Supplementary Figures
A
B
C
D
E
F
G
H
Supplementary Fig.1 Morphological characteristics of the nematode-trapping fungus Arthrobotrys
oligospora XJ-A1 after induction of infective larvae (L3).
A and B: The conidia with one septum each (black arrow); C: Formation of the fungal trap (black arrow) after 6 h
of the fungus/larva interaction; D: Scattered adhesive three-dimensional network traps (black arrow); E and F:
Haemonchus contortus infective larvae (L3) was captured by scattered adhesive three-dimensional network traps
(black arrow) after 12h of interaction with the fungus. G and H: Haemonchus contortus infective larvae (L3) at
48h after the beginning predation by the fungus. Note the nematode (black arrows) that had their inner contents
digested, leaving only the cuticle.
Ac1
Aoz1
Mlx
P186
PⅡ
Pr1
PrA
pSP-3
VCP1
Ver112
MLTNGLISLLAIAGLATNAFAGPIR...KVSNAGAAGAIADKYIVVLKKGLSESAVSKHTNRISSFHTNVARDLT
MLTNGLISLLAIAGLATNAFAGPIR...KVSNAGAAGAIADKYIVVLKKGLSDSAVSKHTNRISSFHSNVARDLT
MILNGLISLLAIAGLA...FAGPIR...KVSNAGAPGTITDKYIVVLKNGLSESAVAKHTNRISSFHSVVARDFT
MHFIPLASLLPLAGLILQVAADLT.....VLGAGSKDKIPNSYIVVLKPSTNQAQVREHTQRISNYHT..RRSLQ
MLTNGLISLLAIAGLATNAFAGPIR...KVSNAGAAGAIADKYIVVLKKGLSDSAVQTYH.RISSFHSNVARDLT
MRLSIIAAALPLAIAAPVVEP......APLIEARG.QTIAGKYIVKHKDTATIGIMD..AASKVPNTE.......
MFPSLLLLNLLPLAIAAPAKRAE...PAPLLVPRGD.TIPDKYIVKYKETFDISAADSTIKEYHAKAE.......
....................A.....RAPLLTPRGASSSSTASTLSSSRTACPSPLSTRLSALCPRRP.......
MQLSVLLTLLPAVLAAPAIVEQRAEPAPLFTPKS..SIIAGKYIVKFKDGVARIAADEATSALSAKAD.......
MRLSIIAAVLPLALAAPVAEPE....IAPLIEARGAQPIAGKYIVKLKDEAKFGIMN..AKSKIPGIE.......
72
72
69
68
71
59
64
43
66
62
Ac1
Aoz1
Mlx
P186
PⅡ
Pr1
PrA
pSP-3
VCP1
Ver112
GARAHGVGKKFRFSSTGFNGYTGGFDKATLQEILNSPEVDYVEQDTVVTT..NAEQTDSTWGLDRISHEEF.DTP
GARAHGVGRKFRFSSTGFNGYVGGFDKATLQEILNSPEVDYVEQDTVVTT..YAEQTDSTWGLDRISHEDY.SAP
GARAHGVGRKFRFAATGFNGYVGGFDKATLQEILNSPEVDYVEQDSVVKI..NAEQLDSTWGLDRISHENY.AAP
.ERGVTTGIRGQFDIQSVKGYTVECDHGTLSQILSSPEVQYVEQEGKTKV..QVTQKPSTWGLSRISWKTLPKAP
GARAHGVGRKFRFSSTGFNGYVGGFDKATLQEILNSPEVDYVEQDTVVTT..YAEQTDSTWGLDRISHEDY.SAP
...........LVYENVLKGFSATLNQEQLDRLRHDP..DTIEQDAIVSINAVVRQAGAPWGLGRISHRAR..GA
...........KTYSHVFNGFAGALNATSIETLRNHPAVDFIENDATVRISAFVEQPGAPWGLSRISHRQR..GG
...........TASTTTFSEASRNLNANDLKTLRDHPDVEYIEQDAIITINAYTQQPGAPWGLGRISHRSK..GS
...........HVYSHLFNGFAGSLTKEELQTLRNHPDVDFIEKDAVMTANAIVEQQGAPWGLGRISNRQK..GS
...........RVYENVLNGFSATLSNEELERLRRDPDVESIEQDAIFSINAITQQQGATWGLTRISHRAR..GS
144
144
141
140
143
119
126
105
128
124
Ac1
Aoz1
Mlx
P186
PⅡ
Pr1
PrA
pSP-3
VCP1
Ver112
YTYEYDETFAGSGTTVYVIDTGIRITHNEFQTENGTSRATWGFNSVDKT.DSDGNGHGTHCAGTIAGKTYGVSKK
YTYEYDETAAGAGTTVYVIDTGIRISHDEFQTVNGSSRATWGFNSVDKT.DSDGNGHGTHCAGTIAGKTYGVSKK
YTYEYDEAAAGAGTTVYVIDTGVQITHDEFKTANGTSRASWGKNFVDSS.DSDGNGHGTHCAGTIGGKTYGVSKK
YSYRYQNTWGGRGTTIYIVDSGVRVSHKEFE.....ARATWGYNAVPGSPNTDNHGHGTHVAGTAAGKTYGVAKY
YTYEYDETAAGAGTTVYVIDTGIRISHDEFQTVNGSSRATWGFNSVDKT.DSDGNGHGTHCAGTIAGKTYGVSKK
TTYDYDSSA.GAGTCVYVIDTGVYDSHPDFEG.....RAKQIKSFVSG..TSDGHGHGTHCAGTIGSKTYGVAKK
SSYAYDDSA.GEGTCAYVIDTGVEASHPEFEG.....RAEFIRSFVAGE.NSDRNGHGTHVAGTIGSKKYGVAKK
TTYEYDTSG.GSGTCAYVIDTGVEASHPEFEG.....RASQIKSFISGQ.NTDGNGHGTHCAGTIGSKTYGVAKK
TTYRYDDSA.GNGACVYVLDTGIETTHPEFEG.....RATWLKSFIDGE.NNDGHGHGTHCAGTVGSKTYGVAKK
TAYAYDTSA.GAGACVYVIDTGVEDTHPDFEG.....RAKQIKSYAST..ARDGHGHGTHCAGTIGSKTWGVAKK
218
218
215
210
217
186
194
173
196
191
Ac1
Aoz1
Mlx
P186
PⅡ
Pr1
PrA
pSP-3
VCP1
Ver112
AKVVAVKVLSPSGSGSLSGVVSGMNWVAENAT....PNFSVASMSLGGSKSAALNTAVDSIFNAGISIVVAAGNE
AKVVAVKVLSAGGSGSTAGVVSGMNWVAENAT....PNFSVASMSLGGSKSTALNAAVDCIFNAGITIVVAAGNE
AKIVAVKVLSAGGSGSTSGVISGMNWVAQNAV....PNFSVASMSLGGGKSTAINSAVDAIFNAGTTIVVAAGNE
ARIVAVKVIGDDGTGQDSYTLAGLNYISRVAK....PGKSVVNMSIGGPKSEAVNAAVEALYKKGIVVVAAAGNE
AKVVAVKVLSAGGSGSTAGVVSGMNWVAENAT....PNFSVASMSLGGSKSAALNTAVDAIFNAGITIVVAAGNE
ASIFGVKVLEDSGSGSLSGVIAGMDFVATDRKSRPCSKGTVASMSLGGGYSATVNQAAARLQASGVFVAVAAGND
TKILGIKVLSDQGSGDYSGILAGMDFAIQDSRTRGCPKGVVANMSLGGGYSAAINQAAAKMIQSNVFLAVAAGND
TKIYGVKVLDNSGSGSYSGIISGMDFAVQDSKSRSCPKGVVANMSLGGGKAQSVNDGAAAMIRAGVFLAVAAGND
AKLLAVKVLDNGGSGSYAGVIAGMEFVSQDYKTRGCPNGAIASMSLGGPFSASVNQAAAAMVSSGVFLSVAAGND
VSIFGVKVLDDSGSGSLSNIVAGMDFVASDRQSRNCPRRTVASMSLGGGYSAALNQAAARLQSSGVFVAVAAGND
289
289
286
281
288
261
269
248
271
266
Ac1
Aoz1
Mlx
P186
PⅡ
Pr1
PrA
pSP-3
VCP1
Ver112
NQDAKNVSPASAPNAITVGAIDSNNKRASFSNWGTLIDVFAPGVSVLSSWATSDTDTKSISGTSMACPHVAGLAA
NQDAKNVSPASAPNAITVGAIDSSNKIASLSNWGTLIDVFAPGVGVLSSWATSDKETKTISGTSMACPHVAGLAA
NQDAKNVSPASAPNAITVGAIDSTNTIASFSNWGTLIDVFAPGVSVLSSWKGSDTATNTISGTSMACPHVAGLAA
NENAGLSSPASARSAITVGATDETDTRASFSNFGSIVDIFAPGVNILSADITSDTATRLDNGTSMASPHVAGLAA
NQDAKNVSPASAPNAITVGAIDSSNKIASFSNWGTLIDVFAPGVGVLSSWATSDKETKTISGTSMACPHVAGLAA
NRDAAQTSPASEPSVCTVGATDSSDRRSSFSNFGKAVDIFAPGTGILSTWNNGG..TNTISGTSMATPHIAGLGA
AKDASQTSPASEPSVCTVGATDSSDRLSSFSNYGAAVDILAPGSDILSTWIGGI..TKSISGTSMATPHIVGLGA
NANAANYSPASEPTVCTVGATTSSDARSSFSNYGNLVDIFAPGSNILSTWIGGT..TNTISGTSMATPHIVGLGA
GADAARYSPASEPSACTVGATTSTDARSSFSNFGKLVDIFAPGSAILSTWINGG..TRSISGTSMATPHVAGLAA
NRDAANTSPASEPTVCTVGATDSNDVRSTFSNYGRVVDIFAPGTSITSTWIGGR..TNTISGTSMATPHIAGLAA
364
364
361
356
363
334
342
321
344
339
Ac1
Aoz1
Mlx
P186
PⅡ
Pr1
PrA
pSP-3
VCP1
Ver112
YYISSFEGAGAKPQVITDRITES..AITGKLNPS..DIKGSPNRIAYNGTA........................
YYISASEG.GADPATITDKITSSRRQWSGHREHPWLPKQDRLQRICLSTHSPKTNHQVTIVAS............
YYISAQGG..ADPASISAKISNS..AVTGQVKG...NIRGSPNKIAYNGAA........................
YFISSRTT.SQSPLNIHSLFITY..SQKGLVKS....PGPSPNRLAYNGWDEYQPYPY.................
YYISASEG.GADPATITDKITSS..AVSGQVTG...NIRGSPNKIAYNGYA........................
YLLALGKGT.AGNLCQTIRTLSTKNVLTGVPSG.......TVNYLAFNGAT........................
YLSSLEGFPGAQALCERIRSLAIRNTISGVPGG.......TVNLLAFNGNPSG......................
YLAGLEGFPGAQALCKRIQTLSTKNVLTGIPSG.......TVNYLAFNGNPSG......................
YLNALQGVVSPAALCKKIQDTAIKNALTGVPAS.......TVNFLAYNGA.........................
YLFGLEGGS.AGAMCGRIQTLSTKNVLTSIPSG.......TVNYLAFNGAT........................
411
426
405
407
408
377
388
367
387
382
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Supplementary Fig.2 Alignment of cuticle-degrading proteases amino acid sequences from A.conoides (Ac1),
A.oligospora (PII, Aoz1and P186), D.varietes (Dv1), P.lilacinus (pSP-3), M.anisopliae (PrA), B.bassiana (Pr1),
P.chlamydosporia (VCP1) and L.psalliotae (Ver112).The GenBank accession numbers of Ac1, PII, Aoz1, P186,
Mlx, pSP-3, PrA, Pr1, VCP1 and Ver112 are AAX54903, CAA63841, AAM93666, AHI12733, AAW21809,
AAA91584, CAB64346 , AAK70804, CAD20578, and AAU01968, respectively. Areas shaded in black are
conserved regions (100% similarity), areas shaded in grey are high degree homology (more than 75% similarity),
areas shaded in yellow are middle degree homology (more than 50% similarity), and unshaded areas are regions
of variability between the proteases. Signal peptide sequences are marked on arrow, and pro-peptides are marked
on the discontinuous arrow. The triple asterisk indicates the N-terminal sequences of mature peptides. Filled
circle indicates the aspartic acid (Asp160)-histidine (His192)-serine (Ser345) (in P186) catalytic triad. Filled triangle
indicates the N-linked glycosylation sites (Asn249, Asn342) (in P186) and the tildes indicate the substrate-binding
S1 (Ser251Ile252Gly253, Ala277Ala278Gly279) (in P186) pocket in subtilisin.
Supplementary Fig.3 Phylogenetic tree showing the relationship between P186 and other fungal subtilases.
Phylogenetic analyses were performed with the MEGA6.0 program package. The data were subjected to
Neighbor-joining (NJ) method of phylogenetic analysis, and the branch support of the NJ tree was evaluated using
bootstrap analysis with 1,000 replications. Aspergillus niger (accession number: AAA32703) was used as
outgroup.
Supplementary Fig.4 SDS-PAGE and Western blot analysis of expressed recombinant protease P186.
Lane 1: Western blot; lane 2, 3, 4 and 5: GS115/pPIC9K-P186 after induction of 24h, 48h, 72h and 96h,
respectively; lane 6: The purified recombinant protease; Lane 7: untransformed GS115; lane 8: GS115/pPIC9K;
M: molecular mass standard.
Supplementary Fig.5 Assay of recombinant protease P186 activity in the cultures of Pichia pastoris strain
GS115.
At the indicated times, a sample of the culture was removed and assayed for P186 activity. The results are the
mean values of three independent assays relative to untransformed P.pastoris GS115 and P.pastoris GS115
transformed with empty vector pPIC9K controls. Specific protease activity was tested using the artificial substrate
Suc-Ala-Ala-Pro-Phe-pNA. Results are expressed as A415/min×10-3(unit).
B
A
Supplementary Fig.6 Effects of pH and temperature on activity of recombinant protease P186.
Relative activity is expressed as a percentage of the maximum activity under standard assay conditions. The
activity of P186 (50 mg/mL) before incubation was defined as 100%. The value is the average of three
replications.
A
B
C
D
A
Supplementary Fig.7
Construction of plasmid pPIC9K-P186 and validation of transformant.
A. PCR amplification of cDNA gene with primers P186-F-SnaBI and P186-R-NotI using fungal
gene/pPIC9K-P186 as a template. Lanes 1 and 2 were the PCR products. M was the DNA marker, following lane
M is the same. B. Results of pPIC9K-P186 digested by SnaBI and NotI. Lane 1, empty vector; lane 2,
recombinant vector; lane 3, product after enzyme digestion.C. PCR amplification of genes (wild-type AOX1 and
vector AOX1 with the gene of interest) from Pichia pastoris GS115/pPIC9K-P186 using primers 5′ AOX1 and 3′
AOX1. Lanes 1,2 and 3 were the PCR products. D. PCR amplification of cDNA gene from P. pastoris
GS115/pPIC9K-P186 using primers P186-F-SnaBI and P186-R-NotI. Lanes 1,2 and 3 were the PCR products.