1. A Genomic and Proteomic Investigation
of the Plant Pathogen
Armillaria mellea: Buried Treasure or
Hidden Threat?
Cassandra Collins B.Sc.
26th July 2013 N.U. I. Maynooth

2. Armillaria mellea major plant pathogen
>600 host species
• Mainly woody plants but also
herbaceous plants and
even weeds.
• Virulent on agronomic crops
– Managed forests
– Fruit crops
• Pathogenicity and virulence
pathways unknown
American Phytopathological Society
– high priority species
Pertot et al. (2008). Crop Protection 27: 1061–1070.
Williams et al. (1989). Forest Insect and Disease Leaflet 78.
American Phythopathological Society (2008). APS Centennial Meeting 2008, pp. 9–181.

3. Natures toolbox for biotechnology
Biomedical
Feedstock
industry
Agro-industrial
waste utilisation
EthanolBio-diesel
Secondary
metabolites
Hydrolytic
enzymes
Peptidases
Cytotoxic
enzymes
Immunostimulatory
enzymes
LaccasesXylanases Cellulases
Bioremediation Antimicrobials
Textile
industry
Food industry
Industrial
enzymes
Anti-
inflammatory
Alves, A. and Record, E. (2004). Applied and Environmental Microbiology 70: 6379–6384.
Bouws et al. (2008). Applied Microbiology and Biotechnology 80: 381–8.
Dwivedi et al. (2011). Journal of Molecular Catalysis B-Enzymatic 68: 117–128.
Erjavec et al. (2012). Trends in biotechnology 30: 259–273.
Grigoriev et al. (2011). Mycology 2: 192–209.
Osma et al. (2010). Enzyme research 2010: 918761.
Paper
industry
Antioxidants AntiviralsAntifungals
Polymer
synthesis
Biosensors
Fungi - potential applications

4. 1. Sequencing of the Armillaria mellea genome, construction
of cDNA database and annotation of the gemome.
2. Development of methods
• culture protein extraction from both mycelia and
secretome
• identification of proteins after LC-MS/MS analysis
• interrogation of the cDNA database followed by
bioinformatic analysis of identified proteins
3. Novel LC-MS/MS methods of analysis of complex A.
mellea protein mixtures from the A. mellea mycelial
proteome and secretome
4. Oxidative stress induction to identify proteins
differentially regulated under two stressors
5. Novel infection model development to identify proteins
which may be uniquely expressed during A. mellea
infection
Thesis objectives

5. A. mellea Genomic Sequence
58.3 Mb
Assembly generated in collaboration by Sanger
sequencing team using Velvet software
Scaffolds Contigs
N50 36,679 5,486
Largest 639,705 154,911
Count 4,377 15,215
Total length 58,385,340 81,738,977
Putative ORF’s located using the gene finding software,
AUGUSTUS
A BLASTP search of ORFs against GenBank
and cDNA database constructed

6. A. Mellea
DSM 3731
L. Bicolor
S238N-H82
C. Cinerea
Okayama 7#130
Sequencing
Institution
NUIM/Sanger
Institute
Joint Genome
Institute
Broad
Institute
Genome Assembly 58.3 Mb 64.9 Mb 37.5 Mb
GC content 48.1% 46.6% 51.6%
No. protein coding
genes
14,473 20,614 13,544
Coding sequence <300
bp
957 2191 838
Avg. CDS length 1,228 bp 1,134 bp 1,352
Avg. exon length 217.5 210.1 251
Avg. intron length 73.6 92.7 75
Avg. No. introns/gene 4.72 4.44 4.66
A. mellea Genome Statisitcs

7. Phylogenetic Supertree
Supertree
A. mellea phylogeny
•Basidiomycotina subphyla
•Agaricomicotina
•Homobasidiomycetes
•Agricarales
Closely related to
Laccaria bicolor -
ectomycorrhizal
Coprinopsis cinerea -
model organism
Agaricus bipsorus – edible
(button mushroom)
Closest relative
Moniliophthora perniciosa
- phytopathogen

8. Biological
Process
Cellular
Component
Molecular
Function
A. mellea Genome Annotation
Biological Process
Molecular Function
Cellular Component

9. Key Gene Families in the A.mellea genome
•CAZys
‒CBM 7 Families 15 genes
‒CE 8 Families 98 genes
‒GH 48 Families, 223 genes
‒GT 23 Families 90 genes
‒PL 4 Families 18 genes
•Expansins 20 genes
•F-box ubiquitin proteosome 71 genes
•Hydrophobins 31 genes
•Metalloenzymes 45 genes
•Oxidoreductases 89 genes
•PPR 10 genes
•Retrotransposable elements 567 genes
•Ribonucleases 424 genes
•Ribosomal proteins 115 genes
•Ribonuclease inhibitor 192 genes
•Hypothetical proteins 2333 genes
•Predicted proteins 921 genes
•A.mellea specific proteins 2721 genes
•Secondary metabolism
–Terpene synthases 38 genes
–NRPS 12 genes
–PKS 11 genes
•Redox
–Cytochrome P450 248 genes
–Oxygenases 92 genes
–Oxidases and peroxidases 118
genes

10. A. mellea morphology
Liquid, solid and shaking cultures

11. Protein Identification
61
2
7
57
26
60
56
16
0
10
19
28
29 30
51
62
13
15
13
2
18
1
40
38
18
2
15
5
15
8
59
19
9
18
5
6564
15
6
18
4
63
66
19
8
16
1
6
768
6
9
15
7
20
16
71
72
20
0
75
14
4
70
73
79
14
9
14
9
20
3
24
14
614
8
20
4
74
14
7
17
317
4
20
5
80
19
3
2
0
6
49
82
14
5
16
4
78
81
76
77
17
5
54
5
12
83
16
6
19
1
19
4
37
97
19
0
6
45
8685
52
21
55
58
22
53
84
87
88 10
892
91
50 4296 10
4
10
3
99
1 1110
0
1
0
1
10
7 10
6
10
5
2
89
11
0
10
911
4
1
1
3
10
2
34 90
11
1
11
2
98
18
7
18
818
9
12
1
93
9
4
95
4
1
12
0
11
9
11
7
1
8
6
1
1
8
11
6
11
5
13
1
12
9 1
3
0
12
513
3
48
47461
9
2
4
4 12
4
12
3
12
2
12
8
12
7
43
39
18
18
0
17
9
17
8
17
6
17
7
18
3
1720
2 16
5
16
7
14
17
235
17
1
343
136
3233
7
9
8
12
6
19
7
15
3
19
5
16
816
917
0
16
2
14
1
19
615
4
15
2
15
1
15
0
14
3 14
2
14
0
16
3
13
9
13
8
13
7
13
4
13
5
13
6
103
pIkDa
150
75
50
20
10
Mycelia Supernatant
Shotgun

12. 2-DE - 279 Mycelial proteins SDS-PAGE - 340 Secreted proteins
Shotgun - 613 proteins
Proteins identified

13. Protein Identification 2-DE
SDS-
PAGE
Shotgun OFFGEL
No. of Proteins 274 340 613 91
Structural features
Signal P 20 141 32 4
Secretome P 128 113 260 44
Hydrophobic proteins 35 82 80 9
Proteins with
transmembrane domains
33 32 63 5
Comparison of A. mellea Proteins
and structural features by Method

14. Protein Identification by protein type
Protein Identification
Mycelial
2-DE
Supernatant
SDS-PAGE
Shotgun
CAZys 7 59 21
Laccase 5
Metalloproteins 2
Oxidoreductase 3 5
Oxygenase, peroxidase, P450 5 12
Peptidase 12
Ribosomal protein 3 63
Ribonuclease 3
Heat Shock proteins 15
Hypothetical proteins 19 30 32
Predicted proteins 6 15 17
A. mellea specific 2 8 15

15. pH 3 pH 11 pH 3 pH 11
Hydrogen peroxide
H2O2 → 2 •OH
Menadione/FeCl3
1. Fe3+ + •O2
− → Fe2+ + O2
2. Fe2+ + H2O2 → Fe3+ + OH− + •OH
•O2
- + H2O2 → •OH + OH- + O2
Fe3+
Oxidative Stress of A. mellea (3 h)
ROS damage - proteins, lipids, carbohydrates and
DNA.
Differentially expressed proteins

16. Protein ID BLAST InterPro H2O2
Menadione
FeCl3
SecretomeP
Score
Am14558 Valosin-containing protein Cytoskeleton-associated protein. ↑ ↓ 0.357
Am17277
5-methyltetrahydropteroyltriglutamate-
homocysteine s-methyltransferase
Vitamin-B12 independent
methionine synthase
↑ ↑
0.472
Am7160 Heat shock protein hsp70 Heat shock protein Hsp70. ↑ 0.333
Am18454 Heat shock protein Heat shock protein 70 ↑ 0.287
Am3211 Pyruvate decarboxylase Thiamine pyrophosphate enzyme ↑ 0.48
Am14050 Saccharopine dehydrogenase Spermine synthase. ↑ 0.587
Am3212
Zinc found in the cytoplasm and
intermembrane space of mitochondria
Peptidase M3A/M3B. ↑ 0.426
Am19877
Glutamic oxaloacetic transaminase
aat1
Aminotransferase. ↑ 0.566
Am16706
a-pheromone processing
metallopeptidase ste23
Peptidase M16. ↓ 0.393
Am7452 Heat shock protein 90 Heat shock protein Hsp90 ↓ 0.147
Am19873 Translation elongation factor 1a
Translation elongation factor
EF1A.
↓ 0.235
Am19381 3-ketoacyl-CoA -thiolase peroxisomal Thiolase-like protein. ↓ 0.548
Am13458 Alcohol oxidase-like protein GMC oxidoreductase ↓ 0.438
Am9629 Glycoside hydrolase family 3 protein Glycoside hydrolase, family 3. ↓ 0.464
A. mellea under oxidative stress
Differentially regulated proteins (n=14)
identified p <0.05

17. Methionine and Polyamine synthesis
SAM cycle

18. Polyamine homeostasis and recycling

19. A. mellea
culture
C. albicans
culture
Incubated for 7 d at
25 °C
Incubated for 21 d
at 25 °C
Cultures harvested
Direct LC-MS/MS
A. mellea
culture
A. mellea /C. albicans co-
culture
C. albicans
culture
Plugs excised,, incubated on
MEA for 30h at 37 °C
Colonies counted Cells stained with FDA/PI
Armillaria mellea
Fungal-fungal interaction

20. 1. C. albicans live
culture (24 h)
2. C. albicans killed by
autoclaving
3. Monoculture of C.
albicans
4. C. albicans following
co-culture with A.
mellea.
Magnification: 20X.
C. albicans cell viability
Simultaneous FDA (live) and PI (dead) cell
staining of C. albicans
Jones, K.H. and Senft, J.A. (1985). Journal of Histochemistry & Cytochemistry 33: 77–79.

21. A. mellea as a model system
Candida albicans 98% mortality
Baumgartner et al. (2011) Molecular Plant Pathology 12: 20.
Losada et al. (2009) Medical Mycology 47: S88-S96.
Mono-culture Co-culture
0
10
20
30
40
50
60
70
80
90
100
Viability (%)
C. albicans cultures from excised co-culture
plugs 30 h post inoculation
***
p=0.0004
Mono-culture
Co-culture

22. Co-cultures of A. mellea and C. albicans
Proteins identified by shotgun proteomics
(n=205)
Protein TCA
Precipitated
Total
Protein Digested
LC-MS/MS
Analysis
Secreted
proteins from agar
suspended in
50 mM potassium
phosphate pH 7;5
Imanaka et al.(2010) J Biosci Bioeng 109:267
Fragner et al. (2009) Electrophoresis 30:2431
Spectrum Mill
search of
A. mellea cDNA
Database
Bioinformatic
analysisUnique, 3
Hypothetical
, 12
Predicted ,
6
Known, 184

23. Proteins Uniquely Identified from
A. mellea/C. albicans co-cultures (n =30)
Enzyme code & GO annotation
Accession
Number
BLAST annotation
Mean
Similarity
Coverage
%
Peptides
GRAVY
score
TM
SigP/
SecP
Enzyme Codes GOs
Am12506 Aryl-alcohol oxidase 64% 2.7 1 -0.1 SigP EC:1.1.99.1 3
Am17545 Coproporphyrinogen iii oxidase 66% 15.7 4 -0.1 2 EC:1.3.3.3 3
Am10593 Cys 2 peroxiredoxin 86% 6.5 1 -0.2 SecP EC:1.11.1.15 5
Am9607 Cytochrome c oxidase subunit v 57% 7.5 1 -0.3 2 SecP EC:1.9.3.1 2
Am12218
Hypothetical protein
SERLADRAFT_459096 [Serpula
lacrymans varS7.9]
66% 4.1 1 -0.2 SigP EC:3.4.11.0 3
Am12353 Iron-sulfur protein subunit 89% 9.7 1 -0.5 SecP EC:1.3.99.1 8
Am19926 Lipoic acid synthase 85% 3.3 1 -0.2 1 EC:2.8.1.8 4
Am18628 Subunit vib of cytochrome c oxidase 84% 15.1 1 -0.8 SecP EC:1.9.3.1 7
Am15086 Succinate-semialdehyde dehydrogenase 74% 2 1 0.1 4 SecP EC:1.2.1.0 2
Am18503 Uracil phosphoribosyltransferase 68% 10.3 1 0.2 SecP EC:2.4.2.9 3
Am14973 Urea hydro-lyase cyanamide hydratase 64% 8.4 1 -0.2 SecP EC:3.1.4.0 2
Lignin
degradation
Peroxiredoxin
Peptidase M28

24. Proteins Uniquely Identified from
A. mellea/C. albicans co-cultures
GO annotation only
Accession
Number
BLAST annotation
Mean
Similarity
Coverage
%
Peptides
GRAVY
score
TM
SigP/
SecP
GOs
Am5344 60s ribosomal protein l10a 97% 10.7 1 -0.7 SecP 6
Am16128 Glycerol-3-phosphate o-acyltransferase 78% 3.3 1 0 3 2
Am13890
Glycosyl hydrolase 53 domain-containing
protein
63% 14.7 1 0 SigP 3
Am13814
Hypothetical protein CC1G_12365
[Coprinopsis cinerea okayama7#130]
69% 18.7 1 -0.3 5
Am13829
Hypothetical protein SCHCODRAFT_107411
[Schizophyllum commune H4-8]
50% 7.9 2 -0.3 SecP 2
Am13379 NADH dehydrogenase 77% 3.2 1 -0.2 2 SecP 3
Am14001 Prohibitin phb1 76% 7.9 1 0.1 SigP 1
Am20343 Predicted protein 77% 21.2 1 -0.3 SecP 3
Am20304 Proteolysis and peptidolysis-related protein 89% 6.6 1 -0.2 SecP 1
Am16124 Sulfide-quinone oxidoreductase 79% 4 2 -0.4 2 3
Am7929 Thioredoxin 69% 15.6 1 0.1 5
Am2793 Ubiquitin domain-containing 70% 6.4 1 -0.2 1
Class II Chitinase
Negative regulator of
cell proliferation
Mitochondrial Protein
Thioredoxin
Thioredoxin

25. Proteins Uniquely Identified from
A. mellea/C. albicans co-cultures
No enzyme or GO annotation
Accession
Number
BLAST annotation
Mean
Similarity
Coverage
%
Peptides
GRAVY
score
TM
SigP/
SecP
Am19980 F1f0-atpsyn f 59% 14.5 1 -0.1 1 SecP
Am18856
Hypothetical protein SCHCODRAFT_237540
[Schizophyllum commune H4-8]
51% 6.1 1 0.4 4 SigP
Am3423
Protein - Haustorially expressed (1→6)-β-
glucan synthesis
63% 18.1 1 0.2 SigP
Am6084 Predicted protein 45% 9.8 1 -0.5 SecP
Am17796 Secreted protein 67% 12.1 2 0
Am14705 Twin-arginine translocation pathway signal 50% 4.9 1 0 SecP
Accession
Number
BLAST annotation
Coverage
%
Peptides
GRAVY
score
TM
SigP/
SecP
Am16692 A. mellea novel protein 6.5 1 -0.4Novel A. mellea
protein
Haustorially
expressed -
avirulence
TAT secretion pathway.
Protein transport.

26. Future work
Conclusions
•Transcriptomics of A. mellea - RNAseq
•Identification hypothetical, predicted and A. mellea
specific protein functions
•Gene expression studies and differential regulation
• Further infection studies
•Quantitative proteomics
•The genome of A. mellea has been sequenced and published on
public databases containing 14473 genes
Methods for culture of A. mellea of nutrient replete/restricted
media were developed
•A novel “Shotgun” proteomic method for protein analysis was
developed
•980 proteins from the genome of A. mellea were identified
•Oxidative stress induction in A. mellea identified 14 differentially
regulated proteins some involved in polyamine biosynthesis
•A novel infection model of A. mellea co-culture with C. albicans
identified 30 proteins uniquely expressed in co-culture

27. This project was funded by: John & Pat Hume Scholarship National University of Ireland Maynooth, National University of Ireland
Travelling Studentship, LC-MS/MS facilities Health Research Board., Higher Education Authority, PRTLI-4.
David Fitzpatrick, Thomas Keane1, Dan Turner1, Grainne O’Keeffe and Sean Doyle.
Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland.
1Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK
Presentations
1. Armillaria mellea – Fruit Producers Nightmare or Buried Treasure? SIPP, NUIM April 2010 – Oral.
2. Armillaria mellea – Fruit Producers Nightmare or Buried Treasure? NUIM, May 2010 - Oral
3. Life is sweet; new insights from protein mass spectrometry of Armillaria mellea. Waters Prize Symposium UCD,
January 2011 – Oral.
4. A shotgun BLAST to Armillaria mellea’s secrets. NUIM, June 2011 - Oral.
5. Sweet secrets from a proteomic investigation of the honey fungus Armillaria mellea. IFS, TCD, June 2011 –
Oral.
6. A Shotgun BLAST reveals Armillaria mellea’s Proteomic Secrets. Computational Biology & Innovation PhD
Symposium, UCD, December 2011 - Oral.
7. Armillaria mellea a plant pathogen under stress. NUIM, July 2012 - Oral.
8. CSI Fungi. Laboratory seminar series, NUIM, February 2013 - Oral.
9. High-throughput DNA Sequencing and Proteomic Analysis of Armillaria mellea – Fruit Producers Nightmare or
Buried Treasure? IMC9, Edinburgh. August 2010. Poster.
10. Armillaria mellea a plant pathogen under stress. 2nd Irish Fungal Society Meeting. Belfast City Hospital, June
2012. Poster. Acknowlegements