASPPGR,GYA(pS)PPPGQFQAGR BC1G13037.1Proteinkinase44,313,430(pS)DPNVMAHQAHQVPGGDAYSVANEDNK,IVWDTQTM(pT)PCGTVGYTAPEIVKDER,GSEEPTYAALDAPPLA(pT)PAVER BC1G13037.1Proteinkinase453,517,537AFNPDLLE(pS)PGAGR,GTAALNPLNPIDDEDFDE(pS)DEENYDENGVPSK,SGQ(pS)DVAQVGDALR BC1G13227.1Proteinkinase64,175,255RP(pS)GNGASTNTPPQPQYTSPLVTTTTTAPVNSR,RAQ(pS)QENQYHER,SHPAGTTSPAGLSHEA(pS)EVLNR BC1G13227.1Proteinkinase175RAQ(pS)QENQYHER BC1G13227.1Proteinkinase255SHPAGTTSPAGLSHEA(pS)EVLNR BC1G13227.1Proteinkinase510/511,733RGDLMQ(t)(s)CGSPCYAAPELVVSDSLYTGR,AG(pS)QGPVEISGAPQGSR BC1G00346.1Proteinkinase97DSQALS(pS)PVR BC1G15362.1RasGEFprotein701/702(t)(s)QPDFSAPAFLAVEDYEK BC1G04274.1Rho-likeGTPase97,101,102/104SNQQNTSSS(pT)PPG(pT)(s)G(s)QGVVIPK BC1G08670.1RhoGDIprotein37MDAEDE(pS)LQR BC1G07505.1BcSTE5Scaffoldprotein12,341AFESGTAYPE(pS)DADDEYER,SDQQP(pT)PGR BC1G12009.1BcSNF1Snf1-likeproteinkinase209,216/217IADFGLSNIM(pT)DGNFLK(t)(s)CGSPNYAAPEVINGK (pS):phosphorylatedsite,(p)S:peptidefoundmonophosphorylatedatdifferentsites,(s),(y),or(t)undistinguishablesite. Theannotationsareaccordingtothegenomedata[1]andnamesofgenesindicatedifknown.

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Figure 1. GO categories (A, biological processes; B, molecular functions) enriched in the B. cinerea andA. brassicicolaphosphoproteomic datasets according to a GOEAST analysis [19] performed on the Ab and Bc datasets (phosphoproteomes vs.

genomic data). The y-scale indicates the percentage of phosphorylated proteins among all annotated proteins in the corresponding GO category. The GO annotation levels are indicated between the parentheses.

this limited dataset, we found some interesting features, such as previously unknown phosphorylation sites in HKs.

Additional growth conditions will now be tested to identify (1) additional phosphoproteins and phosphosites in order to complete the Bc and Ab phosphoproteome datasets, and (2) to compare phosphoproteomic profiles between growth conditions using dimethylformamide labeling and quantitative phosphoproteome comparisons. In particular, this type of approach may be applied to study the impact of external stress conditions on complex protein phosphorylation profiles.

The MS proteomics data in this paper have been deposited in the ProteomeXchange Consortium (http://proteomecentral.

proteomexchange.org) via the PRIDE partner repository [x]:

dataset identifier PXD000817. This work was supported by INRA SPE and INRA AIP Bioressources 2011 (NecroPhos) projects co- ordinated by S.F.

The authors have declared no conflict of interest.

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