Two families of extracellular phospholipase C genes are present in aspergilli

mycological research 110 (2006) 1140–1151

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/mycres

Two families of extracellular phospholipase C genes are present in aspergilli Danny TUCKWELL*, Sandra E. LAVENS, Mike BIRCH F2G Ltd, Lankro Way, Eccles, Manchester M30 0BH, UK

article info

abstract

Article history:

Fungi secrete extracellular enzymes to enable them to harvest nutrients from the environ-

Received 16 May 2006

ment. In the case of pathogenic fungi these enzymes can also be pathogenesis factors. Here

Received in revised form

we report the identification in fungi of a complex family of extracellular phospholipase C

5 July 2006

(PLC) enzymes, homologous to the Pseudomonas aeruginosa PLCH_PSEAE. Database searches

Accepted 28 July 2006

and phylogenetic analysis showed that the PLCs clustered into two groups with different

Published online 2 October 2006

evolutionary histories. One group, subdivided into PLC-A, -B, -C and -D, was found only

Corresponding Editor:

in aspergilli and Neosartorya fischeri. Each species only ever showed three of the four

Paul Hooley

PLCs except N. fischeri which had all four PLCs plus duplicate PLC-A, -B and -C genes. Modelling studies indicated that these PLCs had mechanistic similarities to phosphoesterases

Keywords:

and aryl sulphatases, but that they probably did not differ in substrate specificity. The sec-

Arabidopsis

ond group, PLC-E, was seen in a wider range of fungi including some species of aspergilli

Aspergillus

and was always found in a head-to-head arrangement with a copper oxidase, similar to

Gene transfer

the laccases. The PLC genes appear to have arisen from separate gene transfer events

Neosartorya

from bacteria or lower eukaryotes. Thus, aspergilli have acquired PLCs twice in the course

PLC enzymes

of evolution. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction Fungi of the genus Aspergillus are recognised as being of medical, industrial, environmental, and scientific importance (Latge 2001; Yabe & Nakajima 2004; Ward et al. 2006): A. fumigatus is an important human pathogen, with other Aspergillus species, including A. clavatus and A. flavus, also being of medical significance. In addition, certain strains of A. flavus and A. parasiticus are responsible for the production of aflatoxins. A. oryzae is used in the production of traditional Japanese fermented foods and drinks, such as soy sauce and sake, industrial production of citric acid exploits A. niger, and Aspergillus enzymes are also employed in baking. Most Aspergillus species are saprophytic, and A. fumigatus plays an important role in

the degradation and recycling of organic material in soil (Samson 1994). Finally, A. nidulans has long been used as a model organism in genetic studies. Aspergilli are known to secrete enzymes into their environment. Secreted proteases (Monod et al. 2002) and phospholipase B proteins (Shen et al. 2004) have been identified along with a wide range of other putative secreted enzymes including carbohydrate hydrolases (Robson et al. 2005). These enzymes presumably enable the fungi to control and harvest nutrients from their environment, although in the case of pathogenic fungi, the secreted enzymes could play an important role in the pathogenesis process. Phospholipase C (PLC; EC. 3.1.4.3) enzymes hydrolyse the phosphodiester bond between the phosphate oxygen and

* Corresponding author. E-mail address: [email protected]. 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.07.013

Two families of extracellular phospholipase C genes are present in aspergilli

the glycerol carbon of phospholipids, liberating a diacylglycerol molecule and head group–phosphate complex. PLCs have been recognised as an important pathogenicity factor for many bacteria including Clostridium perfringens, Listeria monocytogenes and Pseudomonas aeruginosa (Titball 1993; Songer 1997) and PLC activity has been reported in mycelial extracts or supernatants of various species of aspergilli including A. fumigatus (Matsuoka et al. 1987; Birch et al. 1996; Birch et al. 2004). Stonehouse et al. (2002) reported a class of PLC proteins typified by the P. aeruginosa PLCH (PLCH_PSEA), identifying homologues in a range of bacteria but also in A. fumigatus and Arabidopsis thaliana. Here we describe the extensive characterisation of this PLC family in fungi, showing that two groups are present and that aspergilli may have acquired these PLCs twice.

Materials and methods Sequence searches Aspergillus clavatus, A. flavus, A. nidulans, A. terreus, Coccidioides immitis, Fusarium graminearum, Neosatorya fischeri and Trichoderma reesei sequence searches were carried out at the NCBI fungal genomes blast website (http://www.ncbi.nlm.nih.gov/ sutils/genom_table.cgi?organism¼fungi) and contigs downloaded from NCBI. A. fumigatus sequence searches were carried out on a locally held copy of the A. fumigatus genome downloaded from CADRE (http://www.cadre.man.ac.uk/; Mabey et al. 2004) or at the at the NCBI fungal genomes blast website as above. Sequences of all other species were searched at the NCBI blast site and protein sequences downloaded from NCBI. Multiple alignments were generated using ClustalW (Higgins et al. 1994) and manually aligned using Align (D. Hepperle, 2001: Multicolor Sequence Alignment Editor, Institute of Freshwater Ecology and Inland Fisheries, D-16775 Stechlin, Germany). The shading of the alignment to show conservation was carried out using Boxshade (http://bioweb.pasteur.fr/ seqanal/interfaces/boxshade.html).

Phylogenetic analysis Phylogenetic analysis was carried out using Treepuzzle 5.2 (Schmidt et al. 2002), MrBayes (Huelsenbeck et al. 2001; Ronquist and Huelsenbeck 2003) and PHYLIP (Felsenstein 2002), using the more conserved and unambiguously aligned regions of the PLC alignment. ML analysis was carried out using Treepuzzle with the JTT model and 25K puzzling steps. Bayesian inference of phylogeny was carried out using MrBayes with a gamma rates setting and a fixed (blosum) amino acid model. Thirty-five thousand generations were run with a sample frequency of 10 and a temperature value of 0.05. The first 10K generations were discarded before analysis. Pairwise distances were calculated using PROTDIST with the JTT model and trees inferred using FITCH with 10 jumbles of input order and global rearrangements. One hundred BS replicates were generated using SEQBOOT, distances were calculated using PROTDIST and trees inferred using NJ and BS values calculated using CONSENSE.

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Gene analysis Codon usage and base composition of Aspergillus fumigatus and A. oryzae PLC genes were analysed using GCUA (McInerney 1998). Correspondence analysis of raw codon scores, correlation analysis of RSCUs and usage of G and C at the third base were carried out and data plotted to identify differences between the PLCs and a reference set of secreted proteins from the appropriate species. The genes flanking the PLCs in different fungi were compared using ACT (Artemis Comparison Tool; Carver et al. 2005; http://www.sanger.ac.uk/Software/ACT/).

Homology modelling Tertiary structure predictions for PLC-A were carried out using Meta (http://bioinfo.pl/Meta/; Ginalski et al. 2003) and Superfamily (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/; Gough et al. 2001). Homology modelling of PLC-A was carried out using Swiss PDB viewer (spdbv) and Swiss model (http:// www.expasy.org/spdbv/; http://www.expasy.org/swissmod/; Guex 1996; Guex & Peitsch 1997) using the human arylsulphatase structure 1E2S as the template (though other templates were investigated) and using the ‘Optimise mode’ approach described at http://www.expasy.org/spdbv/text/modeling. htm. Multiple alignments of PLCs and arylsulphatases were used to map the target to template and a secondary structure prediction for PLC-A, carried out using the Jpred server (http:// www.compbio.dundee.ac.uk/wwww-jpred/submit.html; Cuff et al. 1998) was used to further improve the alignment. A number of different alignments were tested in the loop regions. 1E2S has a C-terminal b-sheet domain that does not match the PLCs and so this domain of the template was not used in modelling.

Results Identification of two classes of PLC genes PLC sequences were identified by exhaustive searching of protein and genome databases, initially with Pseudomonas aeruginosa phospholipase PLC-H as the query (PLCH_PSAEA; Stonehouse et al. 2002), and then with the PLC genes identified from the first searches. Predictions of genes from genomes were optimised by alignment of the resulting proteins. A number of fungal PLCs were identified, chiefly in aspergilli and the Aspergillus anamorph Neosartorya fischeri but also in Coccidioides immitis, Fusarium graminearum and Trichoderma reesei. Only partial sequences could be obtained for N. fischeri sequences Nfis4-7. Significantly, no homologues could be identified in Candida albicans, Cryptococcus neoformans, Neurospora crassa, Saccharomyces cerevisiae, or Ustilago maydis. The fungal PLCs had N-terminal hydrophobic leader sequences, suggesting that the proteins are secreted (Table 1; a sample alignment is shown in Fig 1). PLC homologues were identified in plants, the alvaeolate Tetrahymena thermophila and bacteria, but there were no homologous mammalian PLCs. Phylogenetic analyses were carried out to identify the relationships between the PLCs (Fig 2). ML (TreePuzzle) and

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Table 1 – Sequences used in this study Code AclaA AclaB AclaC AflaA AflaC AflaD AflaE AfumA AfumB AfumC AnidA AnidB AnidC AoryA AoryC AoryD AoryE AterA AterB AterE Atha1 Atha2 Atha3 Atha4 Atha5 Atha6 BpseN Ccre CintE FgraE Iloi MtubA MtubB MtubC MtubD Nfis1 Nfis2 Nfis3 Nfis4 Nfis5 Nfis6 Nfis7 Osat1 Osat2 Osat3 Osat4 Osat5 Osat6 PaerH PaerN Ppat1 Ppat2 Scoe TreE Trur1 Tthe1 Tthe2 Tthe3 Tthe4 Tthe5 Tthe6

Species

Accession code

Aspergillus clavatus A. clavatus A. clavatus A. flavus A. flavus A. flavus A. flavus A. fumigatus A. fumigatus A. fumigatus A. nidulans A. nidulans A. nidulans A. oryzae A. oryzae A. oryzae A. oryzae A. terreus A. terreus A. terreus Arabidopsis thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana Burkholderia pseudomallei Caulobacter crescentus Coccidioides immitis Fusarium graminearum Idiomarina loihiensis Mycobacterium tuberculosis M. tuberculosis M. tuberculosis M. tuberculosis Neosartorya fischeri N. fischeri N. fischeri N. fischeri N. fischeri N. fischeri N. fischeri Oryza sativa O. sativa O. sativa O. sativa O. sativa O. sativa Pseudomonas aeruginosa P. aeruginosa Physcomitrella pateus P. pateus Streptomyces coelicolor Trichoderma reesei Tortula ruralis Tetrahymena thermophila T. thermophila T. thermophila T. thermophila T. thermophila T. thermophila

DQ539344 DQ539343 DQ539345 DQ539348 DQ539349 DQ539347 DQ539346 AAX36075 AAX47073 AAX47074 AN8546.2 AN7691.2 AN2336.2 BAE65551 DQ539350 BAE57699 BAE56070 DQ539351 DQ539353 DQ539352 T02648 BAC22510 BAC22509 E86207 BAC22508 BAC22511 PHLN_BURPS E87624 DQ539341 FG11236.1 YP_156976 PHLA_MYCTU PHLB_MYCTU PHLC_MYCTU PHLD_MYCTU DQ539354 DQ539355 DQ539356 DQ539357 DQ539358 DQ539359 DQ539360 BAB92134 AA066550 XP_549813 NP_908333 XP_463753 XP_470151 PLCH_PSEAE PHLN_PSEAE BJ954474.1 BJ966613 CAD55387 DQ539342 CN204437.1 EAR87010 EAR87012 EAR87011 EAR97886 EAR04836 EAR80609

a The source contig is given for those sequences predicted from contigs. b Only partial sequence was available as these proteins were predicted from short genomic fragments.

Sourcea AAKD02000013 AAKD02000001 AAKD02000011 AAIH01000072 AAIH01000729 AAIH01001658 AAIH01000090

AO090012000083

Contig 1.14 Contig 1.10 Contig 1.4

AEC01000008

AAKE02000011 AAKE02000002 AAKE02000021 AAKE02012936b AAKE02000476b AAKE02004001b AAKE02006182b

AAIL01001087

Two families of extracellular phospholipase C genes are present in aspergilli

1

AflaA AfumA AnidA Nfis1 Nfis7 AfumB AnidB Nfis2 Nfis4 AflaC AfumC AnidC Nfis5 Nfis3 AflaD Nfis6 AoryE CintE FgraE TreeE Atha5 Osat1 Osat3 Osat5 Tthe1 Ppat1 Ppat2 Trur1 MtubA PaerH PaerN

1143

20

40 60 ## ******************** --MSIISLLAFFSLA----------LGINGLPHARSSSPSYGKPVNPIKDTPIKNVVVLVEENLSFDVFAGGLTYNA----------KIDGLVNRE-----MTVLSLAVFFTLG--------------------FGAGAAPHVPHGRLSSPIKNVVVLVQENLSFDNYAGGLTYSP----------SIDGLVNRT------TRVVTLFILLTSSI-----TTVAVVIYLLVAHRLPAPTGPLHHHDSNTPIKNVVVLVQENLSFDTLAGGLNYSR----------DIDNIVNLPPNKR --MTVLSLAVFFTLG--------------------FGAGAAPHVPHSRLSSPIKNVVVLVQENLSFDNYAGGLTYNP----------NIDGLVNRE-----MSIISLLAFFSLA----------LGINGLPHARSSSPSYGKPVNPIKDTPIKNVVVLVEENLSFDVFAGGLTYNA----------KIDGLVNRE-----MHPSALLGLLAFT------------VAASAIPAPPASAHSKHSINNFKSKIKNVVILVMENRSFDNLLGGQKLKG-----------LENPVNTG---P --MHPSALLGLLSFA-------------AAALAVPAGQAPHTARSISNLKSHIKNVVILVMENRSFDNILGGQTTPG-----------LENPLQSG---P --MHPSALLGLLAFV------------VAASAIPAQPANAHSNQSINNLKSKIKNVVILVMENRSFDNLLGGQKLKG-----------LENPVNTG---P -----------------------------------------------------------------------------------------------------MKSTALLTGLGLLASLGLASPVTSEYTSVREAPFGYKPGSKESIENLKDKVENIVWLILENRSFDNILGGVRRQG-----------LDNPINNG---P --MKANTLVPLLGLSSS-----AAAAAVAASGNTPFGYASGSKESIANLKDKIENVVWILLENRSFDNILGGVKRKG-----------LDNVVNNG---P --MKAHTLVPILSLASA-----TAAAAVPPSSKTPFGYASGSKESIANLKDKIENVVWILLENRSFDNILGGVKRKG-----------LDNVVNNG---P -----------------------------------------------------------------------------------------------------MKANTLVPLLGLSSS-----AAAAAVAASSNTPFGYASGSKESIANLKDKIENVVWILLENRSFDNILGGVKRKG-----------LDNVVNNG---P ----------------------------------------------------IKHVVYLMMENHSFSNIAGYWDFHP----------EIDNLRNRK-----MQWTWALITALATAT-----------------SALPHEARKDVWGQLRSNIKHVVYLMMENHSFSNIAGYWDFHP----------EIDNLRNRK--------------------------------MLLGSRPARAGSL--------KDIDHVVIFMQENRSWNNYFGTMAGVRG------------------------------------MKLFIALAIALSLFLTDPGEAEEDTDGREEAARSLKDVDHVIIFMQENRSWNTYFGTMAGTRG---------------------------------------------MLLSRGVGLAIAAFLAAINPAQPASLADIDHVILFMQENRAFDHYFGTMAGVRG---------------------------------------------MPRFVTLAPASATLMAVIGLASAGSLKDIDHVVLFMQENRAFDHYFGTMAGVRG------------------------------------MAFRRVLTTVILFCYLLISSQSIEFKNSQKPHKIQGPIKTIVVVVMENRSFDHILGWLKST---------RPEIDGLTGKE--------------------------------MGRRLLLLFLMLAQAPNSNGDSKIKNVVVLALENRSFDHMLGWMQRLL--------GLPIDGLTGAE--------------------------------MGRRLLLLFLMLAQAPNSNGDSKIKNVVVLALENRSFDHMLGWMQRLL--------GLPIDGLTGAE--------------------------MAVRRRRPGPVAAAVLLLLAVATQAAASPIKTVVVVVMENRSFDHMLGWMKRL---------NPEIDGVTGGE---------------------------------MQKLSALILLIFAVSLTAKESPIKHIVVLMMENRSFDHMLGWMTKG-----GQYGNPNVNGLTGNE------------------------------------------------------------------------------------------------------------------------MAFRARKFFLPFLLLHLLVWPVRPVHPAHTISGPVKTIVVLVMENRSFDHMLGYLNRR---------NPNIDGLSGAE----------------------------MTKTKWCGCWGAGGKETPKAGPVFATPIKTVVILCMENRSFDHMLGFMKRV---------NPEIDGLTGEE---MSASPLLGMSRREFLTKLTGAGAAAFLMDWAAPVIEKAYGAGP--CPGHLTDIEHIVLLMQENRSFDHYFGTLSSTNGFNA-ASPAFQQMGWNPMT---Q --MTENWKFRRRTFLKHGAQAATLAGLSGLFPETLRRALAVEPDIRTGTIQDVQHVVILMQENRSFDHYFGHLNGVRGFND-PRALKRQDGKPVWY---Q -----MISKSRRSFIRLAAGTVGATVATSMLPSSIQAALAIPAHRRHGNLKDVEHVVILMQENRSFDHYFGTLKGVRGFGD-RMAIPLPDGQRVWH---Q

AflaA AfumA AnidA Nfis1 Nfis7 AfumB AnidB Nfis2 Nfis4 AflaC AfumC AnidC Nfis5 Nfis3 AflaD Nfis6 AoryE CintE FgraE TreeE Atha5 Osat1 Osat3 Osat5 Tthe1 Ppat1 Ppat2 Trur1 MtubA PaerH PaerN

65

80 100 120 140 # YCNPSNASD----PFSEKVCAKPIAKNVAPD-DPDHSITGGNQQVYSTYHPNAK-----NDMPG----------MQGFVTEQIVSYGLGSDLSRAAE--YCNPANISD----PHSPMVCAQPTAKNIAPD-DPDHSITGGNQQVYSTYHPDSD-----FDAPN----------MQGFVAEQIAAYGIDGNLSRAGE--FCNPSNVAD----PSSPLICAKPLAKNVAPD-DPDHSIAGGNFQVYGTDHPDLS-----IHKPT----------MQGFVSEQIRSHGINGDLKRAAE--YCNPANVSD----PYSPMVCAKPTAKNIAPD-DPDHSITGGNQQVYSTYHPDYD-----VHAPN----------MQGFVAEQIASYGIEGNLSRAGE--YCNPSNASD----PFSEKVCAK-----------------------------------------------------------------------------YCNPYNLTD----PSQGVACSAAKDFDSITN-DPDHAIYGNNIEFYGTFNPDNTQIAEGKLVPH----------NQGFVHEQVRKYGAKVNKTVLATQ-FCNPFNLTD----PSEGEACTAPKSFDSVIN-DPDHAIYGNNIQFYGEFVPDNAAIASGELKAT----------NKGFAHEQMRLYEDVANKTDLVTE-YCNPYNLTD----PSQGVACSAAKDFDSITN-DPDHAIYGNNIEFYGTFNPDNTQIAEGKLVPH----------NQGFVHEQVRKYGAKVNKTILAAQ----------------------------------------------------------------------------------------------------FCNYKNASD----PSSGKYCTQAKDYDSVFN-DPDHSVTGNNLEFYGTYTPNNGAIASGKVVAD----------QSGFLNAQLNDYPKLAPEEATRQ--FCNPQNVSD----PSSTKWCSVYKDFDSVKH-DPDHSVTGYNMELYGTYTPSNDAIANGTLKPT----------MNGFVEQQLVHHKGLDPKVADEE--FCYPQNVSE----PSSTQRCSVYKDFDSVKH-DPDHSITGYNMELYGTYHPSDEAIRNGTLKPS----------MNGFVQQQLVHHKGMDPKVATEE-----------------------------------------------------------------------------------------------------FCNPQNVSD----SSSTKWCSVYKDFDSVKH-DPDHSVTGYNMELYGTYTPSNDAIADGTLKPS----------MNGFVEQQLVHHKGLDPKVADEE--YCNEYTNPNWTVWGEPLDICAGPYETEVPLE-DPDHEFAGVTYQIYRKWNVTND------DVPN----------MGGFIERQSEKY--QATPGESAF--YCNEYTNPNWTVWGEPLDICAGPYETEVPLE-DPDHEFAGVTYQIYRKWNVTND------DVPN----------MGGFIERQSEKY--QATPGESAF--FNDPNVQV---------NDDGLSVWHQKV---DPSMSENATTLLPWYLGYKG-GDWNDAIQCMVAG--------SNGYEDNQASLNHDLNNNWARNNTPW FQDPNVQV---------NPDGLPVWFQKV---DSDQSTDTKTLLPWHLGYLG-GNWSEAIQCMAAG--------SNGYRANQAALNYGLNNQWVTDNTPW FADPNVQV---------N-DGVPVWKQKV---TAKQSQDTDYITPWYLNYLG-GTWPEATQCMGAG--------SNGWDANQAALNHGANDRWAMNNTPY FADANLQM---------N-DGVPVWKQVT---NSQLTNETDYVTPFYINYLG-GNWTEATQCMVSG--------SNSWQENHAAWNSGRNDRWAVDNSPY -SNPLNVSD----PNSKKIFVSD-DAVFVDM-DPGHSFQAIREQIFG---SNDTSGDPK---------------MNGFAQQSESMEP-------G-MAKN -CNPAPGPG----PADSLLHCVSPDADLVVPDDPAHAFEDVLEQLLGFRPNDSTGAAASPSD------------MSGFVRSAVSVS--------ALLTDA -CNPAPGPG----PADSLLHCVSPDADLVVPDDPAHAFEDVLEQLLGFRPNDSTGAAASPSD------------MSGFVRSAVSVS--------ALLTDA -WNPTNASD----PSSGRVYFGE-GAEYVDP-DPGHSFQEIRQQIFG---SDDASG-PAR--------------MDGFVQQARSLGD-------N-MTAA -CNTALNGT--------QICVAPNAQDCSAY-DPGHGPQTTTERVYNCVYQAQ--NSTSDDPCVNHASLKVPANMQGFVAAAQRGGQ---------DGLT ----------------------------------------------------------------------------------------------------FNVLNDGQ----TGRRTLYVSD-TAEFVDP-DPGHSYQAIEEQVFGPER---KFADPPP--------------MSGFATNAESKLPG--------MS-N -SNPLSLTD----ASAGVATVSD-QAPYIDPKDPGHSYQAIALQIFGSLT--DTSANPPP--------------MSGFAAQAEIEEPG--------LSAT ALDPAGVT-----IPFRLDTTRGPFLDGECVNDPEHQWVGMHLAWNGGANDNWLPAQAT-------------------------------TRAGP-YVPL NYKYEFS-------PYHWDTK---VTSAQWVSSQNHEWSAFHAIWNQGRNDKWMAVQY----------------------------------------PE KGSKGEI------LPYHFDTS---TTSAQRVDGTPHTWPDAQQAWNEGRMDKWLPA----------------------------------------KTER

AflaA AfumA AnidA Nfis1 Nfis7 AfumB AnidB Nfis2 Nfis4 AflaC AfumC AnidC Nfis5 Nfis3 AflaD Nfis6 AoryE CintE FgraE TreeE Atha5 Osat1 Osat3 Osat5 Tthe1 Ppat1 Ppat2 Trur1 MtubA PaerH PaerN

142 160 180 200 ******************************** ****************** *************** VINYYTPDHVPVFNAMAENFVLFDRWFAAVPG------PTNPNRAYLTSGTSHGHGQND--------------------HDFDISNLPQVSIFEQLSAAG VINYYTPEHIPVFNAMAENFVLFDRWFAAVPG------PTNPNRAYLTSGTSHGHGTND--------------------PDFDRSALPQRSIFEQLSEHN VINYYAPEHIPVFNALAENYLLLDRWFASVPG------PTNPNRAYLTSGTSHGHGWND--------------------PSFDHSSLPQVSIFQQLTEAN VINYYTPEHVPVFNAMAENFVLFDRWFAAVPG------PTNPNRAYLTSGTSHGHGTND--------------------PDFDRSALPQRSIFEQLSENN ---------------------------------------------------------------------------------------------------VMNYYTEEQVPVLTSLVQNYLTFNHWHSDIPG------NTNPNRAALVSGTSLGHGLND--------------------AAFTKHEFPQRSIFQQLTETG VMHYYTEEQVPVLTALVKNYLTFNHWHSDIPG------NTNPNRAALVSGSSHGHGLND--------------------DGFTNHELPNRSIFQQLTETG VMNYYTEEQVPVLTSLVQNYLTFNHWHSDIPG------NTNPNRAALVSGTSLGHGLND--------------------AAFSKHQFPQRSIFQQLTETG -----------------------------LPK------PTNPNRAALVSGTSYGHGTND--------------------AAFGLHQFPQRSIFQQLTETN VMGYYTEEEVPTLVDLVDEFTTFNSWFSCVPG------PTNPNRLCALAGTAAGHGKND--------------------DDFLNYGISSKSIFEAANEKG VMGYYSEDEIPTLVNLVDEFTTFNYWHSCVPG------PTNPNRLCALSGTSDGHGTND--------------------NSFDVSGVDISSIFQVASEKD VMGYYSEDEIPTLVNLVDDFTTFNYWHSCIPGFTNVHQPTNPNRLCAVSGTSDGHGEND--------------------NSFDVSGVEISSIFQVASENG ---------------------------------------------------------------------------------------------------VMGYYSEDEIPTLVNLVDEFTTFNYWHSCIPG------PTNPNRLCALSGTSDGHGTND--------------------NSFDVSGVDISSIFQVATEKE VIKAYDEKKSSTLAEIAQNFAFWDSYFAEHPG------PTNPNRQFATSGSTCGFVDNA--------------GQAAGFFNNVTGTTCATSIFEALSNKN VIKAYDEKKSSTLAEIAQNFAFWDSYFAEHPG------PTNPNRQFATSGSTCGFVDNA--------------GQAAGFFNNVTGTTCATSIFEALSNKN SWGYLKRNDIPVQFAIAEGWTAGDMYQESQIT------STNPNRVTLVSGSVNIPGSPQASDQGG-PYIDNNETPGCDTDNINCYPLKWKTIFEIYEEAG SWGYLKRDDIPVQFALAEEWTVADMYQQSQIT------STNPNRVMLVSGSINAPGSPQSPDEGG-VYLDNTETPGCEKPNVNCYPLKWKTIFELYEDAG SMGYYKRQDLPVHFALAEEWTVGDMYQESVIA------STNPNRVMWISGSINVPGSPQTKDEGGYPYIDNNETPGCDKNGINCYPLKWTTAAEKYEAAG SIGFYKRQDIPIQFALADSFVVADMYQEAIVA------ATNPNRVTWLSGSVNVPGGPQTPDQGGNPYIDNNITPGCEPGGFNCYPLKWKTVGEHYEDAG VMSGFKPEVLPVYTELANEFGVFDRWFASVPT------STQPNRFYVHSATSHGCSSNV--------------------KKDLVKGFPQKTIFDSLDENG VMRGFTPSRLPAFSALASSFAVFDRWFSSIPG------PTQPNRLFLYSATSHGAVAHD--------------------KWNLLRGYPQRTIFDSLAADA VMRGFTPSRLPAFSALASSFAVFDRWFSSIPG------PTQPNRLFLYSATSHGAVAHD--------------------KWNLLRGYPQRTIFDSLAADA VMNGFSPDSVAVYRELVGEFAVFDRWFASVPS------STQPNRLFVHSATSGGATSNN--------------------PELLAKGYPQRTIFDNVHDAG EMSGFLPEEVPIITTLANEFALFDNYFVSYPG------CTNPNRMFVHMGTCDGCVQNS----------------------QAIGQIKNTTLQEVLEKNG ---------------------------------------------------------------------------------------------------VMRAFRPEVVPVTTALAMEFAVF----------------------------------------------------------------------------IMSGFAPEHVPVYKALVEEFAVADRWFASVPS------STQTNRQYLHSATSHGLMANN--------------------QQMLVAGLPQKTLFEAVEEGG TMGYYTRQDIPIHYLLADTFTICDGYHCSLLT------GTLPNRLYWLSANIDPAGTDGGPQLVE-------------PGFLPLQQFSWRIMPENLEDAG AMGYFKRGDIPYYYALADAFTLCEAYHQSMMG------PTNPNRLYHMSGRAAPSGDGKDVHIGNDMGDG---------TIGASGTVDWTTYPERLSAAG SLGYYKEQDIAFQFAMANAFTICDAYHCSFQG------GTNPNRLFLWTGTNDPLGQHGGPVTTNDHDS----------NGPVEQGYTWTTYPERLQAAG

Fig 1 – Alignment of representative PLCs. Positions are shaded where they are at least 50% identical (black) or 50% similar (grey). Numbers correspond to AfumA residues. The stars indicate the regions used for phylogenetic analysis. Hashes indicate residues implicated in catalytic function. Abbreviations are as given in Table 1.

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AflaA AfumA AnidA Nfis1 Nfis7 AfumB AnidB Nfis2 Nfis4 AflaC AfumC AnidC Nfis5 Nfis3 AflaD Nfis6 AoryE CintE FgraE TreeE Atha5 Osat1 Osat3 Osat5 Tthe1 Ppat1 Ppat2 Trur1 MtubA PaerH PaerN

216 240 260 ******* ISWINYSNTT-------------------------GFLPDSLFYQWTAKSGKGTTN------------VKSIDQFFNDAKAGTLPQFTWINPECCSYM-ISWINYSNTT-------------------------GFLPDALFYRWTAVSGLGETN------------VRPIDQFYRDAKAGTLPQFTWINPECCSYM-ISWINYSNAT-------------------------GFAPDALFYTWTAKSEAGRNS------------IKSIDQFYSDAAAGNLPQFTWINPECCSYT-ISWINYSNTT-------------------------GFLPDALFYRWTAMSGLGETN------------VRPIDQFYRDAKAGTLPQFTWINPECCSYM----------------------------------------------------------------------------------------------------HSWINYVDPA------------------------GGTGPDAGYFNWTYATNNQDK-------------IQPLAKFYTDAAAGALPELAYLNPSCCGVGTT HSWLNYVDPD------------------------GGTGPDAGFYDWTYATGNDDK-------------IVPLAQFYTDAAAGNLTEFTYINPSCCGVGTT HSWINYVDPA------------------------GGTGPDAGFFNWTYATNNQDK-------------IQPLAKFYTDAAAGALPELAYINPSCCGVGTT HSWINYVDPA------------------------GGTGPDAGFFNWTWATKNNDK-------------IQPLSKFYTDAAAGALPELAYLNPSCCGVGTT VSWLNYDGTN------------------------GEFEPDSLFFTYVNQTSRSN--------------VVPVENFFQDAYLGVLPKFSYINPSCCGTNTN ITWKNYDGTN------------------------GAFLSDALFFNWTAQNAKHN--------------VVPLENFYQDAYLGLLPQLSYINPSCCDLDTN ISWKNYDGTN------------------------GDFLPDALFFNWTSENAQSS--------------VVPLENFYQDAYLGLLPQLSYINPSCCDLDTN -------------------------------------------------------------------------------------------PSCCGLDTN ITWKNYDGTN------------------------GAFLSDALFFNWTAQNAKQN--------------VVPLENFYQDAYLGLLPQLSYINPSCCDLDTN ISWKNVNRST----------------IFGSPIANAGSVLRNGYYRWMDVQDNAIDN------------LAHASDFYRDLEEGTLPTFSYINPECCTID-ISWKNVNRST----------------IFGSPIANAGSVLRNGYYRWMDVQDNAIDN------------LAHASDFYRDLEEGTLPTFSYINP-------VSWQVYQE-----------------------KNNFDDNPLAWFQQYQNASASSPLA-------KKGLSYLGLDAFYKAAANGSLPEVSFIVGPAEL---VSWQVYQG-----------------------EDNFDDNPLAWFEQYQNARPGTPLA-------DKGMAFLGLDKFYKDAAKGTLPQISFIVGPREL---VSWSVFQD-----------------------ADNFDDNPYAWFEQFQDSKSGSKLN-------EKGMKGQSLDAFFAQAASGTLPEVSYIVGPMQL---VTWQVFQD-----------------------ADNFDDNSYARFQQFQDAEPGSSLY-------ERGMKGLSLDTFYAKAANGTLPEVSYIVAPMEL---LSFGIYYQN----------------------------IPATFFFKSLRRLKHLVK------------FHSYALKFKLDAKLGKLPNYSVVEQRYFDIDLF LDYRVYFKT----------------------------IPTTLFYRRLRTVANAARG----------TFRRYDAAFRDHARRGLLPALSVIEPRYFDLTGT LDYRVYFKT----------------------------IPTTLFYRRLRTVANAARG----------TFRRYDAAFRDHARRGLLPALSVIEPRYFDLTGT LSFGVYYQD----------------------------VPAVLFYRNLRKLKYLTK------------FHPFHGAFRDHAARGSLPNYAVVEQHYMDSKSH LSWRYYYED--------------------------QPIDWFLYIEYFNKNFLNRK------------KFSNMEQFYKDAQDGTLANYTFINPSETVRPFL ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------LSFGIY---------------------------------------------------------------------------------------------VSWKVYQN----------------------KGLGRFINTPISNNGLVQAFRQ--AADPRSNLARYGIAPTYPGDFAADVRANRLPKVSWLVPNILQ---VDWRVYQEGGYRSSSLWYLYVDAYWKYRLQEQNNYDCNALAWFRNFKN------APR-DSDLWQRAMLARGVDQLRKDVQENTLPQVSWIVAPYCY---ITWRVYQD----------------------MADNFSDNPLIGFRQYR-------AAAPDSPLIVNGLSTWKLDALKRDVLANSLPQVSWIVAPAKY---277

AflaA AfumA AnidA Nfis1 Nfis7 AfumB AnidB Nfis2 Nfis4 AflaC AfumC AnidC Nfis5 Nfis3 AflaD Nfis6 AoryE CintE FgraE TreeE Atha5 Osat1 Osat3 Osat5 Tthe1 Ppat1 Ppat2 Trur1 MtubA PaerH PaerN

300 320 340 # ## # **** ******************** ************************ ** ----------SFHPPS-PINMGEGFIKSIYEALRSSP-QWNETLFILTFDEHGGFADHVSPP-ENVPAGDNLT-----YTETAKDGQEATFHFD----RL ----------SFHPPS-PINMGEGWIKSVYEALRSSP-QWHETLFILTFDEHGGFADHVPPP-ENVPAGDDLT-----YTETARDGKPSTFAFD----RL ----------SFHPPS-PTNLGEGFVKSVYEALRAGP-QWNETLFILTFDEHGGFADHVPPP-EKVPPGDGIP-----YTEVAKDGKPTTFHFD----RL ----------SFHPPS-PINMGEGWIKSVYEALRSSP-QWHETLFILTFDEHGGFADHVPPP-ENVPAGDDLT-----YTETARDGKASTFAFD----RL -------------------------------------------------------------------------------------------------------------SMHPAG-LISDGEAFIKKVYDALRAGP-QWEETLFILTFDESGGFHDHVSAP--LAPRPDNLT-----FTLSTPSGEDYTFPFD----RL ----------SMHPSG-LISDGETFIKSVYDALRAGP-QWNNTLFVLTFDESGGFHDHVPAP--LAPRPDDLT-----FTLETPNGENYTFPFD----RL ----------SMHPAG-LISDGEAFIKKVYDALRAGP-QWGETLFILTFDESGGFHDHVPAP--LAPRPDNQT-----FTLSTPSGEDYTFPFD----RL ----------SMHPAG-LISDGEAFIKQVYDALRGSP-QWEQTLFILTFDESGGFHDHVPSP--LAPRPDNLT-----YTQTTPSGEPYTLPFD----RL ----------SMHPTG-NVSYGEVFVKQIYDAIRQGP-QWDKTLLFITYDETGGFYDHVPPP--LAVRPDNLT-----YTETAKNGQKYTLHFD----RL ----------SMHPSG-NVSFGQVLVKQIYDAVRTGP-QWDKTLLLVTFDETGGFYDHVEPP--LAVRPDSKT-----YTETAADGSSYTFNFD----RL ----------SMHPSG-NVSFGQVLVKQIYDAVRTGP-QWNKTLLLLTFDETGGFYDHVEPP--LAVRPDDKT-----YVETAADGTDYTFTFD----RL ----------SMHPSG-NVSFGQVLVKQIYDAVRTGP-QWDKTLLLITYDETGGFFDHVQPP--LAVRPDDKT-----YTETAPDGSSYTFNFD----RM ----------SMHPSG-NVSFGQVLVKQIYDAVRTGP-QWDKTLLLVTFDETGGFYDHVQPP--LAVRPDSKT-----YTETAADSSSYTFNFD----RL ----------SMHPKS-NMAAGEQMIKHLYDAVRRSK-YWDNVLIIINFDEHGGFADYVPPP-VNVPRPEDGI-----AFDGESEGRPVTYDFT----RL -------------------------------------------------------------------------------------------------------------SEHPPY-MPKDGAWLQKKVVDAVTKSP-KYSSTLLIISYDETGGFGDHVVPFHSPEDTPGDWM----------TDPYGKFGKI---YVGP ----------SEHAPY-SPKDGAWLQEKVVEAVTKSP-KYGKSVLMISFDESGGWGDHVPPYHSPTDTPGEWI----------DDYLGVFGRI---FTGP ----------SEHPPY-SPHDGSWLQRKVAEAVINSP-KYSKSALIISYDETGGWADHVTPYHAPDGTAGEWI----------DDPYGEVGRT---PTGP ----------SEHPPY-SPHDGAWLQHQVAQAVLNSP-KYNRTALMISYDETGGWFDHVDPYRSPRDTAGEWI----------DDPYGQVGYT---FVGP PAN-------DDHPSH-DVAAGQRFVKEVYETLRSSP-QWKEMALLITYDEHGGFYDHVPTPVKGVPNPDGII-----------GPDPFYFGFD----RL PAD-------DDHPAH-DVANGQRLVKDVYEALRAGP-QWNHTLLIITYDEHGGFYDHVPPPNVGVPSPDAIR-----------GPLPFFFRFD----RL PAD-------DDHPAH-DVANGQRLVKDVYEALRAGP-QWNHTLLIITYDEHGGFYDHVPPPNVGVPSPDAIR-----------GPLPFFFRFD----RL PAN-------DDHPSH-DVFQGQMLVKEVYETLRASP-QWNQTLMVVTYDEHGGFYDHVPTPVTGVPSPDGIV-----------GPPPYNFAFD----RL NHTKSFGLPNDQHPDH-SVKEGERLMKNVYEALRNGP-KWNETLFIITYDEHGGFYDHVPPPQEGVPNPDGKV-----------NAE--GFNFE----RL ----------------------------------------------------------------GVPSPDGVR-----------GPAPHYFNFD----RL ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------SEHPAL-PVALGAVSMVTALRILLSNPAVWEKTALIVSYDENGGFFDHVTPPTAPPGTPGEFV------TVPNIDAVPGSGGIRG-PLGL ----------CEHPWW-GPSFGEYYVTRVLEALTSNPEVWARTVFILNYDEGDGFYDHASAPVPPWKDGVGLS------TVSTAGEIEVSSGL---PIGL ----------SEHPGPSSPIWGAEYTSWVLDALTANPEVWSKTALLVMFDENDGFFDHVAPPAAPSLNKDGTLRGKTTADATLEWHTKGDIRYRNQPYGL

AflaA AfumA AnidA Nfis1 Nfis7 AfumB AnidB Nfis2 Nfis4 AflaC AfumC AnidC Nfis5 Nfis3 AflaD Nfis6 AoryE CintE FgraE TreeE Atha5 Osat1 Osat3 Osat5 Tthe1 Ppat1 Ppat2 Trur1 MtubA PaerH PaerN

355 380 400 420 **************** ************** GIRVPTVLMSPWVGKGVVQNSPAD-QP------NEFTHTSILKYVAELWNLDI---LTPRVDWSPSFRGLITNTFRE-TPEKLPEPADF----------GIRVPTVLMSPWVGKGVVQNRPAD-GN------GEFTHTSILKFVADLWGLEY---LSPRVAWSASFAHLITDTFREDTPATLPEPADF----------GIRVPTLLISPYVQKGAVVHGPKG-KGKKGQAKGEYTHTSILKYVDELWGLDI---LTPRVEWSASFGDLIEKKFREDTPELLPEPVIF----------GIRVPTVLMSPWVGKGVVQNRPTD-GD------GEFTHTSILKFVADLWGLEY---LSPRVTWSASLAHLITDTYREDTPATLPETADF-------------------------------------------------------------------------------------------------------------GGRIPTLLISPWVAKAQVEQKGKNAHGET----VSYSAASILRTLGYLWDFEP---FTPRVEHAASFEHLIRLHARKDTPTALPSAVPFRNRNV-----GGRIPTLLISPWVAKGAVEQKGTNHKNDV----VSYSATSILRTLGYLWDFEP---FNPRVEHAASFEHLITARARDDTIEVLPEPEAFKRRGW-----GGRIPTLLISPWVAKAQVEQKGRNGHGET----VSYSAASILRTLGYLWDFEP---FTPRVEHAASFEHLIRHNARKNTPSALPSAVPFRK--------GGRIPTLLISPWVGKAQVEQKGRNAQGET----VSYSASSVLRTLGYLWDFEP---FTPRVEHAASFEHLIAHHAREDTP-------------------GGRMPTWVISPYSKKGYIEQYGTDPVTGKP---APYSATSVLKTLGYLWDIED---FTPRVAHSPSFDHLIGTTLREDAPIALKTPHTFSV--------GGRMPTWLISPYTPKGHIENYGVDPATGKS---ASYSATSVLKTLGYLWDLDD---FTPRVSHSPAFDHLIGPKKRN-TPATLANPHPFPDAV------GGRMPTWLISPYTPKGHIEDYGVDPVTGQK---ASYSATSALKTLGYLWDLED---FTPRVEHSPAFDHLIGPTLRS-SPETLANPHTFPDAV------GGRMPTWLISPYAPKGHVENYGVDPATGKT---ASYSATSVLKTLGYLWDLED---LTPRVSHSPAFDHLIGPKMRG-APATLANPHPFPDAV------GGRMPTWLISPYTPKGHIENYGIDPVTGKT---TSYSATSVLKTLGYLWDLKD---FTPRVSHSPAFDHLIGPKKRT-APATLANPHPFPDAV------GVRVPAFIISPYIEPNTLIHNDGTNYANN----SAYTHTSMLHFLQELWELEG---LNNRVQWAKTFEHVFSDTKREDTPKTLSTP----------------------------------------------------------------------------------------------------------------GLRVPFYMISPWTRGSRVFTEHADHNSQI----LFIEQWLKARGYENVETPEM---VQWRREHMSDLVSALDL-----------------------DHPD GFRVPFYIISPWTRGGRVLTEHSDHTSQI----LFIEEWLTAKGIENIKSPEV---VPWRREHMSNLVNAFDFENARTQTILFRICLRHVELTLYFQPNL GFRVPFYIISPFTRKGGVYTEHCDHTSQM----TFIERWQAAKGRD-VKSEEV---VPWRRENMADLVNAFDFKNPDYSIPT-----------------GFRLPFYIISPWTRNGGVFSAHSDHNSQI----LFVEKWQAAKGRN-VTTEEM---VYWRRSHMSDLVDAFDFDHPDYSIPQ-----------------GVRVPTFLISSWIEKGTVIHEPEG-PTPH----SQFEHSSIPATVKKLFNLKSH-FLTKRDAWAGTFEKYFRIRDSPRQDCPEKLPEVKLSL-------GVRVPTIMVSPWIRKGTVVGRPPGGPTPT----SEYEHSSIPATIKKIFNLSSD-FLTRRDAWAGTFEHLFTDLDEPRTDCPETLPEIPPPS-------GVRVPTIMVSPWIRKGTVVGRPPGGPTPT----SEYEHSSIPATIKKIFNLSSD-FLTRRDAWAGTFEHLFTDLDEPRTDCPETLPEIPPPS-------GVRVPAIVISPWINKGTVVHGPNGSPTAT----SEYEHSSIPATVKKLFDLPQD-FLTKRDAWAGTFESVVQGRTEPRTDCPEQLPMPMRIR-------GIRVPTIAISPWIEKGQLVKEPKPWQKPF--NTSQFEHSSIISTVMKIFGLEYN--FSKRTEWAATFDDLIS-RTEPRTDCPANLTYIPPPT-------GVRVPTIAISPWIEKGKVEHRAQGPMPS-----SEYEHSSIAATIRKLFNLPQP-PLTAREAWAGNFAHLIS-RTTPRTDTPVELPSPPWSL-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------GFRVPCIVISPYSRGPLMVS-------------DTFDHTSQLKLIRARFGVPVPNMTAWRDGVVGDMTSAFNFATPPNSTRP-----------------GHRVPLIAISPWSKGGKVSA-------------EVFDHTSVLRFLERRFGLVEENISPWRRAVCGDLTSLFDFQGAGDTQVAPDLTNVPQSA-------GPRVPMYVISPWSKGGWVNS-------------QVFDHTSVIRFLEQRFGVMEPNISPWRRAVCGDLTSAFNFANPNNEPFPELPDTS------------

Fig 1 (continued)

Two families of extracellular phospholipase C genes are present in aspergilli

AoryE CintE FgraE TreeE Atha5 Osat1 Osat3 Osat5 Tthe1 Ppat1 Ppat2 Trur1 MtubA PaerH PaerRN

TNLPTLPDAEEPATLLGNYVGSSNCQASH------PTQRPPVPYGQ-QSNVSDALWFEEGYKEVVGYLTEGRYLVFEKSGYALTNAGNATRISSSRTGSG SLPNLPKAELPHRDESGNWDGSAYCESLY------PETRPPVPYDS-QPDRMPTDLVEEGYKECIGTLTEGRFLVFSTTEQSRLLLHSNPDDLTTTDATN ---LPNAP-QPHTNSKGEYDGSSHCSSQY------GSGKPAVPYTGEGANNDTATLAEKGFKPVRGLLTEGRTLVFEASSQALSLGSNGQVVALSKATKM ---LPE-PQTPHTDSNGVYDGSSYCQSLY------SEVQPPVPYTGTGVIADLTTQVERGFKPLRGMLTEGRHLVLEANGFALTQKTSFLGTIVLSRATE --R----PWGAKEDS--KLSEFQVELIQLASQLVGDHLLNSYPDIGKN-----MTVSEGNKYAEDAVQKFLEAGMAALEAGADENTIVTMRPSLTTRTSP ----SSSSSTKKEDGG-WLSDFQRELVQLAAFLNGDYMLSSFAQEYES--RMTMTVKQADAYVRRAVKSFLEASKRAKRLASSLQGISKPEKKKKMGSGD ----SSSSSTKKEDGG-WLSDFQRELVQLAAFLNGDYMLSSFAQEYES--RMTMTVKQADAYVRRAVKSFLEASKRAKRLGANDSAIVTMRPSLTTATTC -------LTEANEEA--KLSEFQQELVQLASVLNGDHQLSSLQDTIRDR----MNVREGIAYMRGAVKRFFETGMSAKRMGVDDEQIVKMRPSLTTRTSP ----------------KAEIERIYSRNVKPTVINAVKRICQEYKP-------NDSECGKKIKTFRDYVNFLEIEIQYKHSY--------------------------RHSPTVESRPLSEFQEELVVLAKSLRRKLGDWATSEASSK-----AEEEANPSGMSVGEANFYVRDAVGSFMRRARSQLTAGRHPDSEAAP---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------NLSHP-LLGALPKLPQCIPNVVLGTTDGALPSIPYRVPYPQVM--PTQETTPVRGTPSGLCS-------------------------------------RKEDAYWQQFYRP-SPKYWSYEPKSLPGQEKGQRPTLAVPYQL--HATLALDIAAGKLRLTLGND--GMSLPGNPQDTAAAVFQVQPREVGNPRFYT ---QADAI-VASQIKLPKPKPPAVAAMPK--QEMGIRPARALPYEL--GVHARYRSGGDALSLTFANT--GKAGAVFQVFDLLDSENPPKRYTVGARKRL

AoryE CintE FgraE TreeE Atha5 Osat1 Osat3 Osat5 Tthe1 Ppat1 Ppat2 Trur1 MtubA PaerH PaerN

YGDKK------------------QRWVIHYSGGQQSGVFHISSALDGKWLGPKGTLLSS---------------NRGSQAADVKITFVGNGQGYTLQYAD ADPDSDSDPDPDLDYNSLYRNSDARWVLHYYNNNSTSSTDVNTTAINQDPPYLLSSGNRRVWLGEEGKLVPRRDAVAIDIDFVRGEGEDKGRGYYIKYHD HDNVK------------------HGWVLHALDIG-GDDFTISSAQNSLYMCSNLKLCKDP---------SSATVFTVDFQPSKGHSFMNKESGQYLAGSA RHDAP-----------------SQRWIAHAVTVG-GDEFTLSDDSGRNYICDNGQLCSDA---------GKAVVFTVGYKSGKGYSFGVKGTGRYLGVGG SEGTNKYIGSY----------------------------------------------------------------------------------------WGPVLIALVLFVLLTPGLLCQIPGSNGRVAEFHSMRTSVASIFVHALLFFAFCAIFMVAVGLHLYAG--------------------------------CP-------------------------------------------------------------------------------------------------AIEQP-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------VTSYPVVQESGEELGRTLNDELDDLLDANGRYAFEVHGPNGFFREFH-GNLHLAAQMARPEVSVTYQR-NGNLQLNIRNLGRLPCRRD-GDAEPGLYPGG HD-SFQGDASGDYHLEVHGPNGFLRVFRGNL-----------------RRDLAERKAPLPEVRIDYEPLFGNLRVQLINRGRHPVKLTVK---DNVYRQG

Nfis6 AoryE CintE FgraE TreeE Atha5 Osat1 Osat3 Osat5 Tthe1 Ppat1 Ppat2 Trur1 MtubA PaerH PaerN

-----------------------------------------------------------------STPIEIDSKGALTLQRREASEEGYKVWSYLAYVCNRRKLHTYRSSARV-----------------RDDGYISVNEDGGVR-VGEEKTKFKVLSVTYHD--------------------------------KKQLSWQD-----------TQSFWKTFSVTYX---------------------------------RGASSYAS--------LNGGLGYWKVYSVSY-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------SRR-YELEPNQAISEVWLLRSSQGWYDLSVTASNTEANYLRRLAGHVETGKPSRSDPLLDIAAT-ERRTVNVPPGQRREVRYSLRSSGNWYDFSVSAQ-GADSFLRRFSGRMEDGRSGFSDPGMGLGTLTF

1145

Fig 1 (continued)

distance (Protdist) analyses gave trees that were identical in the major branches and most minor branches: two major clades of fungal PLCs were seen, one clade comprising four groups, designated PLC-A to -D and one comprising a single group designated PLC-E. In addition, a plant/T. thermophila clade and a bacterial clade were observed. Bayesian inference (MrBayes) gave a tree in which the major branching pattern differed only in that the T. thermophila sequences branched before a plant/PLC-A to -D sequence cluster, rather than clustering the T. thermophila sequences with the plant sequences, away from PLC-A to -D. However, the plant/PLC-A to -D cluster had a low PP value of 0.62 and is therefore less reliable than the well-supported plant/T. thermophila clusters seen in the other two methods. Examination of intron positions provided supported for the presence of distinct PLC-A, -D and -E groups, as each group showed different and characteristic intron patterns. PLC-B and -C could not be distinguished on the basis of intron positions as all, with the exception of AflaC and AoryC, were twoexon genes with an identically located intron. AflaC and AoryC had an additional intron, although this was in the same position in the two sequences (data not shown). Further divisions of the PLC families based on intron position were consistent with the phylogeny. The distinction between AflaC, AoryC and the other PLC-Cs is an example of this. The PLC-As provide a further example; AclaA, AnidA, AfumA, AterA and NfisA were single-exon genes, whereas AflaA and AoryA,

which cluster together by phylogeny, had a single intron in the same position, indicating the appearance of an intron in this lineage.

PLC-A to -D The PLC-A to-D clade was restricted entirely to aspergilli and the Aspergillus anamorph Neosartorya fischeri (Fig 2). Considering PLC-A to -C, it would appear that these arose by two gene duplications, with the duplication of an ancestral sequence giving rise to PLC-A and a PLC-B/-C type gene, which then in turn was duplicated to give PLC-B and –C. However, the evolutionary events that gave rise to PLC-D are not clear, and the long branch connecting these proteins to the rest of the tree complicates accurate assignment of the branch point. With the exception of N. fischeri, none of the species examined showed a representative of all four PLC types. It seems most likely that one of the PLC groups has been lost from each species, although it may be that more sequencing data will complete the picture. N. fischeri had representatives of all four groups, supporting the idea of selective gene loss in other species (Table 1, Fig 1); although only full-length sequences suitable for the phylogenetic analysis were obtained for representatives of PLC-A, -B and -C (Nfis1-3, respectively), Nfis6 was 93.3% identical to the PLC-D sequence AoryD (Table 2). Surprisingly, N. fischeri appeared to have two PLC-A, -B and -C genes; although

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D. Tuckwell et al.

MtubC MtubD MtubA MtubB PaerH Scoe 83,1.00, BpseN 100 PaerN Ccre 72,1.00,89 64,1.00,Iloi

B A C T E R I A

95,1.00,100 97,-,80 99,-,99

81,1.00,89

CintE AterE 100,1.00,98 100,1.00,92 AflaE 100,1.00,98 AoryE 97,1.00,100 FgraE 99,0.89,71 TreE

PLC-E

Tthe5 The4

99,1.00, 100

Tthe1 99,1.00,88 Tthe2 100,1.00,100 90,1.00,87 Tthe3 Osat1 Osat3 Osat4

88,-,91 99,1.00,100

67,0.73,57 93,1.00,99

91,1.00,98 80,0.99,63 53,0.99,95

99,1.00,100

0.1

Atha6 Atha5 Osat6 Atha1 Osat5 Osat2

Tetrahymena thermophilus

P L A N T S

Atha2 Athal3 Atha4 AterB AnidB 95,1.00,100 PLC-B AclaB 54,0.78,97,0.99,84 AfumB 99,1.00,99 Nfis2 76,1.00,81 AflaC 99,1.00,100 AoryC AnidC PLC-C 93,1.00,100 AclaC 96,0.89,100 72,1.00,80 96,-,64 AfumC 99,0.99,87 Nfis3 AflaD PLC-D 99,1.00,100 AoryD AnidA 74,0.84,AfumA Nfis1 55,0.93,100 PLC-A AterA 89,1.00,99 AflaA AoryA 60,0.99,100 AclaA 58,0.67,84 50,1.00,69 81,1.00,83 78,1.00,100

86,1.00,100

Fig 2 – Phylogenetic tree showing the two groups of fungal PLC and their relationships to plant, bacterial and Tetrahymena thermophila PLCs. A ML tree (Treepuzzle; Schmidt et al. 2002) is shown. To the left of each node ML support values are given, followed by Baysian inference posterior probabilities (MrBayes; Huelsenbeck et al. 2001; Ronquist and Huelsenbeck 2003), and bootstrapping values from distance analysis (PROTDIST, Felsenstein 2002), where these are over 0.5 or 50% as appropriate; ‘-’ signifies a value of <0.5 or 50%, or a difference in topology. Sequence identifiers are as given in Table 1. The scale bar corresponds to the branch length for an expected number of 0.1 substitutions per site.

Nfis7, 4 and 5 were all partial sequences, they were seen to be most similar to a PLC-A, -B and -C respectively, while differing from Nfis1, 2 and 3 (Table 2). These observations could be explained by the presence of two distinct PLC-A, -B and -C genes in the genome of N. fischeri. However, as N. fischeri has

a sexual stage, two alleles of each gene could have been sequenced. As the Nfis4, 5 and 7 genes were all on small DNA fragments, it was not possible to determine whether the flanking genes were identical to those in Nfis1, 2 and 3. However, the DNA sequences immediately flanking pairs of PLCs did

Two families of extracellular phospholipase C genes are present in aspergilli

1147

Table 2 – Groupings and near neighbour sequences for Neosatorya fischeri phospolipase Cs (PLCs) Protein Nfis1 Nfis2 Nfis3 Nfis7 Nfis4 Nfis5 Nfis6

Closest sequence AfumA AfumB AfumC AflaA AfumB AfumC AoryD

94.9% 94.3% 96.3% 100.0% 90.9% 92.5% 93.3%

Second closest sequence AclaA AclaB AnidC AoryA Nfis2 Nfis3 AflaD

86.4% 84.4% 86.7% 97.0% 90.6% 92.0% 91.5%

not match and Nfis1 and Nfis7 showed a different intron pattern (data not shown). Completion of the N. fischeri genome sequence may resolve this issue. Phylogenetic analysis shows that the PLC-A to -D group branched before the divergence of the lower eukaryote Tetrahymena thermophila and plants (Viridiplantae), an event that occurred comparatively early in eukaryote evolution (Baldauf et al. 2000). To provide further clarification of the relationship between plant PLCs and the PLC-A to -D group, plant sequences were specifically investigated. Database searches identified expressed sequence tag (ESTs) encoding PLCs from a wide range of plant species including columbine, cotton, lettuce, pine, potato and spruce (data not shown). ESTs were also identified from Bryophyta (mosses), an early-diverging member of Viridiplantae. ESTs from Tortula ruralis (CN204437.1; Trur1) and Physcomitrella pateus (BJ954474.1, Ppat1, BJ966613, Ppat2) were translated and aligned with the PLCs (Fig 1). As the sequences were not full length, they were not suitable for phylogenetic analysis, but pairwise identities (IDs) indicated that the bryophyte PLCs were more similar to plant PLCs than to PLC-A to -D (Table 3), thus a Viridiplantae clade probably exists that is distinct from the PLC-A to -D group. These data, taken together with the phylogenetic trees and the restriction of this PLC set to the Aspergillus genus indicates that the PLC-A to -D group probably arose from a single gene transfer event from a lower eukaryote, or a prokaryote. Codon analysis of the genes for the A. fumigatus and A. oryzae PLCs, comparing them with a set of secreted A. fumigatus or A. oryzae proteins, did not yield any further evidence for gene transfer, either from analysis of GC content at the third base of codons, or calculations of codon usage (data not shown).

PLC-E PLC-E was found in a wider range of fungi than the PLC-A to -D but could only be seen in three species of Aspergillus (Fig 2; Table 1). Examination of the genes flanking this PLC showed that, in all species, the PLC gene was seen head-to-head with a copper oxidase gene (‘divergent orientation’; Fig 3).

Nearest Neosatorya fischeri sequence Nfis2 Nfis3 Nfis2 Nfis1 Nfis2 Nfis3 Nfis1

44.8% 49.1% 48.8% 66.7% 90.6% 92.0% 48.8%

Group PLC-A PLC-B PLC-C PLC-A PLC-B PLC-C PLC-D

Studies of S. cerevisiae and C. albicans have shown that divergently transcribed gene pairs that are conserved in evolution are likely to be co-regulated (Hurst et al. 2002). Therefore it is highly likely that these two genes are functionally associated. Conservation did not extend beyond the two genes. Blast searches with the copper oxidase protein sequence identified homologous fungal laccases and iron transport proteins, as well as plant and bacterial proteins. Phylogenetic analysis indicated that the copper oxidase protein formed a clade within this wider family of fungal, plant and bacterial copper oxidases but there were no orthologues with experimentally derived annotation, and no experimentally annotated proteins clustered with the copper oxidases (data not shown). The similarity to laccases, which are involved in lignin degradation (Baldrian 2006), suggests that the PLC/copper oxidase pair of proteins could be involved in harvesting nutrients from the environment. Codon analysis of A. oryzae PLC-E carried out as described for PLC-A to -D did not provide any further evidence for horizontal gene transfer (data not shown).

Modelling of PLCs A number of lines of evidence indicated that the PLCs were related to alkaline phosphatase and arylsulphatase family (Galperin & Jedrzejas 2001): (1) tertiary structure prediction for PLC-A carried out using Meta (Ginalski et al. 2003) gave the human arylsulphatase C (1auk) as the best consensus prediction; (2) analysis using Superfamily (Gough et al. 2001) indicated a match to the SCOP alkaline phosphatase-like family (E ¼ 9.6e26), of which 1auk is a member; and (3) PLCH_PSEAE has been shown to share homology with alkaline phosphatases (Stonehouse et al. 2002). A preliminary homology modelling study of PLC-A was therefore undertaken using the arylsulphatase structure 1E2S as the template (Figs 4–5). The core structural elements of PLC-A were modelled well (Fig 5 A–B), although there were a number of peripheral loops that could not be modelled, particularly a predicted disulphide-bonded loop between C66 and C80 (marked as arrows on Fig 5), and a large insertion between Q120 and Y145

Table 3 – Percentage identities for translated bryophyte phospholipase C ESTsa Protein BJ954474.1 BJ966613.1 CN204437.1

Arabidopsis thaliana and Oryza sativa

PLC-A to -D

Tetrahymena thermophilus

Bacteria

64.8–71.8% 46.4–59.2% 50.5–63.4%

27.5–51.4% 23.2–36.0% 23.7–42.9%

44.4–62.0% 28.9–37.9% 35.9–43.9%

22.5–38.7% 26.1–31.3% 28.2–34.1%

a Each EST was translated and pairwise IDs calculated for the PLC alignment over the length of the EST.

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Fig 3 – Comparison of the Coccidioides immitis, Fusarium graminearum, and Aspergillus terreus PLC-E and copper oxidase genes, showing that the two genes are conserved whereas the immediately flanking regions are not. The figure was generated using ACT (Carver et al. 2005), comparing the three sections of the genome using tblastx. Lines indicate regions of homology between genes.

(marked as arrows on Fig 5). Comparison of the model with the template structure and known conserved residues in the alkaline phosphatase/arylsulphatase family (Galperin & Jedrzejas 2001) showed that residues involved in the coordination of calcium or magnesium were conserved in the PLCs (E40, N41, D314 and E315, Figs 1, 4 and 5E–F). These data indicate that PLCs should contain a metal binding site. Conserved equivalents of other important residues could also be identified, as the conserved PLC residues and H279 and possibly T348 were comparable with H229 and K302 in E2S. Finally, the catalytic cysteine, threonine or serine of the alkaline phosphatases/arylsulphatases family (A69 in 1E2S) was comparable with T98 of PLC-A, however, the more conserved S96 may be the functional equivalent. These data indicate that the PLCs are likely to have mechanistic similarities to the alkaline phosphatase/arylsulphatase family. Examination of residues conserved across the fungal, plant and bacterial phospholipases indicated that a number were at and close to the predicted active site, and these included some of the predicted coordinating residues above (Fig 5E). The PLC alignment was analysed to identify residues conserved within but not between the PLC-A, -B, -C and -D families. Those on the top magnesium-binding face of the molecule tended to be around rather than at the

active site (Fig 5F). Superimposition of a phospholipid on the active site indicates that these variable regions are not close enough to affect substrate binding (data not shown) suggesting that PLC-A to -D probably do not differ in aspects of substrate recognition, but could interact with membranes in different ways.

Discussion We have identified multiple PLC genes in aspergilli, and shown that they have a complex evolutionary history. It seems most likely that the progenitor of PLC-A to -D and that of PLC-E were introduced into the fungal lineage by two, separate, gene transfer events as the two groups of PLCs fall into distinct clades with different evolutionary histories. Representatives of the PLC-A to -D clade could be found only in aspergilli despite the wealth of fungal genome sequence data available for analysis. The observation that Aspergillus fumigatus and Neosartorya fischeri sequences are nearest neighbours, as are A. oryzae and A. flavus sequences is in agreement with the literature (Geiser et al. 1998a, b). Comparison of our data with the Aspergillus phylogeny of Peterson (2000) indicates that PLCs occur over an evolutionarily widespread of

Two families of extracellular phospholipase C genes are present in aspergilli

jpred PLC-A 1E2S Structure Catalysis

30 40 60 80 100 120 --EEEEEEEE-----------------------------------------EE-----------------HHHHHH----------------------HH PIKNVVVLVQENLSFDNYAGGLTYSP-SIDGLVNRTYCNPANISDPHSPMVCAQPTAKNIAPDDPDHSITGGNQQVYSTYHPDSDFDAPNMQGFVAEQIA RPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLAA---------------GGLRFTDFYVPV----SLATPSRAALLTGRLPVRMGMYPG-----------EEEEEEE------------------HHHHHHHH----------------EEEEE-EE---------HHHHHHHH--------------------------------**---------------------------------------------------------*------------------------------

jpred PLC-A 1E2S Structure Catalysis

129 140 160 180 200 220 HH---------------------HHHHHHHHHHHHH---------------EEEEEE----------------------HHHHHH----EEEEEE----AYGIDGNLSRAGEVINYYTPEHIPVFNAMAENFVLFDRWFAAVPGPTNPNRAYLTSGTSHGHGTNDPDFDRSALPQRSIFEQLSEHNISWINYSNTT--------------------VLVPSSRGGLPLEEVTVAEVLAAR-----GYLTGMAGKWHLGVGPEG-------------AFLPPHQGFHRFLGIPYSHDQG ----------------------EEE---------HHHHHH---------EEEEEEEE---EEE-E-------------EE--------EEEEE----------------------------------------------------------------------------------------------------------

jpred PLC-A 1E2S Structure Catalysis

240 260 280 -------------------------------------------------HHHHHHHHH----------EEEEE---------------------------------------------------GFLPDALFYRWTAVSGLGETNVRPIDQFYRDAKAGT----LPQFTWINPECCSYMSFHPPS-------------PCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLP----GLEARYMAFAHDLMADAQRQDRPFFLYYASH----HTHYPQFSGQSFAERSGRGP ----EEE----EEE------------EEEE-EEE------------HHHHHHHHHHHHHHHHHH---EEEEEEEE------------------------H ----------------------------------------------------------------------------------*-----------------

jpred PLC-A 1E2S Structure Catalysis

300 320 340 360 -----HHHHHHHHHHHHH---------EEEEEEE----------------------------------------------EEEEEEE-----E-E-----PINMGEGWIKSVYEALRSSP--QWHETLFILTFDEHGGFADHVPPPENVPAGDDLTYTETARDGKPSTFAFDRLGIRVPTVLMSPWVGKGVVQNRPADG FGDSLMELDAAVGTLMTAIGDLGLLEETLVIFTADNGPETMRMS-------------RGGCSGLLRCGKGTTYEGGV--REPALAFWPGHIAPGVTHELA HHHHHHHHHHHHHHHHHHHH------EEEEEEEEE-----------------------------------------E--EE-EEEE-------EEE-------------------------------------**--------------------------------*-------------------------------

jpred PLC-A 1E2S Structure Catalysis

-------HHHHHHHHHH---NGEFTHTSILKFVADLWGLEY -SSLDLLPTLAALAGAPLPNV ------HHHHHHHH---------------------------

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Fig 4 – Alignment of PLC-A and arylsulphatase 1E2S showing the predicted secondary structure for PLC alignment (jpred), the secondary structure of 1E2S (Structure), and catalytically important residues of 1E2S (Catalysis). Numbers correspond to AfumA residues.

Aspergillus species, and so the acquisition of the progenitor PLC gene probably occurred either early on in, or not long before speciation in Aspergillus, which may be w10 Myr (Birren et al. 2002). The alvaeolate PLCs and those of Viridiplantae plants clustered separately from the PLC-A to -D set. This places the organism from which the PLC was transferred either among the simplest eukaryotes, or prokaryotes. In contrast to PLC-A to -D, PLC-E was identified in four different fungal genera. Comparison of the PLC-E tree with a fungal phylogeny (Lutzoni et al. 2004) indicates that the branching order of genera is as would be expected. An ancestral gene transfer event could therefore have occurred before the branching of the Eurotomycetidae (aspergilli and Coccidioides immitis) and Sordariomycetes (Fusarium graminearum and Trichoderma reesei) and after the divergence of the saccharomycetes. Although this would explain the absence of PLC-E from Saccharomyces cerevisiae and Candida species, the very patchy distribution of PLC-E, for example within the Sordariomycetes, which includes Neurospora crassa and Magnaporthe grisea for which sequenced genomes are available, implies that widespread gene loss has occurred. Stonehouse et al. (2002) reported a phylogenetic analysis of bacterial PLCs, and our analysis included sequences from the four major groups of bacterial sequences that were described in their tree. Adding more bacterial sequences to our tree would therefore not necessarily help define the points at which the fungal PLCs arose. As an alternative approach to clarifying the origin of PLC-E, bacterial genomes were searched for a copper oxidase–PLC-E gene pair, but none could be identified. However, with the continued increase in the number of available genome sequences that can be anticipated, such a combination may be identified in the future. Sequence analysis and preliminary homology modelling of the PLCs indicated similarities to the alkaline phosphatase/

arylsulphatases family (Galperin & Jedrzejas 2001), and the PLCs can now be included in this family. Although the study was carried out on PLC-A, conservation of a number of the key residues across PLC-A to -D, and PLC-E suggests that all the PLCs are likely to behave in a similar way. In the future, more accurate and detailed modelling studies of the PLCs should determine the extent of the similarity between the mechanism of function of the PLCs and the alkaline phosphatases/arylsulphatases. The purpose of multiple copies of PLCs in aspergilli could not readily be ascertained from the modelling. The lack of differences at the active site suggests that the PLCs differ little in substrate specificity. However, examination of the 50 untranslated sequences indicated a conserved region in the 44 bases upstream of the PLC-A start site in A. fumigatus, A. flavus and A. terreus not seen in PLC-B or -C (D. Tuckwell, unpublished observation). This suggests that one difference between the PLCs lies in their regulation. The PLCs thus join an arsenal of enzymes that potentially enable aspergilli to utilise whatever nutrients are available in the environment. Phospholipase B production has been demonstrated to be an important virulence factor in C. albicans (Leidich et al. 1998) and C. neoformans (Chen et al. 2000). As the natural route of infection of A. fumigatus usually occurs via the lung, which is coated with phospholipid-rich surfactant, the PLCs described here may be found to be pathogenesis factors. In this respect, it is significant that PLC-N from Pseudomonas aeruginosa, which is homologous to the A. fumigatus PLCs, is upregulated some 16-fold during interaction with human airway epithelial cells (Frisk et al. 2004).

Acknowledgements The authors would like to thank Geoff Robson and Catherine Rycroft for their advice and assistance.

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PLC-A

1E2S

B

A

PLC-A

1E2S

D

C H279

H229 T98

A69(C) D28

E40

K302 D29

T348

N41

D282

E315

D281

D314

PLC-A

E

PLC-A

F

Fig 5 – Homology model of PLC-A. Models were generated using Swiss model and viewed using spdbv (Guex 1996; Guex & Peitsch 1997). A–B. View of the modelled structure and template (from the side), arrows mark loops which were not modelled reliably and have been omitted. C–D. Close-up view of magnesium binding/catalytic site (from the top). The magnesium ion in the 1E2S structure is shown in black. E. The PLC-A model (same face as C) showing residues conserved across the fungal, plant and bacterial PLCs. F. The PLC-A model (same face as C) showing residues conserved within the PLC-A, -B, -C and -D groups but not across them. In F the magnesium ion from 1E2S is shown for reference. Numbers in C correspond to AfumA residues, numbers in D correspond to 1E2S residues.

Two families of extracellular phospholipase C genes are present in aspergilli

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