Genome sequence and comparative analysis of clavicipitaceous insect-pathogenic fungus Aschersonia badia with Metarhizium spp.
© Agrawal et al. 2016
Received: 23 December 2015
Accepted: 5 May 2016
Published: 17 May 2016
Aschersonia badia [(Ab) Teleomorph: Hypocrella siamensis] is an entomopathogenic fungus that specifically infects scale insects and whiteflies. We present the whole genome sequence of Ab and its comparison with two clavicipitaceous fungi Metarhizium robertsii (MR: generalist entomopathogen) and M. acridum (MAC: acridid-specific entomopathogen) that exhibit variable host preferences. Here, through comparative analysis of pathogen-host interacting genes, carbohydrate active enzymes, secondary metabolite biosynthesis genes, and sexuality genes, we explore the proteins with possible virulence functions in clavicipitaceous fungi. Comprehensive overview of GH18 family chitinases has been provided to decipher the role of chitinases in claviceptaceous fungi that are either host specific or generalists.
We report the 28.8 Mb draft genome of Ab and its comparative genome analysis with MR and MAC. The comparative analyses suggests expansion in pathogen-host interacting gene families and carbohydrate active enzyme families in MR, whilst their contraction in Ab and MAC genomes. The multi-modular NRPS gene (dtxS1) responsible for biosynthesis of the secondary metabolite destruxin in MR is not conserved in Ab, similar to the specialist pathogen MAC. An additional siderophore biosynthetic gene responsible for acquisition of iron was identified in MR. Further, the domain survey of chitinases suggest that the CBM50 (LysM) domains, which participate in chitin-binding functions, were not observed in MAC, but were present in Ab and MR. However, apparent differences in frequency of CBM50 domains associated with chitinases of Ab and MR was identified, where MR chitinases displayed a higher proportion of associated CBM50 domains than Ab chitinases.
This study suggests differences in distribution of dtxS1 and chitinases in specialists (Ab and MAC) and generalists (MR) fungi. Our analysis also suggests the presence of a siderophore biosynthetic gene in the MR genome which perhaps aids in enhanced virulence potential and host range. The variation in association of CBMs, being higher in generalists (MR) and lower in specialists (Ab and MAC) fungi may further be responsible for the differences in host affiliation.
The fungal family Clavicipitaceae (Hypocreales, Ascomycota) includes many insect-pathogens such as Metarhizium spp. and these fungi have been exploited for their mycoinsecticidal abilities. Family Clavicipitaceae, as defined in modern taxonomy [Clavicipitaceae sensu stricto (s.s.)], includes three lineages specific to scale insects and whiteflies, referred to as Hypocrella, Regiocrella and Torrubiella; and one generalist lineage, referred to as Metacordyceps . In this study, we have selected Aschersonia badia (Ab), an insect-pathogenic fungus belonging to the Hypocrella lineage for whole genome sequencing.
Aschersonia spp. are insect-pathogenic fungi that specifically infect whiteflies (Homoptera, Aleyrodidae) and scale insects (Homoptera, Coccidae). These are predominately found in tropical and sub-tropical areas. Aschersonia spp. [A. aleyrodis (Teleomorph: Hypocrella libera)] were one of the first fungal entomopathogens utilized as a biocontrol agent  and was reported as the cause of epizootics amongst whitefly populations in greenhouses and guava and citrus groves during the 20th century in various parts of the world such as Azerbaijan, Bulgaria, China, Florida, Jamaica, Japan and Russia [3–6]. Moreover, Aschersonia spp. show adaptation to low relative humidity , perseverance on plant exteriors , and compatibility with insect parasitoids  in the management of whitefly pests. However, these fungi take a long time to grow in culture, and are not effective against all host stages and this has limited their successful exploitation against insect pests .
Metarhizium spp. (Clavicipitaceae s.s., Metacordyceps) are well known entomopathogenic fungi and are best suited targets for biocontrol measures. Metarhizium robertsii (MR: previous name M. anisopliae) is a broad-spectrum insect pathogenic fungus that has been approved by the United States Environmental Protection Agency (USEPA) as an active ingredient for pest control [11, 12]. M. acridum (MAC) is a specialist pathogen against locust . These diverse features of clavicipitaceous entomopathogens drive our interest towards comparison of Ab genome with the MAC and MR genomes that are adapted to different host ranges and lifestyles in order to get insights into the evolution of host affiliation of fungal entomopathogenicity.
Fungal entomopathogens are known to exhibit contact-based infection through the host cuticle unlike bacterial and viral entomopathogens which are required to be ingested by the target pest . The primary structural component of host cuticle (arthropod exoskeleton) and cell walls of filamentous fungi is chitin which is the second most abundant natural biopolymer after cellulose. Enzymatic degradation is a crucial step in entry of the fungal spore into the insect body where chitinases play an important role in hydrolyzing the chitin-rich insect cuticle . Metarhizium spp. are known to be prolific producers of chitinases, and many of the MR chitinases have been suggested to be involved in pathogenicity [16, 17]. However, in-depth analysis of chitinases and their relation with pathogenic lifestyles of clavicipitaceous entomopathogens have not been explored. Therefore, in the present study, we explore the diversity of chitinases in Ab, MAC and MR.
Fungal strain and maintenance
Ab strain MTCC 10142 was retrieved from the Microbial Type Culture Collection (MTCC), CSIR-Institute of Microbial Technology, Chandigarh, India. As per MTCC records, the Ab strain was isolated from Homopteran larva. Fungal culture was grown on potato dextrose agar medium and incubated at 25 °C for 20–25 days. DNA isolation from the fresh mycelia was performed using ZR Fungal/Bacterial DNA kit (Zymo Research, Catalogue number D6005) as per the instructions provided in user manual.
Genome sequencing, assembly and annotation
2x100 paired-end shotgun sequencing (average insert size of 350 bp) of the Ab genome was performed using Illumina HiSeq 1000 technology at the Centre for Cellular and Molecular Platforms (C-CAMP) Bangalore, India. The reads were filtered using NGS QC Toolkit v2.2.3  with length cutoff 70 % and base call quality greater than 20; reads were also trimmed for adapters using FastQC  and CLCbio wb6.0 genomics workbench (http://www.clcbio.com). The datasets were filtered for the presence of reads from bacterial DNA that may be present as contaminants. These were then further processed for genome assembly. The high quality reads were assembled into contigs using SPAdes 2.5.1 . Before running SPAdes as an assembler, its 'read-error-correction’ module was used to rectify any orientation issues and InDels in the paired reads. Then its Assembly protocol was run at K-mer range 51–69 with step of +2 and ‘--careful’ option. The contigs obtained from these assemblies were scaffolded using SSPACE v2.0 . Completeness measure was predicted using Core Eukaryotic Genes Mapping Approach (CEGMA) v2.5 with default parameters .
Genome annotation was performed using MAKER pipeline with Augustus as the gene predictor [23, 24]. Fusarium graminearum (Fg) was selected, as a model for gene calling, from the Augustus pre-trained dataset. EST and protein homology evidence of Fg was also provided to MAKER. The repeats were masked using RepeatMasker v4.0.3 (http://www.repeatmasker.org/). MAC CQMa 102 (PRJNA38715) and MR (PRJNA230500) genomes were reannotated using the same methodology for consistency. Both reannotated and the original set of genes/proteins from MAC and MR genomes were used for the computational analyses. The predicted proteins were subjected to BLASTp against the non-redundant (nr) database at the National Centre for Biotechnology Information (NCBI) using an E-value cutoff of 10−5. The draft genome of Ab (Hypocrella siamensis) was deposited at NCBI-GenBank under the accession number JMQE00000000.
To validate the phylogenetic position of Ab, MAC and MR, four gene markers: mitochondrial ATP6 (atp6), the largest and second largest subunits of RNA polymerase ІІ (rpb1 and rpbII) and β-tubulin (tub) genes were retrieved from the Ab, MAC and MR genomes and were concatenated. These concatenated sequences were included in the four gene concatenated sequence dataset of hypocrealean fungi . Maximum Likelihood (ML), Maximum Parsimony (MP) and Neighbor-Joining (NJ) phylogenetic reconstruction was performed using Randomized Axelerated Maximum Likelihood (RAxML) v8.0 , Phylogenetic Analysis Using Parsimony (PAUP) v4.0  and Molecular Evolutionary Genetics Analysis (MEGA) v6.0 , respectively. Ambiguously aligned regions were excluded from the analysis using the trimAl tool . Bootstrap analysis with 1000 replicates was performed.
Cluster of orthologous (COG) groups
The COG clusters were created by subjecting the predicted proteomes to BLASTp against the COG database  2014 update (http://www.ncbi.nlm.nih.gov/COG/) with an E-value threshold of 10−5. For each COG mapping, functional category assignment was given based on COG category letter associations. Reciprocal best BLAST hits (E-value threshold of 10−10) obtained using PERL script of Proteinortho v2.0  were used to characterize the orthologous proteins among all three genomes. Computational Analysis of gene Family Evolution (CAFE) v3.0  was used to estimate the gene family expansions and contractions in Ab, MAC and MR for the CAZymes glycoside hydrolases (GHs, 55 families), glycosyltransferases (GTs, 37 families), carbohydrate esterases (CEs, 10 families), auxiliary activities (AAs, 10 families) and carbohydrate binding modules (CBMs, 17 families) and the trasposase gene families (DNA transposase gene families: 10, Retrotransposase gene families: 7). ML Newick tree prepared through MEGA was used as an input for CAFE. P-value cut-off of 0.01 was selected to calculate the significant changes in gene numbers.
Protein domain search
Secondary metabolite biosynthetic clusters were identified by using the predicted proteome as query on the antiSMASH server . Pathogen Host interacting (PHI) partners were identified by subjecting the predicted proteomes to BLASTp against the PHI database  v3.6 with an E-value threshold of 10−5. The domain architecture of chitinases and destruxins were annotated using the Pfam v27.0 and SMART v7.0 databases [34–36]. Molecular weight was evaluated using the Protparam tool .
Phylogenetic analysis of non ribosomal peptide synthase (NRPS) biosynthetic gene types
NRPS genes identified from the three genomes Ab, MAC and MR were combined with the phylogenomic NRPS dataset reported previously . Their phylogenetic analysis was performed as described above. Multiple sequence alignment file used for the phylogenetic analysis is provided as Additional file 1 (A, B).
Sequence similarity of destruxin gene was calculated using SIAS server (http://imed.med.ucm.es/Tools/sias.html). After running BLASTp [Query: dtxS1 (MR), Database: Ab_Proteome], the alignments of interest (dtxS1 and Ab-Node74-Gene7905) were uploaded in SIAS server and % identities and % similarities were obtained.
Carbohydrate active enzymes (CAZy) search
CAZY classes Glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), auxiliary activities (AAs) and carbohydrate binding modules (CBMs) were searched from the CAZy database. CAZy families were assigned to the proteins by subjecting the predicted proteome of all the three genomes to dbCAN web server .
Transposable elements (TEs) and Repeat induced point mutation (RIP) analysis
TEs were classified by subjecting the genome sequences of Ab, MAC and MR to BLASTn against the Repbase (http://girinst.org) libraries of RepeatMasker (http://www.repeatmasker.org). RIP indices were estimated using the RIPCAL v1.0 .
Identification of MAT idiomorphs
Mating type genes control sexual development in fungi . Putative mating type loci were identified by the presence of conserved domains and the sequence similarities to the corresponding MAT genes in other filamentous fungi. Conserved alpha domain, Proline–Proline–Phenylalanine domain, High–Mobility–Group (HMG) domain with a DNA binding site and MatA HMG box with a DNA binding site were searched to identify the mating type loci MAT1-1-1, MAT1-1-2, MAT1-1-3 and MAT1-2-1, respectively [42, 43]. Synteny for the mating type locus among all the three genomes was analyzed by locating the position of MAT locus and their flanking genes. The absence of any gene was reconfirmed by mapping back the reads on these genes from related fungi such as Metarhizium majus, Trichoderma reesei, Fusarium graminearum, Beauveria bassiana, Cordyceps militaris, Ophiocordyceps sinensis, Hirsutella thompsonii, Isaria farinose, etc. This was done mainly to verify that they were not missing in the three genomes due to assembly errors.
Glycoside hydrolase family 18 (GH18) chitinases search
The chitinase sequences were searched in the predicted proteomes of Ab, MAC and MR genomes using ProfileScan (http://www.csd.hku.hk/bruhk/gcgdoc/profilescan.html) with the conserved active site signature motif [LIVMF]-[DN]-G-[LIVMF]-[DN]-[LIVMF]-[DN]-x-E (Prosite No. PS01095) . The identified chitinase sequences from the three genomes were aligned using PCMA  and profile hidden Markov model was built using HMMER v3.1b1 . This HMMER profile was further used to scan the predicted proteome of the genomes to find other chitinase sequences that have mutations/SNPs at conserved consensus motifs. GH18 chitinase sequences of fungal origin were selected from a previously reported dataset . All retrieved chitinases from the three genomes and GH18 family chitinase sequences of fungal origin were assembled and aligned using MAFFT v7.0 . For subgroup A, B and C fungal chitinases, three separate alignment files were prepared as previously described [47, 49, 50] (Additional file 2).
Phylogenetic relationships of Ab and Metarhizium species
The phylogenetic analysis grouped Ab, MAC and MR within the Clavicipitaceae s.s. clade (Additional file 3: Figure S1). Ab MTCC 10142 strain was clustered within the Hypocrella clade whereas MAC and MR were clustered within Metacordyceps clade (ML, MP and NJ analysis parameters presented 100 % support values) thus validating the phylogenetic identification of the organisms under study. The phylogenetic tree displayed the paraphyletic occurrence of family Clavicipitaceae into clades A, B and C, viz., Clavicipitaceae s.s., Ophiocordycipitaceae and Cordycipitaceae, respectively and the grouping of Clavicipitaceae s.s. clade into four lineages: three specific to scale insects and whiteflies, Hypocrella, Torrubiella and Claviceps; with one as a generalist, Metacordyceps (Additional file 3: Figure S1).
Genome sequencing, assembly and annotated features
High quality reads
Max. contig length
Min. contig length
Total genes annotated
Genes involved in biosynthesis of secondary metabolites
Gene types involved in the biosynthesis of secondary metabolites in Ab, MAC and MR genomes
Unusual HglD/E-like PKS
NRPS are modular enzymes involved in the biosynthesis of diverse low molecular weight bioactive metabolites that participate in various fundamental metabolic functions of fungi such as growth and development, virulence, niche specific functions, stress responses, etc. [38, 55]. A set of three core domains (A-T-C) forms the basic module of NRPS where A is adenylation domain for recognizing and activating the substrate (initiation), T or PCP is thiolation domain or peptidyl carrier protein (PCP) for binding and transferring the activated substrate (elongation) and C is the condensation domain for forming peptide bonds between elongated amino acid (aa) chain (termination) [38, 56]. Additionally, accessory domains like epimerization (E) domain responsible for altering the aa configuration from L to D form, N-methylation domain (nMT) responsible for transfer of the methyl group to the substrate may also be present in the NRPS module [38, 57]. The number and order of these modules on each NRPS genes is known to govern the structural variability of the resulting product [38, 58, 59].
Pathogen-host interacting (PHI) genes
Carbohydrate active enzymes
A total of six classes of carbohydrate active enzymes as provided in CAZy database have been compared. The genes from each class: carbohydrate binding modules (CBMs), glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterase (CE), and auxiliary activities (AAs) are provided in Additional files 6, 7, 8, 9, 10 and 11, respectively.
The prevalence of associated functional modules, CBMs was found to be quite variable in the three fungal genomes (Additional file 6). CBM38 (inulin binding function) and CBM54 (xylan, glucan and chitin binding function) were observed to be unique to Ab. CBM1 (cellulose binding domain) family members were observed in MAC and MR. CBM18 and CBM50 (LysM) family modules known to be associated with chitinase catalytic domains and are implicated to bind chitin were found to be commonly present in all three genomes. However, their frequency was lowest in MAC genome. CAFE analysis suggests a contraction of CBM families in Ab and MAC genomes, whereas they are expanded in the MR genome.
GHs, GTs, PLs, CEs and AAs are the catalytic enzyme classes that direct the lysis, synthesis or alteration of carbohydrate moieties [67–70]. The three genomes show the predominance of GH16 (xyloglucan: xyloglucosyltransferase), GH18 (chitinase) and GH76 (α–1,6-mannanase) family enzymes (Additional file 7). GH9 (endoglucanase) and GH120 (β-xylosidase) were observed to be unique to the Ab genome. GH54 (α-L-arabinofuranosidase; β-xylosidase), GH88 (d-4,5-unsaturated β-glucuronyl hydrolase), GH95 (fucosidase) and GH117 (α-1,3-L-neoagarooligosaccharide hydrolase) were exclusive to the MR genome. GT2 (cellulose/chitin synthase) and GT32 (α-1,6-mannosyltransferase/N-acetylglucosaminyltransferase) were commonly observed in the three genomes (Additional file 8). Ab displayed exclusive occurrence of PL10 (pectate lyase) family enzyme, whereas MAC and MR displayed PL7 (alginate lyase), PL8 (hyaluronate lyase) and PL20 (endo-β-1,4-glucuronan lyase) family enzymes (Additional file 9). CE1 (acetyl xylan esterase; carboxylesterase) and CE10 (arylesterase; carboxyl esterase; acetylcholinesterase; sterol esterase) family enzymes were predominantly present in all the three genomes (Additional file 10). AA3 (glucose-methanol-choline (GMC) oxidoreductases) and AA7 (oligosaccharide oxidase) were the most frequent families in the three genomes (Additional file 11). CAFE analysis indicated the contraction of CAZY families (GHs, GTs, CEs and AAs) in Ab and MAC genomes, whereas their expansion in MR genome.
Repeat induced point mutation (RIP) and diversity of transposable elements (TE)
Repeat-induced point mutation (RIP) is a protection system in fungal genomes that operates on duplicated sequences and checks the development of TEs through hypermutation [71, 72]. A genome-wide RIP analysis was performed on the Ab, MAC and MR genomes. Summary of RIP signatures predicted in the three genomes is provided in Additional file 12. RIP frequency index (CpA + TpG)/(ApC + GpT) was calculated as 0.72 in Ab, 0.94 in MAC and 1.06 in MR (Additional file 13), where the threshold was 1.03 and the RIP index ≤ 1.03 indicated RIP activity. Therefore, Ab and MAC genomes were suggested to present significant RIP activity consistent with earlier reports supporting occurrence of RIPs in specialists genome whereas their absence from non-specialists genomes [13, 60]. The frequency of putative transposase genes suggests their expansion in MR genome as compared to the Ab and MAC genomes (Additional file 14). This observed difference is suggested to be related to the effect of RIP activity [13, 60].
Phylogenetic classification of putative cuticle degrading chitinases
Fungal chitinases are known to belong exclusively to the GH18 family [75–77]. Based on amino acid sequence similarity of their GH18 domains [49, 77], these have been classified into three subgroups A, B and C. These display differences in their enzymatic properties—exo for sg-A and sg-C, whereas endo for sg-B; the presence or absence of carbohydrate binding modules (CBMs), absent in sg-A, whereas present in sg-B at C-terminal and in sg-C at N-terminal—and the architecture of their substrate binding cleft [75–77]. In this study, we identified 16, 23 and 28 chitinase genes in Ab, MAC and MR genomes, respectively. Their properties are presented in Additional file 16. These chitinases were nomenclatured based on their increasing molecular weights and are represented as X Chit-z, where X is the organism label (Ab, MAC or MR) and z is the designated number. A few of the MAC and MR chitinases were named on the basis of sequence similarity between MAC and MR chitinases. The three datasets, viz., subgroup A, B and C were analyzed to reconstruct their phylogenetic relationships.
Subgroup-A (sg-A) dataset
Subgroup-B (sg-B) dataset
This dataset included 68 chitinase sequences, including 29 sequences retrieved from Ab, MAC and MR genomes (Fig. 5b). All chitinases were clustered with H. jecorina chitinases. Interestingly, MAC was found to contain more chitinases than MR. However, Ab contained the least number of sg-B chitinases. Further, Ab Chit-1, MAC Chit-3, MAC Chit-7 and MR Chit-7 were observed to cluster with H. jecorina Chi-15, forming a separate clade in sg-B.
Subgroup-C (Sg-C) dataset
This dataset included 50 chitinase sequences, including 18 sequences retrieved from Ab, MAC and MR genomes (Fig. 5c). The number of sg-C chitinases was higher in the Ab genome as compared to the MR and MAC genomes.
Domain architecture of chitinases
The sg-B chitinases are known to possess CBMs (CBM1) as their C-terminal domains . Our analysis shows CBM1 is present in the MAC and MR genomes but absent in the Ab genome. Multiple (nine) copies of C-terminal CBM50 domains were observed in MR genome.
The sg-C chitinases too displayed some interesting differences. Most of sg-C chitinases contained the N-terminal CBM18 and CBM50 domains in Ab and MAC genomes. However, MR genomes showed regular characteristics of sg-C chitinases, i.e., high molecular weight and presence of multiple domains.
It is often suggested that diversity in pathogen-host relationships contributes to species divergence but the knowledge of genetic mechanisms responsible for speciation and varied host interactions in fungal entomopathogens have been quite limited [80, 81]. The entomopathogenic fungal lineages possess a number of potential pathogenicity associated genes in their genome to acclimatize to varied niches [60, 82–85]. The evolutionary dynamics of host affiliation observed in Clavicipitaceae have been aptly explained by the host-habitat hypothesis where the proximity in the habitat has been related to the acquisition of new hosts . Metacordyceps subfamily in Clavicipitaceae lineage is of interest as it contains pathogenic species varying from specialists (with a narrow host range), transitional (with intermediate host range) to generalists (with broad host range) categories [59, 60]. Comparative analyses of their genomes has already been reported that suggests the association of generalists with loss of RIPs (genome defense) and in turn expansion of gene/protein families, whereas the retention of RIPs in specialists fungi [13, 60]. In the current study, comparative genomics of Ab belonging to the Hypocrella subfamily with Metacordyceps members MAC and MR further aids in understanding the genomic basis for host niche adaptations in the Clavicipitaceae lineage. Ab, being a specialist fungus shows an evolutionary pattern similar to the specialist genome MAC, with a similar occurrence of RIPs and in the evolution of protein families (contraction). This study also supports that during their evolution generalist fungi have lost the active RIP mechanism which may have aided broader host affiliation by the retention of duplicated genes and in turn protein family expansions. Generalist’ pathogens are suggested to have evolve higher numbers of multi-modular NRPS genes (such as dtxS1), siderophores, CBMs and chitinases as contributory factors in acquiring new virulence mechanisms to enhance their pathogenicity potential [13, 60].
Multimodular NRPS, dtxS1 as a virulence factor
The hexamodular NRPS gene responsible for the production of destruxins, identified as dtxS1 gene in MR genome was reported to be involved in the pathogenicity of generalist fungus MR . It was postulated that acquisition of dtxS1 gene in Metarhizium lineages was in coordination with the evolution of host specificity . Our genome analysis suggests that Ab genome does not contain the NRPS dtxS1 multi-domain gene as reported for specialist pathogens [58–60]. The NRPS subfamilies are known to cluster into two main groups: mono/bimodular NRPS (of ancient origin, bacterial and fungal NRPS) and multimodular NRPS (recent origin, exclusively fungal NRPS) . The multimodular NRPS subfamilies were identified to contain variable domain architecture in response to the niche adaptation roles, illustrating frequent incidences of domain gain and loss events, unlike mono/bimodular NRPS that are less variable in architecture [38, 55]. The destruxin biosynthesis NRPS gene belongs to the multimodular NRPS cluster, is likely to have originated via a gene/domain duplication event in MR to form NRPS dtxS1 gene. Gain of multimodular NRPS gene (such as dtxS1) could therefore be suggested as responsible for the generalist behavior and enhanced virulence of MR [59, 60].
Siderophores as virulence factors
Iron is an essential micronutrient for life and the ability of pathogens to cope with limited iron within the host organism represents an evolutionary force for the development of virulence [54, 87]. Siderophores are high-affinity small molecules that function in iron uptake . Pathogenic fungi have evolved the production of these siderophores for acquisition of iron from the host organism they infect, to ensure their own survival [54, 87, 89]. Maintenance of iron homeostasis therefore suggests association of fungal pathogenicity and siderophore biosynthesis [52–54]. There are reports from pathogenic fungi such as Alternaria alternata , Aspergillus fumigatus [53, 54, 91], Cryptococcus neoformans , Fusarium graminearum , Magnaporthe oryzae , Metarhizium robertsii  that correlate iron acquisition and virulence. For example, mutagenesis studies on murine model of invasive aspergillosis caused by Aspergillus fumigatus displayed complete lack of virulence on elimination of the entire siderophore biosynthetic gene (ΔsidA mutant) demonstrating critical role of siderophores in pathogenicity and thereby in fungal-host interactions [91, 96, 97]. Likewise, another mutagenesis study of two NRPS enzymes sidD (intracellular siderophore) and sidC (extracellular siderophore) investigated the involvement of siderophores in pathogenesis in MR . Based on these studies, it could therefore be proposed that fungal entomopathogens have evolved the genes involved in siderophore biosynthesis to raise their virulence potential.
Chitinases as virulence factors
Chitinases have been suggested as the determining factor of virulence in fungal entomopathogens with their involvement in degradation of arthropod cuticle [50, 98]. In the present study, we observe a higher proportion of chitinases in the genomes of the insect pathogens (Ab, MAC and MR) compared to other non-insect pathogens like Aspergillus spp. and Rhizopus spp. [13, 99]. Sg-B and sg-C chitinases are commonly known to be involved in virulent and aggressive functions towards insects in entomopathogenic fungi [100–102] likely due to the presence of additional domains like CBM1, CBM18 and CBM50 responsible for chitin binding functions . Interestingly, only the MR genome is observed to contain chitinases (MR Chit-9 classified under sg-B clade) with multiple CBM50 domains at the C-terminal. Similar observation could also be viewed in sg-C classified MR chitinases with the presence of multiple copies of N-terminal CBM18 and CBM50 domains. The presence of comparatively expanded numbers of accessory domains, CBMs in MR genome may relate to evolution of multiple host adaptations [103, 104]. Moreover, the complete lack of LysM effectors (CBM50 domain) in MAC genome may also be associated with its pathogenicity potential and in turn host-specific behavior [79, 105]. This could also be supported with overall distribution of CBM1, CBM18 and CBM50 domains in Ab, MAC and MR genomes.
Mating type genes are the governing players of sexual functions where the presence or absence of two compatible mating type idiomorphs, MAT1-1 and MAT1-2 decides homothallism (self-fertility) or heterothallism (self-sterility but cross-fertility) in filamentous ascomycetes [41, 106]. Clavicipitaceous fungi too show diversity in their mode of mating [86, 107, 108]. The present analysis based on sequence similarity searches identified putative mating type idiomorphs, MAT1-1 (MAT1-1-1, MAT1-1-2 and MAT1-1-3) in Ab suggesting its potential for sexual fertility, which could also be supported by its known teleomorphic form Hypocrella siamensis . Mating type genes, MAT1-2-3 and MAT1-2-1 in place of MAT1-1-1, MAT1-1-2 and MAT1-1-3 were identified based on synteny analysis in MAC and the conservation of genes flanking the MAT1-1 idiomorph in MAC and MR (Fig. 4).
The present study is an attempt to understand the evolution of entomopathogenicity in Clavicipitaceae s.s. members in relation to their adaptations to host-range. In this study, we suggest the involvement of multimodular NRPS genes, siderophores, CBMs and chitinases in generalist clavicipitacean fungal entomopathogen MR that could have evolved for diverse host affiliations, as compared to specialist entomopathogens Ab and MAC.
Availability of supporting data
Dr. Belle Damodara Shenoy is acknowledged for critical inputs to an earlier draft of the manuscript. This work was financially supported by the project “Expansion and modernization of Microbial Type Culture Collection and Gene Bank (MTCC)” jointly supported by Council of Scientific and Industrial Research (CSIR) Grant No. BSC0402 and Department of Biotechnology (DBT) Govt. of India Grant No. BT/PR7368/INF/22/177/2012. Ab isolate for genome sequencing was obtained from MTCC, Chandigarh, India. NGS data was obtained from C–CAMP next-generation genomics facility, Bangalore, India. YA was supported by senior research fellowship from CSIR [31/050 (0315)/2010-EMR-I] and TN was supported from junior research fellowship from the CSIR XII five-year plan network project Genomics and informatics Solutions for Integrating Biology (GENESIS).
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