Comparative genomic and transcriptomic analyses reveal the hemibiotrophic stage shift of Colletotrichum fungi

Authors


Author for correspondence:

Ken Shirasu

Tel: +81 45 503 9574

Email: ken.shirasu@psc.riken.jp

Summary

  • Hemibiotrophic fungal plant pathogens represent a group of agronomically significant disease-causing agents that grow first on living tissue and then cause host death in later, necrotrophic growth. Among these, Colletotrichum spp. are devastating pathogens of many crops. Identifying expanded classes of genes in the genomes of phytopathogenic Colletotrichum, especially those associated with specific stages of hemibiotrophy, can provide insights on how these pathogens infect a large number of hosts.
  • The genomes of Colletotrichum orbiculare, which infects cucurbits and Nicotiana benthamiana, and C. gloeosporioides, which infects a wide range of crops, were sequenced and analyzed, focusing on features with potential roles in pathogenicity. Regulation of C. orbiculare gene expression was investigated during infection of N. benthamiana using a custom microarray.
  • Genes expanded in both genomes compared to other fungi included sequences encoding small, secreted proteins (SSPs), secondary metabolite synthesis genes, proteases and carbohydrate-degrading enzymes. Many SSP and secondary metabolite synthesis genes were upregulated during initial stages of host colonization, whereas the necrotrophic stage of growth is characterized by upregulation of sequences encoding degradative enzymes.
  • Hemibiotrophy in C. orbiculare is characterized by distinct stage-specific gene expression profiles of expanded classes of potential pathogenicity genes.

Introduction

Members of the ascomycete genus Colletotrichum represent a group of plant pathogenic fungi that can infect a wide range of plant species including many commercially important crops. One example is Colletotrichum orbiculare, which is known as the causal agent of anthracnose on cucurbits including cucumbers, melons and watermelons (Hyde et al., 2009). In addition, Colletotrichum gloeosporioides is a notable pathogen associated with > 470 different host species and is commercially significant as the major causal agent of post-harvest disease in fruits such as avocado, banana, mango, coffee and strawberries (Hyde et al., 2009). For example, C. gloeosporioides contributes to 80–50% of plant deaths in commercially grown strawberry nurseries and 40–50% of yield losses in strawberry cultivation (Xie et al., 2010).

Due to their economic importance, Colletotrichum spp. have become the subject of many studies on fungal pathogenicity (Perfect et al., 1999; Münch et al., 2008). Studies on Colletotrichum have revealed that many phytopathogenic Colletotrichum spp. adopt a hemibiotrophic lifestyle (Fig. 1). In compatible interactions, germinating conidia form melanized appressoria that penetrate epidermal cells directly through the cuticle and cell wall. Following penetration, specialized infection vesicles form and begin the growth of biotrophic intracellular hyphae that develop inside living host cells surrounded by an intact host plasma membrane. The infection then enters a necrotrophic stage where the fungus forms morphologically distinct, secondary, necrotrophic hyphae and obtains nutrients from dead host cells (Perfect et al., 1999; Shen et al., 2001). However, the genus is highly diverse, with different subgroups within a single species complex possessing various host ranges and levels of virulence, from destructive pathogens to putative endophytes (Sharma et al., 2011). Thus, study of this group of pathogens presents a unique opportunity to analyse features underlying a diverse range of plant–pathogen interactions.

Figure 1.

Life-cycle stages of Colletotrichum orbiculare: C. orbiculare infection of Nicotiana benthamiana. Conidia of C. orbiculare (a) germinate and form melanized appressoria to penetrate the epidermal cells of plants (b). The fungus then proliferates as bulbous primary biotrophic hyphae (c) before later developing thin, filamentous secondary hyphae associated with the switch to necrotrophy (d). Images for (b) were taken at 1 d post-inoculation (dpi), for (c) were taken at 3 dpi and (d) at 7 dpi. (e) Biotrophic infection of cucumber epidermal cell by C. orbiculare. The appressorium (A) has a thick melanized cell wall (AW) surrounded by adhesive extracellular matrix (ECM). A penetration peg (PP) emerging from the appressorial pore (AP) has produced an infection vesicle (IV) inside the epidermal cell and is surrounded by an intact host plasma membrane (filled arrowheads). Unfilled arrowheads, hyphae. Bars: (a–c) 20 μm; (d) 100 μm; (e) 1 μm. PW, plant cell wall; V, vacuole.

In the case of other filamentous plant pathogenic hemibiotrophs, colonization and the initial biotrophic interaction with host cells is facilitated by pathogen-encoded small, secreted proteins termed effectors (Sharma et al., 2011). Through such effectors, the pathogen is able to manipulate host metabolism and evade host immune responses that may be triggered by perception of conserved microbe-associated molecular patterns (MAMPS) such as chitin. As part of a ‘molecular-arms race’, plants have, in turn, evolved resistance proteins that can recognize specific effectors secreted by pathogens (Jones & Dangl, 2006; Dodds & Rathjen, 2010). Recognition of effectors by resistance proteins results in the death of infected cells thereby restricting the growth of the pathogen.

The tractability of different Colletotrichum species to in vitro culture and the development of techniques for gene disruption and complementation have facilitated their use as model pathogens (Perfect et al., 1999). In particular, Colletotrichum higginsianum, which invades the model plant Arabidopsis (Narusaka et al., 2004, 2009; O'Connell et al., 2004), and C. orbiculare, which infects Nicotiana benthamiana and Nicotiana tabacum in addition to cucurbits (Shen et al., 2001), have been well characterized. However, studies to identify effector proteins in commercially important phytopathogenic Colletotrichum have lagged behind experiments on pathogen biochemistry, including the analysis of metabolites produced by these fungi, and only a few effector proteins have been characterized so far (Kim et al., 2000; Stephenson et al., 2000; Kleemann et al., 2012; Yoshino et al., 2012). In addition, inventories of putative effectors have been recently predicted from the annotated genomes of C. higginsianum and Colletotrichum graminicola, which infects maize (O'Connell et al., 2012).

This paper describes the sequencing and comparative annotation of the genomes of C. orbiculare 104-T, a strain used extensively as an experimental model for molecular analysis of fungal pathogenicity, and C. gloeosporioides strain Nara-gc5, which was isolated from strawberries (Okayama et al., 2007). Transcriptomic analysis was also performed for C. orbiculare at different stages of plant infection, revealing classes of genes and processes important for infection.

Materials and Methods

Genomic DNA extraction

Genomic DNA from Colletotrichum orbiculare (syn. Colletotrichum lagenarium) strain 104-T (MAFF 240422) and C. gloeosporioides Nara gc5 isolated from strawberry (Akai & Ishida, 1968; Okayama et al., 2007) was obtained from cultures grown in liquid medium (Yoshimi et al., 2004) using the QIAgen DNAeasy Plant Minikit (Qiagen).

Sequencing and assembly

Colletotrichum orbiculare genomic DNA was sequenced to 55× coverage using Illumina Genome Analyzer IIx (34× coverage) and Roche 454 (22× coverage) sequencers (Takara-Bio Inc., Otsu, Japan). Sequences obtained from 454-pyrosequencing were assembled using the GS De Novo Assembler (Newbler, Roche), followed by alignment of reads from Illumina using the BWA (Li & Durbin, 2009) and Bowtie (Langmead et al., 2009) programs to correct homopolymer errors in the 454 data. Whole-genome shotgun (WGS) sequencing of C. gloeosporioides was performed on the Illumina HiSeq 2000 sequencer to 37× coverage with 90 bp paired-end reads from a 500 bp insert library (BGI, Beijing, China). After removal of singletons, low quality and adaptor sequences, reads were assembled using SOAPdenovo (Li et al., 2010). The completeness of each assembly was assessed using CEGMA v2.4 (Parra et al., 2007). All sequences were submitted to NCBI under BioProject accession numbers PRJNA171217 and PRJNA171218.

Repeat finding

De novo repeats were identified using RepeatScout (Price et al., 2005) using default criteria. Repeat elements were then filtered to exclude elements that were > 50% low-complexity and that occurred < 10 times in the respective genome. Consensus sequences containing putative repeat elements were classified using TEclass (Abrusán et al., 2009). Sequences were divided into blocks according to GC content using the program IsoFinder (Oliver et al., 2004). For calculation of RIP indices, dinucleotide frequencies were determined using the RIPCAL program (Hane & Oliver, 2008).

Gene prediction

Genes were predicted using a combination of Augustus (Stanke et al., 2008), GeneMark-ES (Ter-Hovhannisyan et al., 2008) and Conrad (DeCaprio et al., 2007). GeneMark-ES v2 was trained on the unmasked sequence of C. orbiculare, whereas predictions were made using Augustus trained on Magnaporthe oryzae. Gene model predictions were compiled using EvidenceModeler (EVM) including evidence from sequenced transcripts from in vitro-grown C. orbiculare assembled into 96 131 contigs using the Program to Assemble Spliced Alignments feature (Haas et al., 2008). This process was reiterated using Augustus and Conrad trained on the predicted transcripts and on Neurospora crassa. For C. gloeosporioides, gene predictions were made using GeneMark-ES v2 trained on its own genome sequence and Augustus trained on N. crassa, M. oryzae, C. orbiculare and Conrad trained on C. orbiculare. Gene models were compiled using EVM. Nucleotide and amino acid sequences were manually inspected using CLC Genomics Workbench (CLC bio, Tokyo, Japan). Multiple sequence alignments between the C. orbiculare and C. gloeosporioides genomes were performed and visualized using Geneious (Biomatters Ltd, Auckland, New Zealand).

Subcellular localization predictions

The predicted subcellular localizations were determined using SignalP (Nielsen et al., 1997) to identify N-terminal signal peptides and then TMHMM (Krogh et al., 2001) and Fungal BIG-PI (Eisenhaber et al., 2004) to exclude sequences with transmembrane domains and GPI-anchors. Sequences were then submitted to TargetP (Emanuelsson et al., 2000) analysis to identify predicted sequences for extracellular localization.

Functional annotation of predicted genes

Proteins were annotated using BLAST2go on the NCBI nonredundant protein database (Conesa et al., 2005) and using the PFAM server (http://pfam.sanger.ac.uk/). Predicted proteins were classified as proteases by querying the MEROPS database (Rawlings et al., 2012) using a BLASTp cut-off E-value of 1E-10. Sequences with homology for complete protease domains but with mutated active sites were further excluded as proteases. In addition, transporters were annotated by a BLAST search of the transporter classification database (Saier et al., 2009) using a BLASTp E-value cut-off of 1E-5. Potential secondary metabolite clusters were identified using SMURF (Khaldi et al., 2010). Carbohydrate active enzymes were classified using the dbCAN HMMer-based classification system (Yin et al., 2012) applying an E-value cut-off of 10E-5. For comparisons, similar analyses were performed in parallel for other sequenced fungi using sequences of proteins from N. crassa OR74A (version 10) M. oryzae 70-15 (version 6), Fusarium oxysporum f. sp. lycopersici 4287 (version 2) and Fusarium graminearum ph-1 (version 3) downloaded from the Broad Institute of Harvard and MIT. Sequences were clustered with MCL using BioLayout Express 3D (Theocharidis et al., 2009) using BLASTp with 10E-5 as a cutoff. Syntenic regions were detected using the DAGchainer program (Haas et al., 2004) with settings to search for regions of four or more collinear genes.

Prediction of noncoding RNAs

Transfer RNAs were predicted using tRNAScan-SE (Lowe & Eddy, 1997) and ribosomal RNAs were predicted using RNAmmer (Lagesen et al., 2007). Other noncoding RNAs were predicted using Erpin v5.4 (Gautheret & Lambert, 2001) that predicts ncRNAs based on RNA primary and secondary structure signatures from the RFAM 9.1 database.

Microarray analysis of gene expression

A custom 8 × 60 k microarray with 60-mer probes against exons from C. orbiculare predicted genes was designed using the eArray custom microarray design tool (http://earray.chem.agilent.com/earray/) using the best probe methodology and manufactured by Agilent Technologies. Probes were designed against all predicted C. orbiculare exons. In addition, EST sequences of N. benthamiana were used as queries in the design step to reduce the likelihood of designing probes that would cross-hybridize with host sequences. Two to three CDS-specific probes were included for each predicted gene. For 15 probes, five replicates were included as internal controls. Further, 19 493 probes against ESTs from N. benthamiana were also included. A total of 11 152 fungal transcripts were indicated to be ‘detected’ in at least one stage. RNA was extracted from ungerminated conidia, infected epidermal peels after 1 d post-inoculation (dpi) and 3 dpi, from infected leaves at 7 dpi and from vegetative mycelia grown in vitro on complete media. RNA from uninfected leaves and epidermal peels were extracted as negative controls. Total RNA from three biological replicates was extracted using the Plant RNeasy Mini kit with DNaseI treatment (Qiagen) followed by linear amplification and labelling with Cy3 using the Low Input Quick Amp kit (Agilent Technologies, USA). Arrays were scanned using an Agilent Technologies DNA Microarray Scanner with Scan Control software (Agilent) and data were analysed using Feature Extraction Software (Agilent). Signal intensities from probes against fungal sequences were normalized to the 75th percentile and baseline transformed to the median of all samples using the GenespringGX12 software suite (Agilent). Quantitative PCR was performed on selected genes to validate microarray results using the Thunderbird SYBR qPCR mix (ToYoBo). Reactions were run on an Mx3000P QPCR system and analyzed with MxPro QPCR software (Stratagene). The GEO accession number for the microarray data is GSE39714.

Results and Discussion

Genome sequencing and assembly

The Colletotrichum orbiculare genome was sequenced using both the Illumina Genome Analyzer IIx and Roche 454 sequencers and assembled into 525 scaffolds (N50: 428.89 kb) with an estimated total size of 88.3 Mb (Table 1), which is larger than the other sequenced Colletotrichum genomes (57.4 and 53.4 Mb in C. graminicola and C. higginsianum, respectively; O'Connell et al., 2012). The C. gloeosporioides genome was sequenced on the Illumina Hiseq 2000 sequencer and assembled into 1241 scaffolds (N50: 112.81 kb) with an estimated total size of 55.6 Mb. Homologs to a set of conserved eukaryotic genes (Parra et al., 2007) were identified to provide an estimate of assembly completeness. According to this analysis, the assemblies for C. orbiculare and C. gloeosporioides cover 97.98% and 96.37% of the total gene space, respectively.

Table 1. General features of the Colletotrichum orbiculare and C. gloeosporioides genome assemblies
 C. orbiculareC. gloeosporioides
  1. a

    Genome coding region coverage was estimated by CEGs (Core Eukaryotic Genes) using the CEGMA pipeline (Parra et al., 2007).

Assembly size (Mb)88.355.6
Number of scaffolds5251241
Median length (N50)428.89 kb112.81 kb
Contigs105455335
GC%37.52%53.58%
Number of predicted genes13 47915 469
GC% of genes57.09%55.99%
tRNAs459384
rRNAs5858
Repeat elements8.30%0.75%
Coverage of genome coding region (complete/partial)a97.98%/98.8%96.37%/98.8%

Extensive expansion of AT-rich sequences in C. orbiculare

Approximately 8.3% and 0.75% of the assembled C. orbiculare and C. gloeosporioides sequences consist of repeat elements with the majority of elements being the relics of transposable elements rather than encoding active protein-coding sequences. Strikingly, in addition to the annotated interspersed repeat elements, the C. orbiculare genome is enriched with low complexity sequences with higher concentrations of AT nucleotides. By dividing the genome into blocks based on GC content, 5514 blocks with < 50% GC content (AT blocks) were identified in C. orbiculare with an average size of 7.8 kb per block (Table 2). In total, these AT blocks make up 43.4 Mb (49.2%) of the genome, with an average GC% of 19.25% compared to 55.12% in the rest of the genome. In comparison, only 27.4% of the C. gloeosporioides genome assembly consists of AT blocks.

Table 2. Properties of AT and GC blocks in Colletotrichum orbiculare and C. gloeosporioides
 C. orbiculareC. gloeosporioides
AT blockGC blockAT blockGC block
  1. SSP, small, secreted proteins with < 300 amino acids predicted length.

Average GC (%)19.2555.1248.1957.07
Median GC (%)18.2854.7248.9955.74
Total size (bp)43 416 47642 166 8552 014 73053 388 137
Average size (bp)787448 63519 18816 277
Median size (bp)505227 33912 8988508
Number of genes54012 93960214 786
Number of SSP encoding genes3166946729

One possible mechanism that could have given rise to the nonhomogeneous AT-content of the C. orbiculare genome is repeat-induced point mutations (RIP). RIP is a mechanism of defence against transposable elements in ascomycete fungi, in which C and G nucleotides are preferentially mutated to T and A nucleotides in duplicated sequences (Galagan & Selker, 2004). RIP has been hypothesized to generate a similarly compartmentalized genome in the phytopathogenic fungus Leptosphaeria maculans (Rouxel et al., 2011). The components required for RIP are conserved in both Colletotrichum genomes (Supporting Information Table S1), and RIP indices (TpA/ApT dinucleotide ratios) of the consensus sequences of identified repeats in both genomes indicate RIP activity in both species (Fig. 2). It is also possible that an unknown mechanism is responsible for the expansion of AT-rich sequences.

Figure 2.

Analysis of repeats in Colletotrichum orbiculare and C. gloeosporioides. (a) The C. orbiculare genome was shown to be enriched with LTR and DNA transposon-like elements compared to the C. gloeosporioides genome. (b) Fold differences of the dinucleotide frequencies of Colletotrichum repeat elements relative to the control. Nonduplicated genes show that both genomes have an enrichment of TpA and a depletion of CpA dinucleotides, which is characteristic of RIP.

Gene content

A total of 13 479 and 15 469 genes are predicted in the C. orbiculare and C. gloeosporioides genomes, respectively (Table 1), similar to the number of genes predicted in the C. graminicola and C. higginsianum genomes (12 006 and 16 172, respectively; O'Connell et al., 2012). Thus, despite the larger genome size of C. orbiculare relative to the other three genomes, the coding capacity of the genome is not significantly increased. In C. orbiculare, genes are predominantly located in GC blocks (306.9 genes per Mb), with AT blocks being largely gene-sparse regions (12.44 genes per Mb).

Colletotrichum gloeosporioides possesses dispensable, potentially pathogenicity-associated chromosomes that can be horizontally transferred between compatible strains (Masel, 1996). A partial sequence of one of these chromosomes from another isolate of C. gloeosporioides had previously been determined (NCBI Accession AF448489.1) Six scaffolds from the Cgloeosporioides genome sequenced in our study show similarity to this sequence. Notably, the gene densities of these scaffolds are below the genome average. Also, only two of these scaffolds have GC contents of below 50% unlike in Mycosphaerella graminicola where the GC content of small, dispensable, pathogenicity-related chromosomes are lower than the rest of the genome (Goodwin et al., 2011). The majority of genes identified on these scaffolds encode proteins with no homology to known sequences in the NCBI nonredundant database (Table S2). No C. orbiculare scaffolds have significant homology to these sequences, indicating that the region is not conserved and is likely to be species-specific.

The two genomes share many homologous genes, with 8183 (60.7%; C. orbiculare) and 8200 (53.0%; C. gloeosporioides) genes, respectively, grouping into 8166 orthologous groups as defined by an adaptive reciprocal best blast hit strategy (Lechner et al., 2011). Of these, a total of 6769 (50.2%; C. orbiculare) and 6793 (43.9%; C. gloeosporioides) genes group together with 6764 orthologous groups from C. higginsianum and C. graminicola. Furthermore, 6187 (40.0%) C. gloeosporioides genes are organized into syntenic blocks that are collinear with four or more C. orbiculare genes. Among the C. orbiculare genes located in syntenic blocks, the majority are associated with GO terms for primary metabolism and macromolecule biosynthesis, where the conservation of gene order can be important for the regulation of gene co-expression (Fig. S1). A total of 1022 of these collinear genes encode hypothetical proteins identified in other fungal species; however, 59 encode genes with no known homolog in the NCBI nonredundant database.

Expansion of proteases

A total of 287 and 350 protease-encoding sequences are present in C. orbiculare and C. gloeosporioides respectively. These numbers of proteases are higher compared to other fungi including other hemibiotrophic plant pathogens such as F. graminearum and M. oryzae (Fig. 3, Table S3), and is largely due to expansions in the serine protease and metalloprotease families. In C. orbiculare and C. gloeosporioides, 32% and 36.9% protease sequences were predicted to be secreted. The majority of secreted proteases in both genomes are subtilisins (MEROPS family S8A), a family of serine proteases with optimum activity at alkaline pH. This finding is intriguing given that during C. gloeosporioides infections, alkalinisation of host tissue has been associated with virulence (Prusky et al., 2001). Furthermore, subtilisins may be important in penetration and colonization because they could potentially degrade plant cell wall glycoproteins or pathogenesis-related proteins (Olivieri et al., 2002). Subtilisins were similarly found to be expanded in C. higginsianum (O'Connell et al., 2012). Interestingly, clustering analysis revealed that some encoded subtilisins have greater homology to plant rather than fungal subtilisins, suggesting the possible acquisition of these genes by horizontal gene transfer from a host (Fig. S2). Furthermore, metalloproteases, especially from the M43 and M20 families, are highly expanded in both Colletotrichum species. M43 metalloproteases are also expanded in the genome of the endophyte Piriformospora indica, where members are upregulated during colonization of dead roots suggesting a role for these proteases in degrading plant materials (Zuccaro et al., 2011).

Figure 3.

Proteases in Colletotrichum orbiculare and C. gloeosporioides. Number of predicted proteases encoded by the C. orbiculare and C. gloeosporioides genomes in comparison to other fungi. The Colletotrichum genomes have high numbers of putative metalloproteases and serine proteases.

Expansion of carbohydrate active enzymes and LysM-domain containing proteins

The plant cell wall consists mainly of three types of polysaccharides: cellulose; hemicelluloses, consisting of xylans, xyloglucans and mannans; and pectins, consisting of rhamnogalacturonans, homogalacturanans and xylogalacturanans (Caffall & Mohnen, 2009). Carbohydrate active enzymes (CAzymes) that are able to break down these polysaccharides are thus important in establishing infection and also in accessing nutrients during necrotrophic and saprophytic growth. Consistent with this, C. orbiculare and C. gloeosporioides encode a large arsenal of plant cell wall-degrading enzymes (327 and 364, respectively; Table S4). Both genomes contained more cellulose-degrading enzymes compared to other ascomycete fungi, similar to C. higginsianum and C. graminicola (Table 3). In addition, the three dicot-infecting species, C. orbiculare, C. gloeosporioides and C. higginsianum, possess an expanded number of pectin-degrading enzymes, consistent with an adaptation to dicot plant cell walls which contain a greater proportion of pectins compared to monocots (Vogel, 2008). In addition, C. gloeosporioides showed an expansion in the number of genes encoding enzymes involved in the digestion of hemicellulose and/or pectin.

Table 3. Comparisons of the numbers of plant cell wall-degrading enzymes in Colletotrichum orbiculare and C. gloeosporioides and other Ascomycete fungi
Fungal speciesCell wall component
PectinHemicellulose/pectinHemicelluloseCellulose
Colletotrichum orbiculare 103743346
Colletotrichum gloeosporioides 117903544
Colletotrichum higginsianum 79592938
Colletotrichum graminicola 46583242
Fusarium oxysporum 79812327
Fusarium graminearum 49602020
Magnaporthe oryzae 21633035
Neurospora crassa 15331325

Another notable expansion was found in proteins containing the CBM50 chitin-binding module that corresponds to the lysin motif (LysM) domain (Table S4). C. orbiculare and C. gloeosporioides encode a total of 12 and 20 proteins containing this module, respectively, of which 10 and 15, respectively, are predicted to be secreted. A similar expansion of CBM50 domain proteins was also noted in C. higginsianum and C. graminicola (O'Connell et al., 2012). This expansion seems specific to LysM-domain containing proteins because the numbers of proteins containing the chitin-binding module CBM18 is similar to those found in Fusarium spp (Table S4). Interestingly, whereas the CBM18 proteins are associated with chitin-degrading enzymatic domains, the majority of the LysM-domain containing proteins (11 in C. orbiculare and 18 of the C. gloeosporioides genes) are not, indicating that these proteins could be involved in protection against chitin-degradation by plant enzymes or may function to sequester chitin fragments released during infection to evade perception by host chitin receptors (de Jonge & Thomma, 2009; de Jonge et al., 2010).

Greater capacity for secondary metabolite production

Secondary metabolites produced by Colletotrichum are known to contribute to pathogenicity. For example, treatment with the Colletotrichum toxin colletotrichin is sufficient to cause anthracnose disease-like symptoms including plasma membrane damage (Duke et al., 1992) and secondary metabolite profiling has been used to distinguish between pathogenic and nonpathogenic isolates of C. gloeosporioides (Abang et al., 2009). C. orbiculare possesses a number of secondary metabolite backbone-forming proteins, including 24 polyketide synthases (PKS), 1 PKS-like, 11 nonribosomal peptide synthases (NRPS), 9 NRPS-like, 3 PKS-NRPS hybrid backbone synthases and 11 demethylallyl tryptophan synthases (DMAT) (Table 4). Colletotrichum gloeosporioides appears to have an even greater capacity for secondary metabolite production with 34 PKS, 10 PKS-like, 14 NRPS, 10 NRPS-like, 6 PKS-NRPS hybrids and 8 DMAT. As reported for C. higginsianum and C. graminicola (O'Connell et al., 2012), the total number of these key enzymes is much higher than those identified from other fungal genomes such as M. oryzae (32) and F. graminearum (37) indicating that the Colletotrichum genus as a whole has evolved a greater capacity for secondary metabolite production. In addition, both genomes encode a large number of cytochrome P450 proteins, comprising a total of 1.06% and 0.74% of all proteins in C. gloeosporioides and C. orbiculare, respectively. In other fungi, cytochrome P450 enzymes contribute to both primary and secondary metabolism and are involved in the production of mycotoxins and detoxification of host metabolites (Črešnar & Petrič, 2011).

Table 4. Number of genes predicted to be involved in the synthesis of secondary metabolite backbones identified by the secondary metabolite unique regions finder (SMURF)
OrganismPKSPKS-likeNRPSNRPS-likeHybridDMATTotal
  1. NRPS, nonribosomal peptide synthase; PKS, polyketide synthase; DMAT, demethylallyl tryptophan synthase.

  2. a

    Numbers from manual annotations of the C. higginsianum and C. graminicola genomes (O'Connell et al., 2012).

Aspergillus nidulans 24411121658
Stagnospora nodorum 1271150237
Sclerotinia sclerotiorum 162550129
Magnaporthe oryzae 123563332
Neurospora crassa 62330115
Fusarium graminearum 14110111037
Ustilago maydis 32360216
Colletotrichum graminicola 39a7a07a53
Colletotrichum higginsianum 58a12a410a84
Colletotrichum gloeosporioides 34414106876
Colletotrichum orbiculare 2411193654

As in other fungi, many of the genes encoding secondary metabolism key enzymes are organized into clusters together with other genes that may be involved in secondary metabolism such as transporters, oxidoreductases and cytochrome P450s. A total of 38 such clusters were predicted in C. orbiculare and 53 in C. gloeosporioides. Of the 38 C. orbiculare clusters, 25 are in regions syntenic with C. gloeosporioides secondary metabolite gene clusters, as assessed by the conservation of genes (Table S20–23). This analysis showed that C. orbiculare has more secondary metabolism gene clusters in common with C. gloeosporioides than with C. higginsianum or C. graminicola (O'Connell et al., 2012). In total, only seven metabolism gene clusters were found to be common between all four species, including C. higginsianum cluster 10, which is one of the most highly upregulated gene clusters in C. higginsianum during biotrophy (O'Connell et al., 2012).

Potential host manipulation by phytohormone production

The biosynthesis of the auxin indole acetic acid (IAA) and its intermediates has been experimentally demonstrated in C. gloeosporioides and related Colletotrichum species to occur mainly via the indole-3-acetamide (IAM) pathway (Robinson et al., 1998; Chung et al., 2003). Homologs of the iaaM and iaaH genes required for the synthesis of auxin via IAM in Fusarium proliferatum ET1 have also recently been identified in C. gloeosporioides and C. graminicola (Tsavkelova et al., 2012). Interestingly, comparison of the genomic regions containing these genes in the two species revealed a lack of conserved flanking genes. Furthermore, the iaaM and iaaH genes (here identified as Cggc5_4414 and Cggc5_4413) are absent from C. orbiculare despite the conservation of flanking genes in the C. gloeosporioides genome (Fig. S3). This may reflect their distinct host ranges or independent acquisition of these genes in different Colletotrichum lineages. Nevertheless, both genomes contain two genes encoding proteins with homology to members of the auxin efflux carrier superfamily (Cggc5_1694, Cggc5_13472, Cob_5997 and Cob_6211). Thus, IAA may be synthesized via another intermediate in C. orbiculare.

Recently, a secreted chorismate mutase was demonstrated to contribute to Ustilago maydis virulence by reducing the available chorismate, a substrate for salicylic acid (SA) biosynthesis in host cells, which affected SA defense in plants (Djamei et al., 2011). Although no secreted chorismate mutases were found in the two Colletotrichum genomes, a secreted isochorismatase is present in C. orbiculare and two such sequences are predicted in C. gloeosporioides. Like chorismate mutase, isochorismatases also metabolize chorismate and, although present in all filamentous ascomycetes, are predicted to be secreted only in phytopathogens (Soanes et al., 2008).

Comparative secretomes reveal candidate effectors

The secretome of a pathogen includes proteins that are deployed to the host–pathogen interface during invasion of the host and are thus of particular interest for identifying potential virulence factors. A total of 1557 (11.6%) and 2042 (13.2%) proteins in C. orbiculare and C. gloeosporioides, respectively, are predicted to be secreted proteins. An analysis of significantly over-represented GO terms reveals that the majority of annotated secreted proteins have enzymatic activity (Fig. S4), especially in carbohydrate and protein degradation. Consistent with this finding, PFAM domain analysis reveals that out of the 335 and 496 PFAM domains identified in the predicted secreted proteins, only 129 and 190 are not associated with enzymatic functions in C. orbiculare and C. gloeosporioides, respectively (Tables S5–S7).

The Colletotrichum secretomes contain homologs of known effectors from other phytopathogens, indicating the use of conserved infection strategies. These proteins include homologs of the necrosis inducing protein (NPP1) from Phytophthora spp., the Biotrophy-Associated Secreted (BAS) protein 2 from M. oryzae (Mosquera et al., 2009) and the F. oxysporum Secreted In Xylem (SIX) 5 protein (Lievens et al. 2009). Interestingly, the host range determining Fusarium spp. pea pathogenicity protein PEP1 is also present in both species (Coleman et al., 2011). Further, the C. orbiculare genome also encodes small proteins similar to SIX1 and SIX6 proteins of F. oxysporum. A homolog of the Ave1 avirulence effector in the pathogens Verticillium dahliae, F. oxysporum and C. higginsianum that was hypothesized to have been horizontally transferred from plants (De Jonge et al., 2012) was also detected only in C. orbiculare at the end of a region with collinearity to C. gloeosporioides (Fig. S5).

Respectively, Colletotrichum orbiculare and C. gloeosporioides possess 372 and 355 cysteine-rich secreted proteins (cysteine content of 3% or more). This is interesting given that the effectors of many other fungal phytopathogens are cysteine rich and this feature may be important for maintaining protein structure given the relatively small size of these proteins (Stergiopoulos & de Wit, 2009). In comparison, the median cysteine content of all predicted proteins in the C. orbiculare and C. gloeosporioides genomes are 1.1% and 1.2%, respectively. Notably, the majority of cysteine-rich secreted sequences (318 and 288) are SSPs (< 300 amino acids in length). This is consistent with findings that a large proportion of the candidate-secreted effectors identified in C. graminicola and C. higginsianum are cysteine rich (O'Connell et al., 2012).

A total of 700 and 755 SSPs are predicted in the C. orbiculare and C. gloeosporioides genomes respectively. Although some of these sequences are associated with host interaction as predicted secreted proteases and polysaccharide-degrading enzymes, the majority (505 in C. orbiculare and 492 in C. gloeosporioides) are of unknown function. Of these sequences, 276 (54.6%) in C. orbiculare and 243 (49.4%) in C. gloeosporioides encoded cysteine-rich proteins. Amongst all the SSPs, only 142 C. orbiculare SSPs and 134 C. gloeosporioides SSPs could be clustered into tribes containing three or more proteins, representing potential gene families. (Table S8). In both genomes, only four of these genes cluster exclusively with other sequences within the same genome according to TribeMCL analysis. This finding contrasts with the expansion of lineage-specific effectors in the genomes of rust fungi (Duplessis et al., 2011). Possibly this result reflects the diversification of some rust effectors to adapt to specific hosts. Comparison to the other two sequenced Colletotrichum species revealed that 195 (27.9%) C. orbiculare SSPs and 208 (26.8%) C. gloeosporioides SSPs were conserved in all four species (Table 5). Interestingly, only 279 C. orbiculare SSPs possessed homologs in C. higginsianum. This number was similar to the number of SSPs with homologs in C. graminicola despite the fact that C. graminicola has a different host range, infecting only monocot plants. By contrast, 410 SSPs in C. orbiculare were found to have homologs in C. gloeosporioides. This difference may reflect a closer phylogenetic relationship between C. orbiculare and C. gloeosporioides relative to the other two species, which themselves are more closely related, belonging to the graminicola and destructivum sister clades (Cannon et al., 2012).

Table 5. Features of small, secreted proteins (SSPs), < 300 amino acids
 C. orbiculareC. gloeosporioides
  1. BLAST was performed on SSPs from the genomes of Colletotrichum orbiculare, C. gloeosporioides, C. higginsianum and C. graminicola (Cutoff = 10E-5).

Secreted proteins15572042
Small, secreted proteins700775
With homologs in C. orbiculare419
With homologs in C. gloeosporioides410
With homologs in C. higginsianum279277
With homologs in C. graminicola270290
With homologs in C. higginsianum, C. gloeosporioides and C. orbiculare239251
With homologs in C. orbiculare, C. gloeosporioides, C. higginsianum and C. graminicola.195208

Colletotrichum orbiculare transcriptome analysis reveals a significant hemibiotrophic stage shift

A custom microarray was designed to test for the expression of the annotated C. orbiculare genes in planta at different stages of infection on N. benthamiana leaves. The sampled stages (Fig. 1a–d) were as follows: ungerminated conidia, appressorial penetration/early infection (1 dpi), established biotrophic growth (3 dpi), late necrotrophy (7 dpi), as well as in vitro-grown hyphae. A total of 9651 genes were differentially regulated (fold change ≥ 2, ≤ 0.05) in conidia, 1, 3 and 7 dpi compared to in vitro-grown hyphae.

In L. maculans, effector genes are associated with AT blocks, and many SSPs within these blocks are upregulated in planta. Given the similar compartmentalization of the C. orbiculare genome and the greater representation of novel SSP genes in AT-rich regions, we analyzed the expression of genes according to their localization in AT or GC blocks. This revealed that a greater proportion of genes located in AT-rich regions were not upregulated in planta compared to in vitro (Table 6). This was also observed for the genes encoding SSPs within these regions, suggesting the suppression of genes within AT-rich compartments.

Table 6. Percentages of genes in AT blocks and GC blocks upregulated > 10-fold in conidia and at different time points during infection of Colletotrichum orbiculare of Nicotiana benthamiana compared to in vitro
 All genesSSPs
AT blockGC blockAT blockGC block
  1. dpi, days post-inoculation; SSPs, small, secreted proteins.

Upregulated > 10 times at 1 dpi3.54%9.91%7.41%21.82%
Upregulated > 10 times at 3 dpi2.59%5.47%7.41%19.73%
Upregulated > 10 times at 7 dpi3.07%9.24%11.11%14.04%
Upregulated > 10 times in conidia3.54%7.02%3.70%7.97%

GO terms that are over-represented among the sequences upregulated in planta compared to in vitro, are associated with carbohydrate binding and degradation, interactions with the host, protein degradation and transmembrane transport (Tables S9–12). A total of 405 SSPs were upregulated > 2-fold in planta compared to in vitro (P-value ≤ 0.05). Of these, 291 were not associated with any PFAM domain. Further, 165 genes have no known homolog in the NCBI database, indicating that these genes may represent truly novel effectors. The expression patterns of nine selected SSPs were assessed by QPCR (Fig. S6) confirming their individual expression profiles in planta.

Analysis of the transcriptome revealed that the initial colonization stage (1 dpi) is characterized by the upregulation of gene models encoding SSPs (Fig. 4). A total of 28 of the top 100 genes expressed are SSP-encoding genes. In comparison, only 11, 7 and 12 of the top 100 genes expressed in late necrotrophy, conidia and in vitro hyphae, respectively, encode SSPs (Tables S13–S17). Among 69 SSP sequences that were highly upregulated (> 50-fold) during early infection (1 dpi), only 25 were also induced to similar levels at later stages of infection (3 and 7 dpi), indicating that the majority of highly expressed SSP genes are stage-specific. By contrast, relatively few SSP-encoding genes were specifically induced at 3 and 7 dpi. SSPs that had peak expression during early infection (1 dpi) included sequences with homology to the effectors of other phytopathogens such as Fusarium SIX proteins, the Magnaporthe BAS2 protein, as well as two NPP1 domain-containing proteins (Table S18). However, as in C. higginsianum (Kleemann et al., 2012), these two NPP1 homologs do not encode proteins with well-conserved necrosis-inducing consensus motifs.

Figure 4.

Expression of small, secreted proteins (SSPs) in planta. Heatmap of SSPs with at least a 50-fold upregulation in planta compared to in vitro-grown hyphae (VH). This analysis revealed the stage-specific expression of SSPs during infection, particularly during early host invasion (1 d post-inoculation (dpi)). The levels of expression represent normalized probe intensity values (log2). Genes with similar patterns of expression are clustered. Cysteine-rich sequences are denoted with ‘+’ after the gene model name. C, conidia

Among the 10 genes encoding secreted chitin-binding proteins, four had higher expression in planta at the early invasion stage (1 dpi), when they may interfere with the perception of chitin degradation products by host chitin receptors. In addition, three out of four chitin deacetylases (Cob_748, Cob_1749 and Cob_5573) were also upregulated from this early stage of infection onwards. These proteins may potentially function in modifying chitin in fungal cell walls into chitosan to evade host defense responses, as hypothesized for C. graminicola (El Gueddari et al., 2002).

Examination of sequences with homology to MFS transporters revealed that 49 genes (55.7%) were upregulated > 2-fold during penetration/early infection (Table S19). Among these upregulated genes were seven sequences related to hexose transport and six sequences with homology to transporters of other monosaccharides. Other transporters upregulated in planta included proteins related to quinate transport. Four quinate transporters were upregulated > two-fold during initial invasion and biotrophic hyphal growth, but during necrotrophy only one such transporter was upregulated > two-fold. Quinate is a polyol present in decaying plant material that can be used as a carbon source by fungi such as N. crassa (Giles et al., 1985). A recent study suggests that in the early stages of M. oryzae infection, host quinate concentrations are manipulated by the pathogen at the expense of the host shikimate pathway, which synthesizes defense-related phenylpropanoids (Parker et al., 2009; Soanes et al., 2012). The expression pattern of genes in C. orbiculare encoding quinate transporters is consistent with the importance of quinate utilization during early infection, although genes in the quinate utilization cluster of C. orbiculare had peak expression during late necrotrophy. This finding indicated that quinate may also be important as a carbon source on dying host tissue.

Interestingly, the early invasion stage (1 dpi; Fig. 1b) is also characterized by the upregulation of many secondary metabolite backbone synthesis genes (Fig. 5). Although expression of secondary metabolite synthesis genes is commonly associated with necrotrophic fungi (Pinedo et al., 2008; Amselem et al., 2011), among the 54 C. orbiculare secondary metabolite backbone synthesis genes, 22.2% were upregulated > 10-fold at this stage compared to in vitro hyphae, four of which did not have detectable expression in vitro. By contrast, only 9.3% secondary metabolism genes were upregulated to the same extent during the necrotrophic stage. This result indicates that the products of these enzymes are not necessarily toxins and that the fungus may be manipulating its host via secondary metabolites during biotrophic growth. Secondary metabolite backbone synthesis genes were also among the top 100 most highly expressed genes during early penetration and biotrophic growth in C. higginisianum (O'Connell et al., 2012), suggesting that this strategy may be common in other Colletotrichum species. Alternatively, these secondary metabolites may play important roles within the fungus to aid infection. One gene that falls into this category is the PKS1 gene (Cob_9513), required for melanin biosynthesis, which was upregulated > 90 times at this stage. This result is consistent with the importance of melanin in appressorial penetration of host epidermal cells.

Figure 5.

Expression of secondary metabolite backbone synthesis genes in planta. Heatmap of secondary metabolite backbone synthesis genes showing differential expression in planta and in conidia compared to in vitro-grown hyphae (VH). This analysis revealed the increased expression of secondary metabolite backbone synthesis genes during early invasion. The levels of expression represent normalized probe intensity values (log2). Genes with similar patterns of expression are clustered. dpi, days post-inoculation.

The 3 dpi time point corresponds to a period of intracellular biotrophic hyphal growth (Fig. 1c). Transcriptionally, this stage is similar to the early invasion stage although in general genes were not as highly upregulated relative to in vitro-grown hyphae. As in the early invasion stage, a large number of SSPs were highly expressed. In particular, 32 of the top 100 expressed genes were SSPs (Table S14). Furthermore, as in the earlier stage of infection (1 dpi), many secondary metabolite backbone synthesis genes were also upregulated at 3 dpi. Interestingly, these included a gene with homology to an NRPS, one of the most highly expressed genes during biotrophic hyphal growth. This gene is in a predicted secondary metabolite cluster and another member of the cluster, encoding an MFS drug efflux transporter, was also upregulated at this phase suggesting that the product of the enzyme may be targeted to the host. This cluster was not identified in the other three Colletotrichum species, indicating that the product could be lineage-specific to C. orbiculare.

The necrotrophic phase of growth at 7 dpi (Fig. 1d) was notable for the expression of degradative enzymes such as carbohydrate-degrading enzymes and proteases. Specifically, in contrast to the early invasion and biotrophic phases of growth, where only 8% of genes encoding plant cell wall-degrading enzymes were upregulated 10-fold or more compared to in vitro hyphae, the proportion of such enzymes upregulated at 7 dpi was 15%. In particular, this increase can be attributed to the upregulation of pectin-degrading enzyme sequences, consistent with the abundance of pectins in dicot cell walls (Fig. 6).

Figure 6.

Plant cell wall-degrading genes upregulated in planta compared to in vitro-grown hyphae. Number of predicted plant cell wall-degrading enzyme genes upregulated 10-fold or more compared to in vitro-grown hyphae at different time points during infection of Nicotiana benthamiana and in conidia, classified according to carbohydrate substrate. dpi, days post-inoculation.

Analysis of the expression profiles of genes encoding secreted proteases in C. orbiculare revealed that although some proteases were expressed from early infection at 1 dpi, many sequences had peak expression only at the necrotrophic stage (Fig. 7). Furthermore, the most upregulated protease-encoding sequences in planta belong to the metallo- and serine-protease families. Three out of five zinc carboxypeptidases from the M14 family were upregulated > 10-fold in planta from 1 dpi, with expression peaking at the late necrotrophic stage. In addition, four out of five M43 metalloproteases were upregulated > 10-fold during infection, with expression also peaking during late necrotrophy, consistent with roles in the breakdown of plant material and the drastic change from biotrophic to necrotrophic growth. However, this expression pattern does not apply to all protease-encoding genes. For example, among the 12 subtilisin genes that were upregulated two-fold or more in planta, four showed highest expression during early infection, three showed peak expression during biotrophic growth, whereas three others were preferentially expressed during necrotrophy. Such contrasting expression profiles suggest that particular subtilisins may have specific roles at different points in infection.

Figure 7.

Expression of proteases in planta. Heatmap of proteases showing at least two-fold upregulation in planta compared to in vitro-grown hyphae (VH). The expression of proteases during infection shows that the majority of upregulated protease-encoding genes have peak expression during late necrotrophy. The levels of expression represent normalized probe intensity values (log2). Genes with similar patterns of expression are clustered. The putative class of each protease is indicated after the gene ID. dpi, days post-inoculation.

In C. gloeosporioides and Colletotrichum coccodes infections, accumulation and secretion of ammonia are important for virulence through the alkalinization of host tissue (Prusky et al., 2001; Alkan et al., 2008; Miyara et al., 2010). Analysis of predicted ammonia transporter genes provides evidence for the upregulation of all four genes in planta relative to in vitro-grown hyphae (Table S19); however, the highest expression levels of all four genes were at the conidial stage. Appressorium formation by germinating C. gloeosporioides conidia is stimulated by exogenous ammonium (Miyara et al., 2010) and the high expression of ammonium transporters in C. orbiculare conidia may reflect the importance of the perception and accumulation of external nitrogen sources before conidial germination.

Concluding remarks

Recent studies have indicated that genome expansion via the action of repeat elements in filamentous plant pathogens can drive the replication and diversification of pathogen-encoded virulence factors. This can result in irregular organization of the genome, as well as the enrichment of virulence factors within regions or compartments of the genome (Raffaele & Kamoun, 2012). One of the remarkable findings of our study is that within the same fungal genus, the genome organizations of two Colletotrichum pathogens are quite different. The C. orbiculare genome is significantly expanded relative to that of C. gloeosporioides and is organized into blocks of sequences with strikingly distinct AT and GC contents, with gene-sparse AT blocks potentially arising as the result of transposable element inactivation via RIP. In L. maculans, RIP-affected regions are sources of novel effectors where the mutation of duplicated sequences drives effector diversification (Rouxel et al., 2011). Although the C. orbiculare genes located within AT blocks had higher sequence variation compared to the rest of the genome, as indicated by the lack of known homologs, very few of these genes are highly upregulated in planta, suggesting that these genomic regions are not enriched with effector genes. However, we cannot exclude the possibility that these genes are expressed in hosts other than N. benthamiana.

The genome-wide comparative analyses presented here revealed a number of genes that are potentially involved in pathogenesis. In particular, gene families encoding proteases and carbohydrate-degrading enzymes that are able to target a wide range of substrates, are highly expanded, possibly reflecting the broad host ranges of both species. The presence of large numbers of secondary metabolism genes, similar to the genomes of C. higginsianum and C. graminicola (O'Connell et al., 2012), highlights the potential capacity of Colletotrichum species metabolites for host manipulation and/or detoxification of host antifungal compounds. In particular, the upregulation of C. orbiculare secondary metabolism-related genes during early infection of N. benthamiana coupled with the observation that many such genes are also upregulated in C. higginsianum during biotrophy (O'Connell et al., 2012), suggests that small molecules are important in the establishment and/or maintenance of biotrophy. A number of SSP effector candidates are also present in the two genomes described in this study. Different expression profiles of secreted proteins during infection were observed, with more SSPs upregulated during the establishment of biotrophy followed by a shift to the upregulation of genes associated with degradative enzymes during necrotrophic growth. A similar shift in expression patterns was also noted in other Colletotrichum species, particularly in C. higginsianum (O'Connell et al., 2012), underscoring the importance of these different groups of genes for the different stages of hemibiotrophic pathogen growth. Importantly, the list of SSPs that are highly upregulated in planta represents a base for future studies on effector biology of C. orbiculare. Finally, the genomic information presented in this study will be a valuable resource for relating data from functional studies to pathogen genetics. This information is especially significant because C. gloeosporioides and C. orbiculare not only serve as model systems for plant–pathogen interactions, but also represent two economically significant pathogens where a deeper understanding of pathogen biology can directly inform strategies for targeted disease control.

Acknowledgements

This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry to K.S., Y.T., Y.N.; Grant-in-Aid for Scientific Research (KAKENHI) (24228008 to K.S, 21380031 to Y.K.) and by The Strategic Research Funds in Kyoto Prefectural University to Y.K. We thank Yoshihiko Hirayama for kindly providing C. gloeosporioides strain Nara gc5.

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