Insight into different environmental niches adaptation and allergenicity from the Cladosporium sphaerospermum genome, a common human allergy-eliciting Dothideomycetes

Cladosporium sphaerospermum, a dematiaceous saprophytic fungus commonly found in diverse environments, has been reported to cause allergy and other occasional diseases in humans. However, its basic biology and genetic information are largely unexplored. A clinical isolate C. sphaerospermum genome, UM 843, was re-sequenced and combined with previously generated sequences to form a model 26.89 Mb genome containing 9,652 predicted genes. Functional annotation on predicted genes suggests the ability of this fungus to degrade carbohydrate and protein complexes. Several putative peptidases responsible for lung tissue hydrolysis were identified. These genes shared high similarity with the Aspergillus peptidases. The UM 843 genome encodes a wide array of proteins involved in the biosynthesis of melanin, siderophores, cladosins and survival in high salinity environment. In addition, a total of 28 genes were predicted to be associated with allergy. Orthologous gene analysis together with 22 other Dothideomycetes showed genes uniquely present in UM 843 that encode four class 1 hydrophobins which may be allergens specific to Cladosporium. The mRNA of these hydrophobins were detected by RT-PCR. The genomic analysis of UM 843 contributes to the understanding of the biology and allergenicity of this widely-prevalent species.

. A total of 5,215 predicted proteins were annotated redundantly into 5,853 KOG classifications (Fig. 3a). Among the highest annotated groups, posttranslational modifications of proteins appeared significant in cellular regulation, development and adaptation to stress 12 . We identified 69 putative genes encoding chaperones in group O (Posttranslational modification, protein turnover, chaperons) which may be associated with stress adaptation, misfolded proteins degradation via the ubiquitin-proteasome system, regulatory degradation of metabolic enzymes and cell viability. In group G (Carbohydrate transport and metabolism), the largest number of genes (77) is for a putative permease of the major facilitator superfamily. Furthermore, 26 were putative monocarboxylate transporters which are important to energy utilisation, intracellular pH regulation, and virulence in some pathogenic fungi 13 .
The top five metabolic pathways of the genes annotated in KEGG pathways were carbohydrate metabolism (575), amino acid metabolism (413), lipid metabolism (281), energy metabolism (255), and nucleotide metabolism (240) (Fig. 3b). It is not surprising for UM 843 to contain many genes involved in carbohydrate metabolism since carbon source is an essential nutrient for fungal growth, conidiation and virulence 14 .
Based on the GO classifications, 6,065 predicted genes received a GO assignment (Fig. 3c). A total of 1,733 and 1,080 genes were assigned to the response to stimulus category (GO: 0050896) and the response to stress category (GO: 0006950), respectively. The genes were further annotated to various stress responses. The highest number of predicted genes were assigned to the response to osmotic stress category (139). This distribution of genes might be reflective of the habitats of C. sphaerospermum in which the fungus has to combat with osmotic imbalance 1 . The osmotic responses of C. sphaerospermum UM 843 are further discussed in the subsection of "Fungal adaptation and stress responses". Gene families. We performed all-against-all BLASTP for 303,264 proteins from 23 Dothideomycetes and two Sordariomycetes as the outgroup (Supplementary Table S1), obtaining 24,581 orthologous clusters with 2,203 single-copy orthologues (one copy of gene from each species). Maximum likelihood and Bayesian trees were constructed using concatenated alignments generated from 10% of the single-copy orthologues identified (220 single-copy orthologues). The topology of the maximum likelihood tree built by RaxML was identical to that of the Bayesian tree. The Dothideomycetes were categorised into four orders encompassing Pleosporales, Capnodiales, Botryosphaeriales and Hysteriales. The UM 843 genome is within the order Capnodiales where it forms a sister-group relationship with two clusters (Fig. 4).
Of the 23,800 orthologous clusters generated from the 23 dematiaceous Dothideomycetes, 3,333 clusters (14%) were conserved within the class while 51 clusters contained 125 UM 843 unique genes (recent paralogues) in this study set (Supplementary Table S2). Hydrophobins (DOTH 13561), mitogen-activated protein kinases (MAPKs) (DOTH 14960) and other metabolism related genes were among the UM 843-specific putative genes found. The three putative genes from DOTH 14960 shared ≥ 60% similarity with Schizosaccharomyces pombe MAPK Spk1 (Fus3 orthologue) (GenBank: P27638). This protein is responsible for appressorium formation and pathogenicity of certain plant pathogenic fungi, sexual/asexual reproduction, hyphal growth and Scientific RepoRts | 6:27008 | DOI: 10.1038/srep27008 conidial germination in filamentous fungi 15 . However, these genes might be atypical MAPKs that were identified in Fusarium graminearum 16 as the typical T-X-Y motif in the activation loop of MAPK was absent. However, the exact role of these atypical MAPKs remain unknown.

Sexual reproduction.
To date, Cladosporium herbarum and Cladosporium silenes are species with established anamorph-teleomorph stages while Cladosporium grevileae is only known with a sexual stage 17 . Most of the genes involved in sexual reproduction were identified in UM 843 (Supplementary Table S3). This strain showing (e,f,g) conidiophores bearing conidium (e, × 2000 magnification, bar 3 μ m), periclinal rim (f, × 5000 magnification bar 1 μ m) and verruculose surface of conidia (g, × 5000 magnification, bar 2 μ m). might be a heterothallic fungus with a predicted high-mobility-group (HMG) domain containing Mat1-2 gene (UM843_3044), sharing 65.05% identity with Dothistroma septosporum Mat1-2 (GenBank: ABK91354). This suggested that sexual reproduction might occur in C. sphaerospermum. The gene was found in adjacent to genes encoding DNA lyase (Apn2), anaphase promoting complex protein and cytochrome c oxidase subunit Vla (Cox13). The presence of Apn2 and Cox13 nearby Mat1-2 is similar to the previously reported mating type cluster 18 (Supplementary Fig. S1). Furthermore, although the genes involved in mating and cell cycle in fungi are initiated by Fus3 MAPK signalling pathway which is stimulated by pheromones 19 , no pheromone genes were predicted in UM 843. These features posed a possibility that this fungal strain is unable to carry out mating process despite the presence of sexual reproduction genes. Nonetheless, it has been shown that some fungi can undergo sexual reproduction without the activation of pheromone response pathway by pheromone ligand 19 . Hence, it is still possible that UM 843 can mate by a different mechanism of pheromone activation or without pheromone activation.
Carbohydrate Active enZymes (CAZymes). Fungal CAZymes play an important role in the degradation of the plant cell wall into carbon sources required for fungal growth or the infection of the plant host 20 . In this study, a total of 605 putative CAZyme catalytic domains comprising 261 glycoside hydrolases (GH), 98 glycosyltransferases (GT), 114 carbohydrate esterases (CE), 14 polysaccharide lyases (PL), 77 auxiliary activities (AA), and 41 carbohydrate-binding modules (CBM) were identified in UM 843 (Supplementary Table S4). The CAZymes predicted in all Dothideomycetes genomes were compared with those in UM 843 for correlation with its possible lifestyle ( Supplementary Fig. S2). Zhao et al. 20 revealed that CE11, GH73, GH80 and GH82 families were absent in saprophytic fungi. This absence was also observed in UM 843 suggesting it to be a saprophyte. However, at this stage, no conclusive inference can be drawn that UM 843 belongs to the saprophytic group.
UM 843 contains at least 171 CAZymes that are involved in plant cell wall degradation (Supplementary Table S5). The presence of a high number of hemicellulose and pectin degrading CAZymes suggested the preference of this fungus for soft plant tissue 21 (Supplementary Table S5). Nevertheless, UM 843 has the highest number of predicted CBM1 (carbohydrate-binding modules 1) among the Capnodiales (Supplementary Table S6). Apart from associating cellulases in ensuring contact between catalytic domain and substrate, CBM1 has been shown to be able to disrupt the crystalline structure of cellulose by non-hydrolytic cleavage of inter-and intra-hydrogen bonds of polysaccharide chains 22 . The weakened cellulose structure allows easy accessibility of other enzymes such as hemicellulolytic enzymes to carry out catalytic reactions.
Peptidases. In UM 843, 130 predicted peptidases were identified with no predominance of any particular enzyme family (Supplementary Fig. S3; Supplementary Table S7). This observation is consistent with the saprophytic lifestyle to degrade different types of substrate complexes available in the environment into smaller residues to be absorbed into the fungal cell 23 .
The small sized (2-5 × 2-4 μ m) C. sphaerospermum conidia is easily disseminated, and hence, may be inhaled by humans to reach the lung alveoli 1 . We postulated reactions involving secreted peptidases to take place when the conidia of C. sphaerospermum reach the lungs of humans. Five of the 31 secreted (two A01, two S09, one M36) and one non-secreted peptidase from the A01 family in UM 843 were shown to be putative peptidases involved in lung tissues disruption. These are the A01 secreted aspartic peptidases (UM843_1326 and UM843_4966) belonging to the holotype peptidase F (51.70% and 61.99% identity, respectively) known to hydrolyse elastin and laminin, which are the components of lung 24 ; a secreted metallopeptidase from family M36 (UM843_2925) showing 67.86% identity to a holotype fungalysin involved in elastin hydrolysis 25 ; and a cell wall-associated aspartic   peptidase, PEP2. This PEP2 peptidase (UM843_5823; 76.15% identity) facilitates the penetration of young hyphae into the host connective tissue 26 . Two peptidases (UM843_2883 and UM843_1649) belonging to the family S09 showed 56.24% and 50.07% identity to dipeptidyl peptidase (DPP) IV and DPP V, respectively. These two peptidases have the Gly-X-Ser-X-Gly conserved sequence motif and catalytic triad Ser 631, Asp 711, His 746 and Ser 565, Asp 646, His 678 in UM843_2883 and UM843_1649 respectively. The predicted catalytic sites are conserved with other reported DPPs (Supplementary Figs S4 and S5). DPP IV has been shown to facilitate colonisation of the lung by binding and subsequently degrading the dipeptide of collagen 27 , whereas DPP V is the elicitor of host defence mechanisms 28 . These peptidases might work in concert to disrupt lung tissues. Table S8). One polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) hybrid, five PKS or PKS-like and ten NRPS or NRPS-like enzymes were annotated using SMURF analysis. Among the PKS, two reducing PKS were predicted to contain the domain arrangements of ketosynthase (KS)-acyltransferase (AT)-dehydratase (DH)-methyltransferase (ME)-enoyl reductase (ER)-ketoreductase (KR)-acyl carrier protein (ACP) (UM843_7344) and KS-AT-DH-ER-KR-ACP (UM843_9325). One of the non-reducing PKS (UM843_1729) was found likely to be involved in pigment synthesis and two NRPS (UM843_7306 and UM843_8410) are likely to be responsible for siderophore biosynthesis.

Secondary metabolites. We predicted 16 secondary metabolite backbone genes in UM 843 (Supplementary
Melanin. Most fungi synthesise melanin via the 1,8-dihydroxynaphthalene (DHN) biosynthesis pathway 29 to protect them from UV irradiation, desiccation, high temperatures, and oxidants. Evidence for this pathway in C. sphaerospermum was shown by the generation of melanin-deficient C. sphaerospermum from cultures in medium containing tricyclazole, an inhibitor of DHN melanin biosynthesis 30 . The PKS gene (UM843_1729) we predicted in UM 843 is best matched to a characterised conidial yellow pigment biosynthesis PKS from Aspergillus fumigatus (alb-1) (GenBank: Q03149) 31 and shows high identity to a predicted Cladosporium phlei Cppks1 protein (GenBank: AFP89389) involved in pigment biosynthesis 32 (Supplementary Table S9). A starter unit: ACP transcylase (SAT) domain (PF16073) typical of non-reducing PKS was identified in the gene 33 . Also predicted were domains in the order of SAT-KS-AT-DH-ACP-ACP-TE which was similar to that of Alb-1 and Cppks1. We also predicted The phylogenomic tree was constructed with 23 Dothideomycetes spp. including UM 843 and two outgroups from Sodariomycetes spp. using Bayesian and maximum likelihood analysis. The first number at the node is Bayesian posterior probability followed by the maximum likelihood bootstrap number. Values less than 1 or 100 for posterior probability and maximum likelihood bootstrap number, respectively, were shown on branches.
Siderophores. Iron plays important roles in cellular processes but excessive of iron in cells is dangerous to the organism. To overcome the bioavailable scarcity and cytotoxic effect of iron, fungi have developed different strategies for iron uptake and regulation. Siderophore-mediated Fe 3+ uptake is one of the mechanisms for iron homeostasis 37 . We found putative genes that are essential in the synthesis of siderophores (Supplementary Table S10). UM843_8412 and UM843_7304 are putative genes encoding L-ornithine-N 5 -monooxygenese involved in the first committed step in siderophore biosynthesis while UM 843_7306 encodes NRPS SidD that is responsible for fusarinine-type siderophore biosynthesis 38 . As previously reported by Schrettl et al. 38 , our analysis showed that UM843_7306 has a domain arrangement of adenylation (A)-thiolation (T)-condensation (C)-T-C, similar to that in A. fumigatus SidD (GenBank: Q4WF53). The clustering of siderophore biosynthesis genes in UM 843 appears to be different from the gene clusters reported by others 39 , probably as a result of rearrangement during genome evolution and speciation in fungi. In UM 843, the putative gene cluster encompasses genes encoding L-ornithine-N 5 -monooxygenase, esterase, NRPS SidD, ABC transporter SitT, carnitinyl-CoA dehydratase and acetyltransferase SidF (Supplementary Fig. S7). However, the orthologue of SidG gene that is responsible for the synthesis of triacetylfusarinine C (TAFC) was absent. Thus, UM 843 may be synthesising only the TAFC precursor, fusarinine C, but not TAFC itself. Nevertheless, UM 843 possess the ability to utilise TAFC produced by other organisms. A gene encoding siderophore esterase (UM843_1515) similar to EstB of A. fumigatus (GenBank: XP748686) that hydrolyses iron-chelated TAFC was identified. This gene was located adjacent to the TAFC transporter MirB (UM843_1516) (Supplementary Fig. S8), showing an organisation similar to that reported previously 39 .
Another putative gene UM843_8410 was found to be involved in the production of ferrichrome-type siderophores. Ferrichrome NRPSs have a diversity of domain architectures consisting of two to four complete A-T-C modules which are usually followed by a T-C repeat 37 . The gene UM843_8410 has the order of domains A-T-C-A-T-C-T-C-A-T-C-T-C-T-C. The gene organisation of the ferrichrome-type siderophore in UM 843 is similar to that in C. heterostrophus 37 , where the gene encoding an ABC transporter spans between L-ornithine-N 5 -monooxygenase and ferrichrome NRPS encoding genes ( Supplementary Fig. S9).

PKS-NRPS Hybrid.
Recently, an isolate of C. sphaerospermum was reported to synthesise polyketide hybrid cladosins such as Cladosin C showing mild antiviral activity 40 . In UM 843, a putative PKS-NRPS hybrid, UM843_7284, shared 40% and 41% identity with Talaromyces stipitatus putative PKS (GenBank: XP_002478535) and Aspergillus clavatus PKS-NRPS cytochalasin (GenBank: A1CY8), respectively. The domain arrangement of the gene revealed an arrangement of KS-AT-KR-ACP-C-A-T-reductive domain (R), which is different from that of T. stipitatus and A. clavatus. Further inspection of the predicted gene cluster revealed neighbouring genes that might be associated with the synthesis and transport of the product, such as genes encoding transporters, cytochrome P450, alpha beta hydrolase, thioesterase and transcription factor domain containing proteins (Supplementary Table S11). As only one putative PKS-NRPS hybrid was identified in the genome, further studies need to be carried out to confirm the function of this gene in the synthesis of cladosins.

Fungal adaptation and stress responses.
Most fungi have their own system to respond to multiple stresses from their ecological niche for adaptation and survival. In order to survive in hostile environments, they have to be able to detect stress, transduce stress signals and respond to the stress 41 . We found 340 genes involved in stress responses including amino acid starvation, nitrogen starvation, iron starvation, osmotic stress, oxidative stress, and heat stress (Supplementary Table S12).
Apart from biosynthesis of sidorephores in iron acquisition, UM 843 might also be employing another high-affinity iron acquisition system, the reductive iron assimilation mechanism (RIA), to regulate iron homeostasis. We identified a ferroxidase and iron permease similar to FetC (UM843_5150, 69.12% identity to P38993) and Ftr1 (UM843_5151, 59.69% identity to P40088), respectively, that are involved in the RIA. They were located adjacent to each other in the UM 843 genome (Supplementary Fig. S10). The ferroxidase and iron permease encoding gene cluster was also found in other fungi 37 .
Adaptation in hypersaline environment. C. sphaerospermum has also been isolated consistently from hypersaline environment 1 . In this study, UM 843 is shown able to grow at a high concentration of NaCl (20% w/v) ( Supplementary Fig. S11). UM 843 was predicted to contain 21 genes encoding plasma membrane and intracellular cation transporters which are involved in cation homeostasis by maintaining low intracellular Na + concentration. Also identified were the plasma membrane H + -ATPases that supply energy to the secondary transporters (Table 3; Supplementary Table S13) and all the subunits of V-type ATPase complex that play an important role in acidification of vacuolar lumen and correct functioning of other organelles (Supplementary Table S14). The Scientific RepoRts | 6:27008 | DOI: 10.1038/srep27008 V-type ATPase complex also works together with the plasma membrane H + -ATPase to maintain the cytosolic pH homeostasis 42 . As seen in Table 3, UM 843 contains more Ena genes than Nha1 genes. This might confer the ability to survive in the near neutral to alkaline pH of a hypersaline environment, as Ena is important in the export of Na + ions at alkaline pH 43 . However, Nha1 is also required by the fungus as this gene is critical in the immediate response to osmotic shock 44 .
A comparison of the UM 843 transporters with those reported in halotolerant Hortaea werneckii, halophilic Wallemia ichthyophaga and non-halotolerant Mycosphaerella graminicola was carried out to determine the genes confer to the ability of UM 843 to survive in high salinity environment (Table 3). UM 843 contains more genes encoding Trk, Tok, Ena and Pho89 compared to M. graminicola although it has lesser genes compared to H. werneckii which had undergone recent whole genome duplication 45 (Table 3). In addition, we managed to identify genes encoding K + (Na + )-ATPase (alkali cation uptake, Acu transporters) and K + -H + symporter (Hak symporters) in UM 843 which were not found in H. werneckii. These transporters function in high affinity uptake of K + ions which might be beneficial for organisms to adapt in a hypersaline environment 46 . It has been reported that different strategies were used by H. weneckii and W. ichthyophage to counteract high salinity 45,47 . The diversity of cation transporters found in UM 843 indicates that this fungus possibly uses strategies that are different from those used by H. werneckii and W. ichthyophaga in their response to dynamic changes in salinity.
Besides the accumulation of nontoxic ions to overcome osmotic stress, the accumulation of compatible solutes is another strategy employed by microorganisms in osmo-adaptation. Glycerol was reported as the main solute used by H. werneckii and W. ichthyophaga to maintain cell turgor pressure 48 . The synthesis of glycerol is carried out by NAD-dependent glycerol-3-phosphate dehydrogenase (Gpd) and glycerol-3-phosphatase (Gpp), acting on the glycolysis intermediate dihydroxyacetone phosphate. The putative glycerol metabolism-related genes in UM 843 are listed in Supplementary Table S12. The Gpd gene (UM843_9164) shared 74.22% and 73.29% identity with H. werneckii Gpd1A and Gpd1B, respectively. Similar to the Gpd of H. werneckii and W. ichthyophaga, UM843_9164 does not have the N-terminal type 2 peroxisomal targeting sequence (PTS2) (Supplementary Fig. S12). The absence of PTS2 in the gene is suggested to be an advantage for survival in high salinity environments 49 .
In addition, six putative genes were identified to encode glycerol/H + symporter Stl1 that is essential for active glycerol uptake. All the genes appeared to contain 12 transmembrane domains, a typical characteristic of Stl1 50 (Supplementary Fig. 13). The glycerol/H + symporter located in the plasma membrane actively imports glycerol into the cell during hyperosmotic shock 50 . Moreover, three genes encoding aquaglyceroporin (UM843_5638, UM843_4817 and UM843_4886) that function in the efflux of glycerol during hypoosmotic conditions were also identified. The presence of sugar/H + symporters and aquaglyceroporins in large numbers suggests that UM 843 can mount a rapid response to combat a highly dynamic concentration of external NaCl. Additionally, UM 843 might synthesise taurine to act as an osmoregulant ( Supplementary Fig. S14), as suggested in the acidophile Acidomyces richmondensis 51 .
The high osmolarity glycerol (HOG) pathway is an important mitogen activated protein kinase (MAPK) signalling pathway that is involved in osmoregulation activated under osmotic and cationic stress 15 . We detected the genes involved in the HOG signalling pathway in UM 843 (Supplementary Table S12). This pathway is activated by two types of osmosensors, the Sho1 (UM843_8679) and Sln1 (UM843_4487). The histidine kinase Sln1 together with the phosphorelay molecule Ypd1 (UM843_8084) and response regulator Ssk1 (UM843_8730) form the final receptor that activates the MAPK kinase Pbs2 (UM843_2812) and in turn, phosphorylates MAPK Hog1 (UM843_5411). In the activation via the Sho1 branch, Sho1 activates Pbs2 through Ste11 and Ste20 (UM843_3491) 15    Fungal allergens. Allergy is one of the main concerns in medical mycology as numerous fungi such as Aspergillus, Alternaria, Penicillium, and Cladosporium are known to cause allergic reactions 6 . Among Cladosporium species, C. herbarum is the best studied with a total of 60 antigens, of which at least 36 having reactions with IgE antibodies from patients' sera have been identified, as reported in the latest Thermo Scientific allergen database (http://www.phadia.com/en/Products/Allergy-testing-products/ ImmunoCAP-Allergen-Information/Molds-and-other-Microorganisms/Allergens/Cladosporium-herbarum-/). Although C. sphaerospermum has been reported to be an allergy-causing mould, specific allergens have not been identified 55 . BLASTX similarity searches using the predicted gene models identified 28 genes functionally annotated as allergens showing > 50% identity to the respective allergen (Table 4). Although not all the predicted genes will encode allergenic proteins, this feature indicates a high potential for serum cross-reactivity in patients sensitised with these fungal allergens, as proteins with > 50% identity to known allergens are likely to cross-react 56 . For instance, cross reactivity among fungal allergens has been reported between Cla h 8 and Alt a 8 57 ; and Cand b 2 and Asp f 3 58 . Compared to other Dothideomycetes, four predicted gene families encoding allergens, viz. Cla h 10, Cla h HCh 1, Asp f 23 and Cand a 1, have higher copy numbers of genes in UM 843 (Supplementary Table S15). Interestingly, Cla h HCh 1 was only found in UM 843 (UM843_1201, UM843_6061, UM843_4115 and UM843_3639). These putative proteins were similar to C. herbarum Cla h HCh 1 (GenBank: Q8NIN9) ( Table 3). The mRNAs of the four genes (UM843_1201, UM843_6061, UM843_4115 and UM843_3639) were detected by RT-PCR ( Supplementary  Fig. S16). The extracted mRNA used for cDNA synthesis were of high quality (Supplementary Fig. S17) and the RT-PCR products corresponding to the size of open reading frames (ORF) (~300 bp) were successfully amplified from the converted cDNA. This finding revealed that these predicted allergen-encoding genes are not pseudogenes but were expressed in UM 843 (Supplementary Figs S16, S18-S21). Weichel et al. 59 showed that Cla h HCh1 was the only hydrophobin in C. herbarum that elicited a specific IgE-dependent allergic reaction. As previously reported 60 , we noticed that UM843_1201, UM843_6061, UM843_4115 and UM843_3639 contain eight conserved cysteine residues each ( Supplementary Fig. S22). It would be interesting to determine the allergenicity of Cla h HCh1 in C. sphaerospermum as it might be a Cladosporium-specific allergen.
The C. sphaerospermum UM 843 genome provides the first glimpse into the genetic basis of important adaptation traits for the fungus to survive in diverse environmental niches. The same genes that enable adaptation to adverse environmental conditions may also confer a selective advantage for survival and adaptation in adverse  microenvironments in human hosts. The isolation of UM 843 from the peripheral blood sample of a patient foretells the emergence of C. sphaerospermum as an important opportunistic pathogen in susceptible human populations. Furthermore, allergen-encoding genes identified in this study could be further validated at the protein level and tested for specific allergenicity. These candidate allergens could be useful for the development of immunotherapeutic vaccines against allergic fungal reactions in humans.

Methods
Ethics statement. The genome used in this study was obtained from a fungal isolate routinely cultured and archived by the mycology laboratory in a teaching hospital 7  Fungal isolate. UM 843 was isolated from the peripheral blood sample of a patient in UMMC, Malaysia. The isolate was sub-cultured on Sabouraud Dextrose Agar (SDA). For the scanning electron microscopy (SEM), a 7-days old culture on SDA was processed and viewed under SEM (Phillips XL30 ESEM, the Netherlands). To test the ability of UM 843 to grow in high salt medium, the isolate was cultured in Sabouraud Dextrose Broth (SDB) supplemented with 5%, 10%, 15%, 20% and 25% (w/v) of NaCl. The growth of the fungus was observed up to 14 days of incubation. Molecular identification. ITS-based molecular identification was carried out using ITS region. The isolate was subjected to DNA extraction, amplification, DNA sequencing and ITS-based phylogenetic analysis was performed as previously described with slight modification 7 . The selection of species to be included in the phylogenetic analysis was limited to species closely related to C. sphaerospermum. Complete ITS1-5.8S-ITS2 sequences for 18 C. sphaerospermum species complexes and two outgroup strains were obtained from GenBank for phylogenetic tree construction. Bayesian tree analyses were performed using MrBayes. Bayesian Markov Chain Monte Carlo (MCMC) analysis was conducted by sampling across the entire general time reversible (GTR) model space. A total of 150,000 generations were run with a sampling frequency of 100, and diagnostics were calculated for every 1,000 generations. A burn-in setting of 25% was used to discard the first 375 trees.
Genome analysis workflow of Cladosporium sphaerospermum UM 843. The genomic DNA extraction, sequencing, assembly, gene model prediction, and functional annotation of C. sphaerospermum UM 843 was conducted as previously described 61 with some modifications. The 5-kb insert library was sequenced using the Illumina HiSeq 2000 system. The sequenced reads were then combined with the 500-bp Illumina sequenced reads 8 for further processing. Genes associated with stress responses were identified by performing a local BLASTP search against a database built from the Fungal Stress Response Database (FSRD, http://internal. med.unideb.hu/fsrd) using the criteria of e-value threshold ≤ 1e-5, identity exceeding 50% and subject coverage exceeding 70%.
The protein sequences of all current publicly-available dematiaceous Dothideomycetes genomes were downloaded from different databases to determine the orthologues in UM 843 for the orthologous genes and genome comparative analysis (Supplementary Table S1). A phylogenomic tree was constructed using all proteome clusters of Dothideomycetes generated from comparative analysis with inclusion of another two proteomes from class Sordariomycetes as outgroup strains (Supplementary Table S1). A total of 220 single-copy orthologous genes containing one member in each species was subjected to individual sequence alignments and removal of spurious sequences or poorly aligned regions with trimAL (with the automated option). The filtered multiple alignments were then concatenated into a superalignment with 110,781 characters. Subsequently, the best-fit substitution model was selected by running ProtTest version 3.2 62 with AIC calculation on the alignment. The phylogenomic tree was constructed by using MrBayes as previously described 61 and RAxML version 7.7.9 63 . The MCMC was run with a sampling frequency of 100 for 250,000 generations, and burn-in setting of 25%. RAxML was run with model PROTGAMMALGF to search for the best-scoring maximum likelihood tree, followed by 100 bootstrap replicates. Convergence was observed after 50 replicates using -I autoMRE option in RAxML.

RNA extraction and Reverse Transcription PCR of hydrophobin genes. The 7-days old mycelia
were scraped off from the agar surface and 100 mg of samples were crushed into fine powder with liquid nitrogen. Total RNA were then isolated from the frozen samples using RNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. The concentration of RNA was determined using spectrophotometer at the wavelength of 260 nm. The purity of RNA sample was assessed using the ratio of absorbance at 260 nm and 280 nm. The size distribution and integrity of the RNA were checked by 1% (w/v) agarose gel electrophoresis. The RNA bands were visualised by RedSafe nucleic acid staining solution (Intron Biotechnology, Korea) staining and observed under UV light. The RNA samples with the ratio of absorbance at 260 nm and 280 nm was between 1.8 and 2.1 and high integrity were used for subsequent cDNA synthesis. cDNA was synthesised from the extracted total RNA using RevertAid H Minus first strand cDNA synthesis kit (Fermentas, Germany) according to manufacturer's protocol with only modification in using a primer mixture of 0.5 μ L oligo (dT) 18 primer and 0.5 μ L random hexamer in the cDNA synthesis. The primers used for amplification were listed in the Supplementary Table S16. PCR was performed with an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 58 °C for 30 seconds, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min. The PCR product was then electrophoresed in 1% (w/v) agarose gel at 90V for 30 min, purified and sent for Sanger sequencing (First Base Laboratories, Malaysia).