For the HH3220SMU Final Project Report

HH3220SMU Appendices

The following pages encompass Tables, Figures, Additional Data Notes and References (Literature Cited) for the HH3220SMU final project report

TABLES

Table 1. Screening of cDNA infection library for Agaricus and Verticillium sequences. ABVF clone number is location in macro- arrayed cDNA library. Probe (blot) refers to SSH sequence used for library screening. Some probes identified more than one (closely related) gene type during hybridisations. Likely host or pathogen origin is indicated; ‘???’ remain ambiguous or undetermined.

Library Number (ABVF) size Organism Sequence homology Full length Probe (blot) Macro-array location bp host / pathogen Database searching

107O5 362 ??? ??? ??? ras1p / exo 1,3 gluconase (P4E8/P1G6) MJS1 106E15 618 Agaricus NADH oxidase (CAA43221.1 ) YES NADH oxidase/DAHP synthase (P4C3/P2F8) MJS2 142G15 614 Agaricus NADH oxidase (CAA43221.1 ) YES NADH oxidase/DAHP synthase (P4C3/P2F8) MJS2 142K9 408 ??? ??? ??? ras1p / exo 1,3 gluconase (P4E8/P1G6) MJS1 105I24 604 Verticllium H4 histone (XP_658338.1) YES ras1p / exo 1,3 gluconase (P4E8/P1G6) MJS1 125F18 217 ??? ??? ??? NADH oxidase/DAHP synthase (P4C3/P2F8) MJS2 110L18 795 Agaricus chitin deacetylase No Chitin deacetylase MJS3 126B14 827 Agaricus chitin deacetylase No Chitin deacetylase MJS3 142D2 1072 Agaricus chitin deacetylase YES Chitin deacetylase MJS3 136I23 669 Agaricus chitin deacetylase No Chitin deacetylase MJS3 dag11/exo 1,3 gluconase/carbomyl posphate synthetase 110P14 222 ??? ??? ??? (P4C3/P1G6/P1D9) MJS4 dag11/exo 1,3 gluconase/carbomyl posphate synthetase 124H13 421 Verticllium metallopeptidase (XP_748917) No (P4C3/P1G6/P1D9) MJS4 dag11/exo 1,3 gluconase/carbomyl posphate synthetase 108O6 628 Agaricus dag11 YES (P4C3/P1G6/P1D9) MJS4 dag11/exo 1,3 gluconase/carbomyl posphate synthetase 110N12 510 Agaricus dag11 No (P4C3/P1G6/P1D9) MJS4 dag11/exo 1,3 gluconase/carbomyl posphate synthetase 121N13 640 Agaricus dag11 YES (P4C3/P1G6/P1D9) MJS4 dag11/exo 1,3 gluconase/carbomyl posphate synthetase 124E14 508 Agaricus dag11 No (P4C3/P1G6/P1D9) MJS4 dag11/exo 1,3 gluconase/carbomyl posphate synthetase 125O2 554 Agaricus dag11 YES (P4C3/P1G6/P1D9) MJS4 dag11/exo 1,3 gluconase/carbomyl posphate synthetase 126F15 312 Agaricus dag11 No (P4C3/P1G6/P1D9) MJS4 dag11/exo 1,3 gluconase/carbomyl posphate synthetase 141A5 659 Agaricus dag11 YES (P4C3/P1G6/P1D9) MJS4

17G9 302 Agaricus hydrophobin (CAA61530) No ras1p/map1k/DAHP MJS5 17H11 543 Verticllium hypothetical protein (CAD70512) YES ras1p/map1k/DAHP MJS5 18G9 536 Agaricus cruciform DNA recognition protein (CAB85690) YES ras1p/map1k/DAHP MJS5 44I23 ?? Verticllium enoyl-CoA hydratase/isomerase XP_369757.1 Possibly ras1p/map1k/DAHP MJS5 45I23 ?? Verticllium enoyl-CoA hydratase/isomerase XP_369757.2 Possibly ras1p/map1k/DAHP MJS5 33N8 681 Agaricus polyubuquitin (AAC15225.1) No polyubuiquitin MJS6 37M11 1088 Agaricus polyubuquitin (AAC15225.1) YES polyubuiquitin MJS6 39A23 324 Agaricus polyubuquitin (AAC15225.1) No polyubuiquitin MJS7 38F1 337 Verticllium glucose-repressible gene (XP_753236.1) YES polyubuiquitin MJS8 17D5 481 Verticllium Metarhizium anisopliaec DNA clone Ma#1891 YES MA#1891/ras1p/map1k??? MJS7 17G1 487 Verticllium Metarhizium anisopliaec DNA clone Ma#1891 YES MA#1891/ras1p/map1k??? MJS7 22A5 482 Verticllium Metarhizium anisopliaec DNA clone Ma#1891 YES MA#1891/ras1p/map1k??? MJS7 22E23 479 Verticllium Metarhizium anisopliaec DNA clone Ma#1891 YES MA#1891/ras1p/map1k??? MJS7 23G21 482 Verticllium Metarhizium anisopliaec DNA clone Ma#1891 YES MA#1891/ras1p/map1k??? MJS7 24D7 486 Verticllium Metarhizium anisopliaec DNA clone Ma#1891 YES MA#1891/ras1p/map1k??? MJS7 24P3 484 Verticllium Metarhizium anisopliaec DNA clone Ma#1891 YES MA#1891/ras1p/map1k??? MJS7 17N3 459 Agaricus map1k??? ??? MA#1891/ras1p/map1k??? MJS7 23C7 484 Agaricus map1k??? ??? MA#1891/ras1p/map1k??? MJS7 21K15 493 Agaricus AB494 Basidiome cDNA library YES MA#1891/ras1p/map1k??? MJS7 37M3 1502 Agaricus DAHP YES DAHP/Carbomyl phosphatase MJS8 3J13 488 Agaricus Carbomyl phosphate synthase No DAHP/Carbomyl phosphatase MJS8 15G20 614 Agaricus Carbomyl phosphate synthase No DAHP/Carbomyl phosphatase MJS8 31N22 1577 Agaricus Carbomyl phosphate synthase YES DAHP/Carbomyl phosphatase MJS8 40F4 1352 Agaricus Carbomyl phosphate synthase No DAHP/Carbomyl phosphatase MJS8 36P10 1017 Agaricus Carbomyl phosphate synthase No DAHP/Carbomyl phosphatase MJS8 41D12 788 Agaricus Carbomyl phosphate synthase No DAHP/Carbomyl phosphatase MJS8 31O23 405 Verticllium stress response RCI peptide (EAL92348.1) Yes DAHP/Carbomyl phosphatase MJS8 34E24 611 Agaricus Ras1p No Ras1p MJS9 7C17 597 Agaricus L10 ribosomal protein No Ras1p MJS9 47G4 431 Agaricus hypthetical protein (EAK81924.1) Yes Ras1p MJS9 4I14 303 Agaricus Ras1p No Ras1p MJS9

Table 1 (continued above)

Table 2. Relative expression of A. bisporus infection response genes identified from SSH. mRNA levels were measured using Q-PCR of lesion and healthy mushroom tissue. Values were normalised against Agaricus 18S. Three replicates for each gene were used and the confidence limits reflect the variance within the three replicates

95 % confidence A. bisporus Q-PCR expression limits Gene / notes fold increase lower upper exo-1,3-beta-glucanase 2.0 1.3 3.2 chitin deacetylase 999.0 633.8 1574.5 3-deoxy-7-phosphoheptulonate synthase 3.5 2.0 6.1 MAP kinase (very low homology - if at all) 3.0 2.4 3.7 NADH-ubiquinone oxidoreductase 2.0 1.5 2.7 polyubiquitin 1.5 1.2 1.8 Ras-like protein (small G-protein) 1.0 0.8 1.4 carbamoyl-phosphate synthase 1.8 0.8 4.0 chitin synthase 1 1.5 1.0 2.1 dag-11 18.3 13.3 25.1 AB134 (5' similar to IIdD1) 11.8 7.0 20.0 AB494 (Basidiome cDNA library) 2.5 1.7 3.6 ribosomal L10 protein 3.3 1.4 7.6 60S (very low homology - if at all) 33.5 17.8 62.8 glyceraldehyde-3-phosphate dehydrogenase 1.5 1.2 1.9 cruciform DNA binding protein 4.8 2.6 8.9 putative 1,4-beta-cellobiosidase 1.3 0.8 2.0 O42713 Tyrosinase (EC 1.14.18.1) 0.8 0.4 1.4 C. elegans ABC6 protein 1.8 1.1 2.9 N. crassa probable sterol C-24 reductase. 0.8 0.4 1.3 (PAB1) Para-aminobenzoic acid synthetase 0.6 0.5 0.8 1-pyrroline-5-carboxylate dehydrogenase 0.5 0.4 0.8 elongation factor 1-alpha 2.1 1.1 3.9 Beta-tubulin 1.2 0.7 2.1 argininosuccinate lyase 1.2 0.8 1.6 Carbamoyl-phosphate synthase, arginine-specific, large chain 0.5 0.4 0.7 peroxisomal 3-ketoacyl-coA thiolase Kat1 0.6 0.4 1.0 Cell division control protein 10 (MIPS) 0.9 0.6 1.4 priA protein - shiitake mushroom 1.3 0.8 2.2

Table 3. Relative expression of Verticillium fungicola infection genes identified from SSH and MCW libraries. mRNA levels were measured using Q-PCR from Verticillium plate cultures and Verticillium in Agaricus lesions. Values were normalised against Verticillium 18S. Three replicates for each gene were used and the confidence limits reflect the variance within the three replicates

confidence limits GENE Warwick ID Bristol ID fold increase lower upper small oligopeptide transporter E2(P5) - 34.9 29.1 42.0 Ma#1891 contig 18 AB04A07 5.1 3.9 6.6 60S-L44 D5(2) AB01D07 4.5 3.8 5.4 hypothetical protein F610888.1 contig 17 AB01F02 3.9 3.0 5.1 DUF895 domain membrane protein contig 17 AB03B04 3.8 3.0 4.9 60S-L12 contig 22 AB03F06 3.2 2.6 4.0 antigenic cell wall protein MP2 contig 9 - 2.2 1.6 3.0 cell wall mannoprotein MnpA contig 13b AB03A11 1.4 1.0 1.9 ADP Ribosylation Factor contig 15 - 1.3 1.0 1.6 Beta Glucocidase F7(2) - 1.2 0.8 1.6 cyanovirin N like protein contig 20a AB02E08 1.0 0.9 1.1 Histone 3 contig 30 AB01D05 0.8 0.7 1.0 hypothetical protein FG02852.1 (v313) - AB07A05 0.7 0.5 1.0 clock controlled protein contig 3 AB01E10 0.6 0.3 0.9 - : no additional identifiers

Table 4. Summary of pRNAi silencing vectors built for URA3 and CBX model genes. Different regions of URA3 (F1, F2 and F3) and SDH-CBX (F1 and F2) genes were used to make RNAi constructs. Each RNAi expression cassette was cloned into pGreen-hph1 in two different orientations with respect to the hph marker; (i) Unidirectional transcribed tandem unit (unidirectional – U) or (ii) divergently transcribed tandem unit (divergent – D) generating different binaries.

RNAi Target Sequence Size Cassette Binary code construct gene region fragments orientation code (bp) F1 URA U URA3 1293-1787 529 Unidirectional pRNAiJT001

F1 URA D URA3 1293-1787 529 Divergent pRNAiJT002

F2 URA U URA3 1584-2016 467 Unidirectional pRNAiJT003

F2 URA D URA3 1584-2016 467 Divergent pRNAiJT004

F3 URA U URA3 1715-2095 401 Unidirectional pRNAiJT005

F3 URA D URA3 1715-2095 401 Divergent pRNAiJT006

F1 CBX U CBX 1068-1412 446 Unidirectional pRNAiAC001

F1 CBX D CBX 1068-1412 446 Divergent pRNAiAC002

F2 CBX U CBX 1474-1867 398 Unidirectional pRNAiAC003

F2 CBX D CBX 1474-1867 398 Divergent pRNAiAC004

FIGURES

Figure 1. Verticillium fungicola infections of Agaricus bisporus. From left to right: healthy mushrooms, cap lesions, stipe blowout and dry bubble.

Figure 2. Putative biological function of Agaricus bisporus response genes. SSH cloning was used to recover genes up- and down-regulated during infection with Verticillium fungicola.

Figure 3. Putative biological function of Verticillium fungicola response genes expressed during infection of Agaricus bisporus and recovered from SSH library.

Figure 4. Relative expression levels for specific Agaricus bisporus genes identified in healthy mushroom and infected lesion tissues. Expression was measured by Q-PCR and normalised against 18S. The standard employed for each gene consisted of dilutions of pooled cDNA from lesion samples. Control gene is beta-tubulin, where there is no significant difference between lesion and healthy tissues. Five genes identified by SSH are all up regulated on infection. Relative comparisons between genes are not appropriate.

Figure 5. Verticillium fungicola cDNA - Expressed Sequence Tags identified during growth on Mushroom Cell Wall agar. Percentage values of ‘0’ are less than 1%.

Figure 6. Schematic representation of pGreen-hph1RNAi silencing binaries. RNAi hairpins units (KpnI fragment) were cloned in two orientations. pGreen-hph1RNAi has gpdA promotor and trpC terminator from Aspergillus nidulans. Selection in Agrobacterium tumefaciens uses kanamycin resistance: Agaricus bisporus transformants are selected using resistance to hygromycin.

Figure 7. Q-PCR analyses of URA3 hairpin transformants of Agaricus bisporus. Left panel: Amplification profiles URA3 and 18S transcripts. At cycle 32, product generated both 18S and URA3 primers were similar. Right panel: Relative expression level of URA3 gene on A. bisporus RNAi transformants (T numbers). Target URA3 transcripts were normalised against A. bisporus 18S. T5, T7, T18 and T26 showed reduced level of URA3 transcript compared with non-silenced controls (C1, C2).

Figure 8. Q-PCR determined relative expression of CBX gene in Agaricus bisporus RNAi transformants (T numbers). All transformants tested showed reduced level of CBX transcript compared with non-silenced control (C1 = C43-carb.9)

Figure 10. Improved cloning vector for RNAi hairpin constructs. Sense and antisense target regions are cloned in multiple cloning sites under the control of A. bisporus GPD promoter and A. nidulans trpC terminator. Full hairpin unit is excised as KpnI fragment and introduced into the pGreen_hph1 binary vector prior to Agro-transfection (Leach et al., 2004)

Figure 11. RNAi hairpin vectors for knockdown of A. bisporus chitin deacetylase (CDA) and 3-deoxy-7-phosphoheptulonate synthase (DAHP synthetase) target genes.

Figure 12. Schematic showing the position of Q-PCR primers used to quantify transcriptional levels of chitin deacetylase (CDA) and DAHP synthetase in hairpin transformants. Primer pairs annealed either inside (PCR1) or outside (PCR2) the hairpin target region

Figure 13. Q-PCR analysis of chitin deacetylase (chit; upper panel) and DAHP synthetase (dahp; lower panel) expression in mycelial cultures. Relative expression in 10 hairpin transformants is compared with two, no hairpin control hygR A15 transformants. Expression levels are relative to mean A15 (wild type) expression. White bars are expression of the hairpin target region. Black bars are expression measured outside the target (native gene).

Figure 14. Q-PCR comparison of chitin deacetylase (chi; upper panel) and DAHP synthetase (dahp; lower panel) gene expression in A. bisporus healthy (control) and V. fungicola (infected) lesion tissues. Error bars (standard error) are derived from three independent experiments. Transformants where tissue cultures did not yield hygromycin resistant mycelia are indicated (*).

Figure 15. Variation in Verticillium fungicola lesion morphology in Agaricus bisporus RNAi-hairpin transformants down-regulated for chitin deactylase (CDA). Right panel: lesions in wild-type A15 Left panel: CDA RNAi transformant chi-A16. Lesions on A16, were often deeper and more pronounced than the equivalent on A15.

Figure 16. Gene-disruption strategy for Verticillium fungicola MAPK gene (VFMK1). Upper panel: Primer map of primers used for confirmation of transformation and disruption of VFMK1. Primers hygA and hygB were used in combination with one another to confirm the presence of the hygromycin resistance cassette among V. fungicola lines that displayed hygromycin resistance. Primers LAFKO and hygoutC2 were used in combination with one another to confirm homologous integration of the gene disruption cassette into VFMK1. Lower panel: Agarose gel electrophoresis of PCR products amplified using LAFKO with hygoutC2 (to confirm homologous integration) and hygA with hygB (to confirm genuine hygromycin resistance). WT lanes are the results of PCR from 150-1 (wild type) DNA. 37MK1, 37MK3 and 38MK1 are PCR results from DNA extracted from lines displaying resistance to hygromycin after transformation. Far left lane contains the molecular marker (Hyperladder I, Bioline).

ADDITIONAL DATA NOTES

NOTABLE GENES / GENE FAMILIES A number of interesting genes and functional classes were observed during our bioinformatic analyses. The following detailed notes on functional characterisation and pathogenesis are provided in addition to the classifications summarised in the main report (Objective 1).

Two up-regulated genes were selected for gene suppression studies (Objective 3). Notes appear in the main report and are duplicated (highlighted) below.

Putative Up Regulated Host (A. bisporus) Genes

Metabolism: • Carbamoyl phosphate synthase • DAHP synthase 3-deoxyarabinoheprulosonate 7-phosphate (DAHP) synthase is the first committed enzyme of aromatic amino acid biosynthesis. Two DAHP synthase enzymes have been described in the filamentous fungus Aspergillus nidulans and three in Neurospora crassa (Hartmann et al., 2001). Plants have been shown to duplicate their DAHP synthase enzymes. In the model plant Arabidopsis thaliana there are two copies of DAHP synthase, one induced in response to wounding or pathogen attack and one constitutively expressed (Keith et al., 1991). In plants the aromatic amino acids are the building blocks of a range of secondary compounds including antimicrobials such as phytoalexins and glucosinolates (Jones et al., 1995).

Energy: • NADH ubiquinone oxidoreductase The NADH ubiquinone oxidoreductase (Complex I) provides the input to the respiratory chain from the NAD-linked dehydrogenases of the citric acid cycle. The complex couples the oxidation of NADH and the reduction of ubiquinone, to the generation of a proton gradient that is then used for ATP synthesis. The complex occurs in the mitochondria of eukaryotes. NADH ubiquinone oxidoreductase has been shown to be up-regulated in the basidiomycete yeast Cryptococcus neoformans in response to reactive nitrogen species (nitric oxide, NO). Nitiric oxide inactivates mitochondrial NADH:ubiquinone oxidoreductase by binding the iron-sulfur center (Missall et al., 2006). In addition, the exogenous addition of dibenzo-p- dioxin to the basidiomycete Phanerochaete chrysosporium led to the isolation of six stress- responsive genes expressed against the exogenous addition of dibenzo-p-dioxin from the lignin-degrading basidiomycete, one of which was NADH ubiquinone oxidoreductase. Apart from being a response gene to oxidative stress, NADH ubiquinone oxidoreductase has been suggested to play a role in the production of the superoxide radical. This is due to the low redox potential required for one electron reduction of dioxygen to superoxide (Joseph-Horne et al., 2001). An alternative (or perhaps additional) function for the G. trabeum reductase cannot be ruled out yet. This enzyme belongs to a widely distributed family of flavoprotein quinone reductases that are generally thought to detoxify intracellular quinones by maintaining them in the reduced form (Jensen et al., 2002). Quinones are cytotoxic in part

because they readily undergo intracellular one-electron reduction to semiquinones, which react rapidly with O2 to produce superoxide. By contrast, hydroquinones are relatively nontoxic because they undergo rapid one-electron oxidation to semiquinones only in the presence of transition metal oxidants, such as Fe3+, which are generally unavailable inside cells because they are sequestered in redox-inactive complexes (Jensen et al., 2002). Since G. trabeum produces large amounts of quinones as natural metabolites, it may have an unusually high requirement for a quinone detoxification system. The NADH ubiquinone oxidoreductase identified in this study has functional similarity to the Gloeophyllum trabeum qrd2 that is responsive to stress (Cohen et al., 2004).

Protein synthesis: • Ribosomal Protein L10 • Ornithine oxo acid transaminase (OAT) OAT is involved in the production of amino acid proline from arginine. Upstream the presence of carbamoyl phosphate synthase within the up-regulated pool, would suggest an increase in arginine production. Combined with the down-regulation of pruA this would suggest that more proline is being produced. Proline has been shown to be a potent reactive oxygen species (ROS) scavenger within fungi (Chen and Dickman, 2005).

Transport: • Monosacharide transporter

Cell rescue, defence, death and ageing: • A. bisporus dag11 • Glutathione-s-transferase • hmp1 (CDBP) • peroxidase • chloroperoxidase There are numerous articles on the use of peroxidases as the means of defence by plants to pathogens (De Jesús-Berríos et al., 2003). They have even been transformed in to plants to control against fungal pathogens (Rajasekaran et al.).

Protein destination: • Ubiquitin precursor

Cellular communication/ signal transduction: • Ras1p Ras1p is considered a critical element in the adenylyl cyclase signalling pathway (Crechet et al., 2003)

Cellular Biogenensis: • exo-1,3-beta-glucanase (Agaricus bisporus) (van de Rhee et al., 1996) Most information on beta glucanases is presented from the perspective of the pathogen. However, they have been produced by resistant plant cultivars (Munch-Garthoff et al., 1997). • glucan 1,3 beta-glucosidase protein putative (homology with Cryptococcus neoformans var. neoformans)

• Chitin synthase (homology with Cryptococcus neoformans var. neoformans CHS) (Valdivia and Schekman, 2003) • Chitin deacetylase (CDA from Cryptococcus neoformans var. neoformans) Chitin deacetylases (CDAs) from different species have the same catalytic activity and stringent specificity for chitinous substrates but may be involved in a variety of biological processes in different organisms. In the fungi M. rouxii and Absidia coerula, CDAs are constitutively expressed enzymes (Kafetzopoulos et al., 1993a) (Gao et al., 1995), localized in the periplasmic space in order to perform their task in cell wall construction, whereas in the plant pathogen Colletotrichum lindemuthianum and in Aspergillus nidulans CDAs are secreted enzymes postulated to act on chitin oligomers released by fungal cell walls, in order to promote plant invasion (Tsigos and Bouriotis, 1995) or cell wall degradation (Alfonso et al., 1995). In procaryotes, the NodB family of genes encodes CDA, which participates in the biosynthesis of the Nod factors that promote plant nodulation (Kafetzopoulos et al., 1993a; Kafetzopoulos et al., 1993b), while putative CDA homologs are found in nonendosymbiotic bacteria. Given this diversity in the biological functions of CDA, the availability of the S. cerevisiae !cda mutant phenotype as well as the easily measurable CDA activity offer important tools not only toward understanding of cell wall formation but also toward the elucidation of the structure-function relationships of chitin deacetylases (Christodoulidou et al., 1996). In C. neoformans chitin deacetylase was down-regulated in response to NO stress (Missall et al., 2006). The role of an A. bisporus CDA could be deacetylation of chitin oligosaccharides during autolysis, after action of endochitinase on cell walls. Interestingly the A. bisporus CDA was massively up-regulated in lesions. • Glucan 1,3, beta-glucosidase The expression in tobacco of barley glucan 1,3, !-glucosidase and chitinase led to enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level (Jach et al., 1995). Extracellular !-1,3-glucanase has been shown to disrupt !-1,3-glucan, the molecule present in fungal cell wall (Vazquez-Garciduenas et al., 1998). !-1,3-glucanase is designated as a PR or pathogenesis-related protein that takes part in defense, as an extended group of proteins (Cutt and Klessig, 1992). Several glucanases have been described from different species (Meins et al., 1992). The involvement of !-1,3-glucanase in plant defense has been demonstrated using a direct approach and transgenic plants expressing the gene (Cornelissen and Melchers, 1993). Production of extracellular !-1,3-glucanases, chitinases, and a proteinase increases significantly when a Trichoderma species is grown in a medium supplemented with either autoclaved mycelium or host fungal cell walls (Carsolio et al., 1994) (Vazquez-Garciduenas et al., 1998) (Geremia et al., 1993). These observations, together with the fact that chitin, !-1,3-glucan, and protein are the main structural components of most fungal cell walls (Peberdy, 1990) are the basis for the suggestion that lytic enzymes produced by some Trichoderma species play an important role in the biocontrol and destruction of plant pathogens (Chet and Baker, 1980) (Chet et al., 1979). • Exo 1,3 beta-glucanase An enzyme previously implicated in another fungal–fungal interaction (Pichia vs Botrytis) (Jijakli and Lepoivre, 1998)

Pathogen (Verticillium) genes

Protein synthesis: • 60S genes • 40S ribosomal protein • S30 • Translation elongation factor, 1 gamma • Translation elongation factor, 1 alpha

Cellular communication/signalling: • ADP ribosylation factor • Rho-GDP dissociation inhibitor • Cellular organisation • Beta-tubulin • Histone H3

Protein destination: • Cyclophilin like peptidyl-prolyl cis trans isomerase

Transport facilitation: • Glucose transporters • Porin • Oligopeptide transporter protein

Cell wall Biogenesis: • Beta-glucosidase • Cell wall glycoprotein • Cell wall protein

Transposon and insertion sequences: • Env homologue • Intron endonuclease

Cell rescue defence, death and ageing: • Glucooligosaccharide oxidase • Snodprot1 • glucosyl hydrolase

Metabolism: • Succinate dehydrogenase • Gluconate kinase • Phosphomevalonate kinase

Miscellaneous: • Antigenic cell wall protein

• Clock controlled protein 6 This V. fungicola sequence was very abundant in our SSH library (32 copies). It had no significant homology in database searches but did match a Verticillium EST obtained from the Agaricus cell wall material library with similarity to a clock-controlled protein from Neurospora crassa. It is possible that circadian clocks may also control genes regulated during pathogenesis and respond to stimuli such as light.

Putative Down Regulated Host (A. bisporus) Genes

Transport: • Monosaccharide transporter

Metabolism: • 3-ketoacyl CoA thiolase (KAT) KAT catalyses a key step in fatty acid "-oxidation. KAT genes may be up-regulated in response to pathogens in a resistance response in incompatible reactions. In the compatible response between Arabidopsis and Pseudomonas syringae pv. tomato the gene was not up- regulated, it was however up-regulated when the pathogen included an avirulence gene (Almeras et al., 2003). • Tyrosinase A polyphenol oxidase (PPO) enzyme. Studies into A. bisporus tyrosinase expression in response to infection with a ‘brown blotch’ pathogen also revealed a decrease in activity. However, a reduction in total tyrosinase was not observed in more resistant strains (Soler- Rivas et al., 1997). Over-expression of polyphenol oxidase in tomato plants resulted in enhanced bacterial disease resistance (Li and Steffens, 2002). Furthermore, antisense gene down-regulation of the Tomato polyphenol oxidase gene resulted in increased disease susceptibility (Thipyapong et al., 2004). • Catechol oxidase Another polyphenol oxidase (PPO), these enzymes are considered a primitive, non- specific eukaryotic defence system. PPOs contain copper and catalyse the oxidation of phenols in plant cells. They are brought into contact with air and their substrate when cells are damaged (e.g. browning). The products of this reaction can be converted into quinones that will stop the growth of fungi. This reaction is more readily carried out by fungal-resistant varieties of plants than their non-resistant counterparts. The reaction may enable resistant plants to localise the infection of pathogenic fungi. It is conceivable that Agaricus is using similar PPO mediated defence pathways.

Cellular organisation: • Histone H4 Down-regulation of the histone H4 indicates a change in chromatin structure during infection. Such changes bought on by stress have been seen in other fungi (Steen et al., 2002). • Sterol C24 reductase • Plastocyanin domain Plastocyanin domains have been found within fungal laccases (Piontek et al., 2002)

Transport facilitation: • ABC transporter The ATP dependant ABC transporter superfamily contains membrane proteins that translocate a wide variety of substrates across extra- and intracellular membranes, including metabolic products, lipids and sterols. Using ATP driven efflux they have been shown to remove toxic compounds from fungi (Schoonbeek et al., 2001). Thus it is possible that the down-regulation of an ABC transporter could be important in the compatible interaction between the two fungi.

Cell rescue, stress and defence: • Anaphylatoxin Within the mouse - Candida albicans pathosystem, C5 anaphylatoxin plays a critical role in host resistance mechanisms (Mullick et al., 2004). • Cytochrome P450 The ubiquitous Cytochrome P450s are heme-dependent mixed-function oxidase systems that utilize NADPH and/or NADH to reductively cleave dioxygen to produce a functionalized organic substrate and a molecule of water. P450s figure prominently in general plant defence due to their multiplicity in the highly complex phenylpropanoid, terpenoid, and alkaloid biosynthetic pathways synthesizing cell wall components and common defence agents (Whitbred and Schuler, 2000). They have also been observed to play a role in mushroom morphogenesis (Arima et al., 2004) and post-harvest development (Eastwood et al., 2001). • PriA protein One SSH transcript was similar to the developmentally regulated PriA from Lentinus edodes (shiitake mushroom) associated with fruiting-body formation (Kamada et al., 1980). Both sequences are similar to the C. neoformans DHA1 involved in delayed type hypersensitivity (Mandel et al., 2000). • Glucose-6-phosphate amidotransferase • Septin The septins were first discovered in the budding yeast Saccharomyces cerevisiae and were named for their role in cytokinesis and septum formation (Longtine et al., 1996 ). Septins are highly conserved in fungi and animals, although absent in plants and many protozoans (e.g., Plasmodium fasciculatum and Dictyostelium discoideum). Septin proteins are characterized by their distinct GTPase domains and an ability to form filaments (Douglas et al., 2005). Septin’s can be regulated by the Rho-family GTPase Cdc42, phosphorylation, and GTP binding. Analysis of specific Cdc42 point mutations showed that Cdc42 carries out a special role in septin ring formation that is independent of its role in actin polarization. A genetic link has been found between septins and chitin deposition in the yeast Saccharomyces (DeMarini et al., 1997). Septin localization appears to depend on a signal from the Cdc42p GTPase. In the heterobasidiomycete Ustilago maydis a septin (sep3) is required for normal cellular morphology and division (DeMarini et al., 1997) • Glutathione-s-tranferase Glutathione-s-transferases are dimeric enzymes that catalyse the conjugation of electrophilic molecules to glutathione. Their expression is a well-known stress response to invading fungi within plants. They can act as carriers of various factors and as signalling

molecules, and can also detoxify reactive oxygen induced organic hydroperoxides of fatty and nucleic acids. • EGF domain EGF is a polypeptide of about 50 amino acids with three internal disulfide bridges. It first binds with high affinity to specific cell-surface receptors and then induces their dimerization, which is essential for activating the tyrosine kinase in the receptor cytoplasmic domain, initiating a signal transduction that results in DNA synthesis and cell proliferation. • Glutamate dehydrogenase • PruA gene • Glucoamylase

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