A Genomic and Proteomic Investigation of the Plant Pathogen Armillaria mellea: Buried Treasure or Hidden Threat? Cassandra Collins B.Sc. 26 th July 2013 N.U. I. Maynooth
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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
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 speciesPertot 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.
Natures toolbox for biotechnology
Biomedical
Feedstock industry
Agro-industrial waste
utilisation
EthanolBio-diesel
Secondary metabolites
Hydrolytic enzymes
PeptidasesCytotoxic 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 AntiviralsAntifungalsPolymer
synthesis Biosensors
Fungi - potential applications
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 proteins3. 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
A. mellea Genomic Sequence 58.3 Mb
Assembly generated in collaboration by Sanger sequencing team using Velvet
softwareScaffolds Contigs
N50 36,679 5,486Largest 639,705 154,911Count 4,377 15,215
Total length 58,385,340 81,738,977Putative ORF’s located using the gene finding
software, AUGUSTUSA BLASTP search of ORFs against GenBank
and cDNA database constructed
A. MelleaDSM 3731
L. BicolorS238N-H82
C. Cinerea Okayama
7#130Sequencing Institution
NUIM/Sanger Institute
Joint Genome Institute
BroadInstitute
Genome Assembly 58.3 Mb 64.9 Mb 37.5 MbGC 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 1,352Avg. exon length 217.5 210.1 251Avg. intron length 73.6 92.7 75Avg. No. introns/gene
4.72 4.44 4.66
A. mellea Genome Statisitcs
Phylogenetic SupertreeSupertree
A. mellea phylogeny•Basidiomycotina subphyla•Agaricomicotina•Homobasidiomycetes•Agricarales Closely related to Laccaria bicolor - ectomycorrhizalCoprinopsis cinerea -model organismAgaricus bipsorus – edible (button mushroom)
Closest relative Moniliophthora perniciosa - phytopathogen
Biological Process
Cellular Component
Molecular Function
A. mellea Genome AnnotationBiological ProcessMolecular Function
Cellular Component
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
A. mellea morphologyLiquid, solid and shaking cultures
Protein Identification
61
27
57
26
60
56
160
10
19
28
29
30
51
62
1315
132
181
4038
182
155158
59
199
185
65
64
156
184
63
66
198
161
6768
69
157
20
16
71
72
200
75144
70
73
79
149
149
203
24
14614
8
204
74
147
173174
205
80
193
206
49
82
145
164
78
81
76 77
175
54
5
12
83
166
191
194
37
97
190 6
45
868552
21
5558
225384
8788 10
892 91
50 4296 104
103
99
1 11100
101
107 10
6
105
289
110
10911
4113
10234 90
111
112
98
187
18818
9121
9394
95 41
120119
117
186
118
116
115
131
129 1
30
12513
3
4847461
92
44 12
4123
122
128
127
43
39
18180
179
178
176
177
183
17202 16
5
167
14
17235
171
343136
3233
79
8
126
197
153
195
16816
9170
162
141
19615
4
152
151
150
143 14
2140
163
139
138137
13413
5136
103pIkDa
150
75
50
20
10
Mycelia Supernatant
Shotgun
2-DE - 279 Mycelial proteins SDS-PAGE - 340 Secreted proteins
Shotgun - 613 proteins
Proteins identified
Protein Identification 2-DE SDS-
PAGEShotgu
n OFFGEL
No. of Proteins 274 340 613 91Structural featuresSignal P 20 141 32 4Secretome P 128 113 260 44Hydrophobic proteins 35 82 80 9Proteins with transmembrane domains
33 32 63 5
Comparison of A. mellea Proteins and structural features by Method
Protein Identification by protein type
Protein Identification
Mycelial
2-DE
Supernatant
SDS-PAGE
Shotgun
CAZys 7 59 21Laccase 5Metalloproteins 2Oxidoreductase 3 5Oxygenase, peroxidase, P450 5 12Peptidase 12Ribosomal protein 3 63Ribonuclease 3Heat Shock proteins 15Hypothetical proteins 19 30 32Predicted proteins 6 15 17A. mellea specific 2 8 15
pH 3 pH 11 pH 3 pH 11
Hydrogen peroxideH2O2 → 2 •OH
Menadione/FeCl31. 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
Protein ID BLAST InterPro H2O2 Menadione
FeCl3 SecretomeP
Score
Am14558 Valosin-containing protein Cytoskeleton-associated protein. ↑ ↓ 0.357
Am172775-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
Methionine and Polyamine synthesisSAM cycle
Polyamine homeostasis and recycling
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
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.
A. mellea as a model systemCandida albicans 98% mortality
Baumgartner et al. (2011) Molecular Plant Pathology 12: 20.Losada et al. (2009) Medical Mycology 47: S88-S96.
Series10
102030405060708090
100
Mono-culture Co-culture
C. albicans cultures from excised co-culture plugs 30 h post inoculation
Viability (%) ***
p=0.0004
Mono-culture
Co-culture
Co-cultures of A. mellea and C. albicans Proteins identified by shotgun proteomics (n=205)
Protein TCAPrecipitated
TotalProtein 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 109:267Fragner et al. (2009) Electrophoresis 30:2431
Spectrum Millsearch of
A. mellea cDNA Database
Bioinformatic analysisUnique; 3Hypothetical; 12
Predicted ; 6
Known; 184
Proteins Uniquely Identified from A. mellea/C. albicans co-cultures (n
=30)Enzyme code & GO annotation
Accession Number BLAST annotation Mean
SimilarityCoverage
% 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
Proteins Uniquely Identified from A. mellea/C. albicans co-cultures
GO annotation only Accession Number BLAST annotation Mean
SimilarityCoverage
% 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
Proteins Uniquely Identified from A. mellea/C. albicans co-cultures
No enzyme or GO annotationAccession Number BLAST annotation Mean
SimilarityCoverage
% 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.
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
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
Presentations1. 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 - Oral3. 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