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Proteomic analysis of the knob-producingnematode-trapping fungus Monacrosporium lysipagum
Alamgir KHANa,*, Keith L. WILLIAMSa, Julie SOONb, Helena K. M. NEVALAINENc,d
aProteome Systems Ltd., 1/35-41 Waterloo Road, North Ryde, NSW 2113, AustraliabAustralian Proteome Analysis Facility (APAF), Level 4, Building F7B, Research Park Drive,
Macquarie University, Sydney, NSW 2109, AustraliacDepartment of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, AustraliadMacquarie University Biotechnology Institute, Macquarie University, Sydney, NSW 2109, Australia
a r t i c l e i n f o
Article history:
Received 20 December 2007
Received in revised form
26 March 2008
Accepted 11 June 2008
Corresponding Editor: Judith K. Pell
Keywords:
Cross-species matching
2D gel electrophoresis
Nematophagous fungi
Soil fungi
* Corresponding author. Australian Proteomney, NSW 2109, Australia Tel.: þ61 2 9850 62
E-mail addresses: [email protected]/$ – see front matter ª 2008 The Bdoi:10.1016/j.mycres.2008.06.003
a b s t r a c t
The soil-inhabiting, nematode-trapping fungus, Monacrosporium lysipagum, captures mobile
stages of nematodes using specialized morphological structures, sticky knobs, that arise
from mycelia. A study was conducted to separate the proteome of M. lysipagum mycelia
containing knobs on two-dimensional (2D) gels resulting in a partial map of the proteome.
The fungus was grown in a liquid soy peptone medium supplemented with the amino
acids phenylalanine and valine to produce mycelia with knobs. Proteins extracted from
the mycelia were separated by 2D gel electrophoresis and relatively high abundant proteins
were identified by peptide mass fingerprinting (PMF). Out of the 250 proteins analysed by
PMF, 51 (20 %) were identified by cross-species matching due to unavailability of genomic
information from M. lysipagum. This is the first published report on a proteomic analysis of
a nematode-trapping fungus.
ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction 2006b; Supplementary Material Movie Clip S2: fungus digests
The soil-inhabiting fungus Monacrosporium lysipagum is
a knob-producing nematode trapper and a potential biocon-
trol agent of plant parasitic nematodes (Khan et al. 2006a). M.
lysipagum produces sticky knobs on the apex of hyphal
branches and captures nematodes during their random mi-
gration in the soil using these knobs (Rubner 1996). The fungus
catches nematodes in a split second (Supplementary Material
Movie Clip S1: fungus catches nematodes) and kills them
quickly regardless of differences in the degree of knob attach-
ment (Khan et al. 2006b). Once attached, the knobs of
M. lysipagum germinate as trophic hyphae, penetrate the
nematode cuticle and digest the body contents (Khan et al.
e Analysis Facility, Leve04.u, [email protected] Mycological Society
nematodes). Knob-free vegetative hyphae of M. lysipagum do
not capture or kill nematodes.
Although a number of interactions between nematode-
trapping fungi and nematodes have been described in some
detail for Arthrobotrys oligospora (Tunlid et al. 1992), which
catches nematodes with adhesive hyphal structures, consid-
erably less is known about the events involved in nematode
trapping by the knob-producing fungus M. lysipagum. The
knob produced by M. lysipagum penetrates the nematode cuti-
cle at the place of contact regardless of its location (Khan et al.
2006b). It has been shown that free nematodes are attracted
towards nematodes caught by a massive number of fungal
knobs on agar plates (Supplementary Material Movie Clip S3:
l 4, Building F7B, Research Park Drive, Macquarie University, Syd-
. Published by Elsevier Ltd. All rights reserved.
1448 A. Khan et al.
fungus signals nematodes). This indicates the possibility of
a chemical signal emanating from the knob to attract the
nematodes. Therefore, active compounds involved in the fun-
gus–nematode interactions may be specifically associated
with the fungal knobs. This notion is supported by a substan-
tial difference (23 %) in the gene expression pattern between
knob-producing and mycelial cells in M. haptotylum (Ahren
et al. 2005). Another nematode trapper, Dactylaria spp., catches
nematodes with sticky knobs, which are similar to the knobs
of Monacrosporium spp. In this case, antifungal antibiotics des-
ignated dactylfungins A and B, active against Candida pseudo-
tropicalis, were isolated from a liquid culture of D. paravispora
D500 (Xaio et al. 1993). Most of the work with proteins related
to fungus–nematode interactions reported so far has been
carried out with proteins secreted in liquid culture. Consider-
ably less information is available on the identification or
characterization of proteins extracted from the fungal mycelia
or specific trapping structures.
A unique, yet unidentified, carbohydrate-binding protein
not present in the vegetative cells has been isolated from
the adhesive cells of A. oligospora (Borrebaeck et al. 1984). In
another study, surface polymers from the trap cells of A. oligo-
spora were visualized using TEM and neutral sugars, uronic
acid, and proteins were extracted from these polymers (Tunlid
et al. 1991). There were more polymers present in the trap cells
than in vegetative hyphae, suggesting synthesis of yet uniden-
tified trap-specific compounds.
Current knowledge of genes and proteins of nematode-
trapping fungi that may be involved in the adhesion or
infection processes is not sufficient to understand the molec-
ular basis of the infection process. At the date of submission of
this manuscript, only 13 proteins (total 50 entries) from 13
Monacrosporium species were recorded in the TrEMBL database
(none in Swiss-Prot database) with nine from M. haptotylum
but none from M. lysipagum. Other than this, there is no
information available on the identification of any proteins
from Monacrosporium spp. Here we have undertaken a proteo-
mic approach to extract and separate proteins on two-
dimensional (2D) gels from mycelia containing knobs and
attempted identification of the proteins by peptide mass
fingerprinting to create an initial 2D gel map for M. lysipagum.
Fig 1 – Light micrograph show mycelia of Monacrosporium
lysipagum with knobs and a 2D gel image of the proteome.
(A) mycelia with 62 knobs on a slide. The arrows indicate
knobs that are observed as swellings on the mycelia.
Bar [ 20 mm. Proteins from M. lysipagum mycelia were ex-
tracted and separated on 2D gels on a linear pH range 4–7
(left to right) as shown in (B). Microscope slides were pre-
pared from the samples at the end of the growth period by
placing some mycelia in a drop of lactophenol cotton blue
on a slide, after which the sample was enclosed with
a coverslip and examined. Photographs were taken under
a stereomicroscope (Olympus).
Materials and methods
Culture of fungus
Monacrosporium lysipagum (IMI 375301) was isolated from an egg
mass of the root-knot nematode Meloidogyne javanica grown on
tomato in a glasshouse at the Macquarie University. The fungus
was grown on a medium containing 125 mg l�1 soy peptone
(Oxoid,Basingstoke, Hampshire, UK),and50 mg l�1 ofeachphe-
nylalanine (Sigma, St Louis, MO) and valine (Sigma) at pH 7.4 for
the production of knobs (Fig 1A) as described (Friman 1993). Cul-
ture medium (250 ml in a 1 l flask) was inoculated with 7 ml fun-
gal spores (6� 105 spores ml�1) harvested in sterile water from
a three-week-old culture grown on potato–carrot agar (PCA)
plates (Khan et al. 2006a). Inoculated flasks were incubated at
21 �C (�1 �C) at 125 rev min�1 for 10 d and the mycelia were har-
vested by centrifugation at 600 g for 15 min. Formation of knobs
on the mycelia were observed under a microscope (see Supple-
mentary Material for additional information). The mycelia were
then aliquoted to samples with approximately 200 mg wet
weight, dried under vacuum and stored at �20 �C until used. A
specimen of M. lysipaum (IMI 375301) used in this work has
been deposited in the Genetic Resources Collection of CABI Bio-
science (IMI), Egham, UK.
Extraction of proteins from mycelia
Proteins were extracted from 30 mg dried mycelia by
dissolving the samples in 2 ml of multiple chaotrope sample
solution containing 7 M urea, 2 M thiourea, 4 % (w/v) CHAPS
(3-[(3-Cholamidopropyl)-dimethyl-amino]-1 propanesulfo-
nate), 40 mM Tris, 5 mM TBP (Tri-n-butylphosphine), 10 mM
Proteomic analysis of a knob-producing nematode-trapping fungus 1449
acrylamide and a protease inhibitor cocktail as recommended
(Sigma). Mycelia in the sample solution were sonicated by an
ultrasonication probe (Branson, Danbury, CT, USA) for 3� 10 s
at 70 % amplitude followed by 10 min sonication in a water-
bath (Transsonic T 700 H, Elma, Germany). The sonicated
mycelial suspension was centrifuged at 20 000 g for 20 min
and the supernatant incubated at room temperature for
90 min for the reduction and alkylation of proteins. The alkyl-
ation reaction was then quenched by adding 10 mM DTT and
incubated for another 10 min. This protein solution was
aliquoted and stored at �70 �C until used.
2D gel electrophoresis
2D gel electrophoresis was carried out following standard
methods (Herbert et al. 2001) to separate the proteins
extracted from mycelia. For separation in the first dimen-
sion, approximately 200 mg protein (determined by the Brad-
ford assay using BSA as a standard) was loaded on an 11 cm
linear pH 4–7 immobilized pH gradient (IPG) strip (GE
Healthcare, Uppsala) by in-gel rehydration method. Gel di-
mensions and the gradient were 13� 8 cm and 6–15 % T, re-
spectively. The gels were poured in our laboratory using the
Tris–acetate buffer at pH 7. Triplicate gels were run and
stained with colloidal Coomassie G-250 as described (Her-
bert et al. 2001).
Detection of protein spots on the gels
Gel images were captured in tagged image file format (TIFF)
with a UMAX PowerLook III flatbed scanner (UMAX Technolo-
gies, Dallas, TX, USA) at 300 dpi. Images were pre-warped using
TT900 S2S v.2006 (Nonlinear Dynamics, Newcastle upon Tyne,
UK) with 90 warp vectors. The images were uploaded into Pro-
genesis Discovery 2005 image analysis software (Nonlinear Dy-
namics) using the analysis wizard and spots were manually
edited using Progenesis PG240, v.2006. Manual editing included
spot add, delete, join, and split. Artefactual spots (streak and
smear) and spots that were not possible to analyse were erased
from the images.
Sample preparation for mass spectrometric analysis
We have considered relatively dark spots on the gel for
identification of proteins because the genome of Monacrospo-
rium lysipagum is unknown hence the chances are smaller
for getting identification of faint spots due to a low number
of peptide matches is likely. Proteins from the gel were
excised (250 spots), washed, dried, and digested with trypsin
as reported earlier (Khan et al. 2005) and zip tipped automati-
cally on 96-well plates using Xcise�, a bench-top robotic
protein processing system for mass spectrometric analysis
(Shimadzu Biotech, Nakagyo-ku, Kyoto, Japan). Trypsin used
in this work was porcine sequencing-grade modified trypsin
(Promega, San Luis Obispo, CA, USA).
Peptide mass fingerprinting
Matrix-assisted laser desorption ionization time-of-flight
mass spectrometry (MALDI-TOF MS) was performed on an
Axima CFR instrument (Shimadzu Kratos, Manchester, UK),
equipped with an N2 laser (337 nm, 10 Hz repetition rate) as
described (Wilson et al. 2002). Internal two-point mass calibra-
tion was automatically performed on two auto-digested
tryptic peptides (mono-isotopic masses 842.51 and 2211.10).
Peaks of peptide masses were harvested by using a peak-
picking tool Peak Harvester V 1.5 (Breen et al. 2000). Two blank
gel plugs were cut and digested from an area outside of the IPG
strip and the masses generated from these plugs were sub-
tracted from the peptide mass list of each protein spot. This
removed autolysed trypsin, matrix, and Coomassie peaks.
Database search and protein identification
The peptide mass range of 600–3000 Da (with mass tolerance
of 50 ppm) was used for the Swiss-Prot database (Swiss-Prot
2006.02.21) search using the MS-Fit tool in Protein Prospector
(http://prospector.ucsf.edu/) to identify proteins. The genome
of Monacrosporium lysipagum is unknown, therefore, the data-
base was searched against all data from all species. The search
parameters were restricted by the apparent molecular weight
(�20 %) and pI (�1 unit) of the proteins displayed on the gel
and one missed cleavage of a peptide was allowed. Cysteine
alkylation (by acrylamide), methionine oxidation, and lysine
methylation were considered for modification of peptides in
the primary level search. When the peptide masses (at least
four) were matched to protein sequences in the database,
a number of parameters were considered such as number of
missed cleavage peptides and modified peptides, intensities
of matched peptides and sequence coverage for the
identification of proteins in the secondary level search as
described earlier (Khan & Packer 2006). Additionally, positions
of matched peptides in the matched protein sequence were
also considered. For example, if the majority of the peptides
matched in a scattered position rather than to peptides close
to each other in the protein sequence, this was not considered
to be identification.
Results
Separation and detection of protein spots on 2D gels
Multiple chaotrope sample solution that contained the CHAPS
detergent was used for global proteome extraction. Final con-
centration of protein in the extracted solution was 2 mg ml�1
and this equals to 13 % (approximately) of the initial dry myce-
lia used for protein extraction. In this work we did not pursue
optimization of the sample preparation, for example, for
membrane associated proteins. Despite this limitation, a sig-
nificant number of proteins were resolved with an average
of 1027 spots (present in all three gels) on the gel (n¼ 3,
S.D.� 9) detected in the pH range 4–7 (Fig 1).
Identification of proteins in mycelia with knobsPeptide masses were carefully obtained by eliminating the
background signals and peptide masses ranging from 600–
3000 Da were used for database search. A conservative view
was taken to assign an identification of a protein as all the pro-
teins were matched to organisms other than Monacrosporium
Table 1 – Summary of the proteins identified from Monacrosporium lysipagum mycelia with knobs
Spotno.a
Accessionno.
Organism Name of the protein Matchedpeptides
Sequencecoverage
Mass pI
1 P10443 Escherichia coli DNA polymerase III alpha subunit 14 17 129906 5.2
2 P15716 E. coli ATP-dependent clp protease clpA 10 20 84208 5.9
3 Q03587 Thermoplasma acidophilum DNA-directed RNA polymerase B 20 27 134692 6.5
4 P10443 E. coli DNA polymerase III alpha subunit 10 14 129906 5.2
5 P46598 Candida albicans Heat shock protein 90 homologue 18 27 80824 4.8
6 P46598 C. albicans Heat shock protein 90 homologue 15 21 80824 4.8
7 P53623 Pichia angusta Heat shock protein 70 2 16 27 70073 4.9
8 P53623 P. angusta Heat shock protein 70 2 22 44 70073 4.9
9 P32590 Saccharomyces cerevisiae Heat shock protein homologue SSE2 10 18 77621 5.5
10 P46817 Mycobacterium bovis Peroxidase/catalase 12 18 80577 5.1
11 P40747 Bacillus subtilis Hypothetical oxidoreductase yuxG 12 25 76021 5.8
12 Q18486 Caenorhabditis elegans Ubiquinone biosynthesis protein coq-8 12 25 83615 6.8
13 P48373 Streptococcus pneumoniae DNA gyrase subunit B 11 24 72238 5.4
14 P38315 Saccharomyces cerevisiae YAP1-binding protein 1 13 23 77741 5.5
15 O69460 M. leprae ATP-dependent DNA helicase recG 11 18 81505 6.7
16 P46493 Haemophilus influenzae Threonine dehydratase biosynthetic 16 29 56663 6
17 P46493 H. influenzae Threonine dehydratase biosynthetic 17 31 56663 6
18 Q9UVW9 Acremonium sp. Physcomitrella patens Actin, gamma 14 42 41608 4.5
19 Q9XFG3 Alternaria alternata Tubulin gamma chain 7 18 53292 5.9
20 Q9HDT3 Methanococcus jannaschii Enolase (2-phosphoglycerate dehydratase) 8 19 47206 5.2
21 Q57689 Alternaria alternata Arginyl-tRNA synthetase 22 40 64980 6
22 Q9HDT3 Botrytis cinerea Enolase (2-phosphoglycerate dehydratase) 8 17 47206 5.2
23 O13419 M. jannaschii Actin 26 71 41640 5.4
24 Q57880 S. cerevisiae Hypothetical protein MJ0438 9 37 43739 6.4
25 P15019 Botrytis cinerea Transaldolase 13 32 37037 6.1
26 O13419 Penicillium citrinum Actin 27 72 41640 5.4
27 P33161 Schizosaccharomyces pombe Phosphoglycerate kinase 13 30 44074 6.1
28 P31411 Penicillium chrysogenum Vacuolar ATP synthase B 8 15 55842 5.2
29 Q9URS0 Saccharomyces cerevisiae Actin, gamma 7 30 41757 5.4
30 P00830 S. cerevisiae ATP synthase beta chain 12 29 54924 5.7
31 P00830 S. cerevisiae ATP synthase beta chain 15 33 54924 5.7
32 P00830 S. cerevisiae ATP synthase beta chain 17 36 54924 5.7
33 P00830 S. cerevisiae ATP synthase beta chain 13 29 54924 5.7
34 P00830 S. cerevisiae ATP synthase beta chain 8 18 54924 5.7
35 P00830 Botrytis cinerea ATP synthase beta chain 8 18 54924 5.7
36 P53373 Botrytis cinerea Tubulin beta chain 14 28 49798 4.9
37 P53373 Botrytis cinerea Tubulin beta chain 17 28 49798 4.9
38 P53373 Mycosphaerella graminicola Tubulin beta chain 16 25 49798 4.9
39 O94128 Pestalotiopsis microspora Tubulin alpha chain 11 25 50009 4.9
40 Q9UV72 M. graminicola Tubulin beta chain 5 18 49836 4.8
41 O94128 Phaeosphaeria nodorum Tubulin alpha chain 14 30 50009 4.9
42 P41799 M. graminicola Tubulin beta chain 9 19 49939 4.9
43 O94128 Schizosaccharomyces pombe Tubulin alpha chain 9 21 50009 4.9
44 Q9P546 Trichoderma reesei 40S ribosomal protein S0-B 6 22 31421 5
45 Q9HEM9 T. reesei 14-3-3 like protein 9 27 30422 4.8
46 Q9HEM9 Arabidopsis thaliana 14-3-3 like protein 4 14 30422 4.8
47 Q96299 Saccharomyces cerevisiae 14-3-3-like protein GF14 mu 6 23 29520 4.9
48 P15019 S. cerevisiae Transaldolase 17 43 37037 6.1
49 P15019 T. harzianum Transaldolase 12 27 37037 6.1
50 Q99002 T. harzianum 14-3-3 protein homologue 13 37 29998 5.8
51 Q99002 14-3-3 protein homologue 12 39 29998 5.8
a A 2D gel map of these identified proteins is shown in Fig 2.
1450 A. Khan et al.
spp. After peptide masses were matched to proteins at the
primary level search, additional parameters were considered
in the secondary level search to increase the confidence prior
to assigning an identification of a protein.
Out of the 250 protein spots analysed, 51 proteins (20 %)
encoded by 31 genes were identified by MALDI-MS (Table 1,
Fig 2) using a cross-species identification method. This dataset
represents one of the largest 2D gel maps of proteins from
a filamentous fungus for which the genome has not been
sequenced, and is the first report on proteomic analysis of
Monacrosporium spp. The majority of the proteins identified
from mycelia with knobs were either house-keeping proteins
or enzymes and membrane-associated proteins. However, we
identified two membrane-associated proteins, including one
mitochondrial protein, enolase, and an ATP synthase beta
chain from a fungus and yeast, respectively. Cytoplasmic
Fig 2 – A 2D gel map of proteins from the mycelia of Mona-
crosporium lysipagum with knobs. The proteins were iden-
tified using MALDI-TOF MS analysis and a summary of the
identified proteins numbered on the gel is presented in
Table 1.
Proteomic analysis of a knob-producing nematode-trapping fungus 1451
proteins are more conserved across species and, therefore,
there is a better chance to identify them by a cross-species
identification method. Conversely, structural proteins are
more specific to fungal species, particularly the fungal cell
wall, and they are difficult to identify by a cross-species iden-
tification method.
Discussion
The key objective of this study was to create an initial 2D gel
map of the proteome of Monacrosporium lysipagum with protein
identifications. This proteome map will aid identification of
proteins from Monacrosporium spp. to facilitate further studies
into the infection process. Initially, we did attempt to isolate
knobs from the mycelia of M. lysipagum following the method
described (Friman 1993) for extraction of proteins from the
knobs but it was found that some mycelium remained at-
tached with the knobs and it was not possible to collect
enough material for the proteomic study (see Supplementary
Material for additional information). Therefore, proteins were
extracted and identified from the mycelia with knobs.
We followed a method for global proteome extraction with
no further modifications and were able to separate 1027 pro-
teins at a high resolution. The number of proteins separated
and the resolution obtained were comparable with those
reported by Grinyer et al. (2004) for a mycoparasitic biocontrol
fungus, Trichoderma harzianum. Despite obtaining good-quality
mass spectrometry data, just over 20 % of the proteins were
identified using cross-species matching. Even so, success of
protein identification here is comparable with other studies
involving fungi for which there is no genomic information
available. For example, in the work of Grinyer et al. (2004),
out of the 96 spots analysed, 25 spots related to 22 genes
were identified from the total protein extract of T. harzianum
using a cross-species identification method. In another study,
20 spots (out of 56 spots analysed) were identified from the cell
envelope of T. reesei using cross-species identification before
the genome sequence was disclosed (Lim et al. 2001). It was
beyond the scope of this study to generate, for example, a large
number of antibodies against the identified proteins for vali-
dation of the identification process.
The majority of the proteins identified from mycelia with
knobs were either house-keeping proteins or enzymes and
membrane-associated proteins. Our findings are in concert
with the study by Ahren et al. (2005) who carried out a gene
expression microarray study with M. haptotylum. Homologues
for several genes that were up-regulated in knob cells in-
cluded genes encoding glycogen phosphorylase, ubiquinol,
cytochrome c oxireductase, alkaline serine protease, cuticle-
degrading serine protease, ribosomal proteins, heat-shock
proteins, and ATP synthase (Ahren et al. 2005). Actin, a com-
monly identified and well-conserved protein across species
is involved in cell polarity (Ahren et al. 2005). This protein,
also identified in our work, may be involved in quick forma-
tion of knobs of M. lysipagum in the presence of prey or culture
substrate, such as the two amino acids added in the culture
medium. We also identified a clp protease in mycelia produc-
ing knobs, similar to a fungal secreted serine protease.
In summary, we were able to separate 1027 proteins on the
gel pH range 4–7 and 51 proteins were identified. The missing
genome data is a major factor for the relatively modest iden-
tification rate of proteins of M. lysipagum. The low success
may also imply that there are more differences in the proteins
from different fungal species than has been anticipated.
Acknowledgement
We thank Mark P. Molloy (Australian Proteome Analysis
Facility) for preliminary review of the manuscript.
Supplementary material
Supplementary data associated with this article can be found,
in the online version, at doi: 10.1016/j.mycres.2008.06.003.
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