Mycologia, 106(4), 2014, pp. 629–641. DOI: 10.3852/13–262 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

Transposable elements belonging to the Tc1-Mariner superfamily are heavily mutated in Colletotrichum graminicola Raı´ssa Mesquita Braga

with three characteristic conserved motifs. However, this sequence is interrupted by five stop codons. Genomic DNA from various isolates was analyzed by hybridization with an internal region of TCg1. All of the isolates featured transposable elements that were similar to TCg1, and several hybridization profiles were identified. C. graminicola has many Tc1-Mariner transposable elements that have been degenerated by characteristic RIP mutations. It is unlikely that any of the characterized elements are autonomous in the sequenced isolate. The possible existence of active copies in field isolates from Brazil was shown. The TCg1 element is present in several C. graminicola isolates and is a potentially useful molecular marker for population studies of this phytopathogen. Key words: RIP, transposase, transposon

Departamento de Microbiologia, Instituto de Cieˆncias Biome´dicas II, Universidade de Sa˜o Paulo, Avenida Professor Lineu Prestes 1374, Cidade Universita´ria, Sa˜o Paulo, Brasil. CEP: 05508-900

Mateus Ferreira Santana Departamento de Microbiologia, Universidade Federal de Vic¸osa, Avenida Peter Henry Rolfs s/n, Campus Universita´rio, Vic¸osa, Brasil. CEP: 36570-000

Rodrigo Veras da Costa Embrapa Milho e Sorgo, Rod MG 424 Km 65, Sete Lagoas, Minas Gerais, Brasil. CEP: 35701-970

Sergio Herminio Brommonschenkel Departamento de Fitopatologia, Universidade Federal de Vic¸osa, Avenida Peter Henry Rolfs s/n, Campus Universita´rio, Vic¸osa, Brasil. CEP: 36570-000

Elza Fernandes de Arau´jo INTRODUCTION

Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais, Rua Raul Pompeia 101, Sa˜o Pedro, Belo Horizonte, Brasil. CEP: 30330-080

The ascomycete fungus Colletotrichum graminicola (Cesati) Wilson (teleomorph Glomerella graminicola [Politis]) the causative agent of leaf anthracnose and stalk rot in maize (Zea mays L.) (Munch et al. 2008) and one of the most common and economically important pathogens of maize (Bergstrom and Nicholson 1999). This fungus has a hemibiotrophic life cycle that is characterized by a short biotrophic phase followed by a necrotrophic phase (Munch et al. 2008). Its sexual cycle is rare and has not been reported in the wild (Crouch and Beirn 2009). Colletotrichum graminicola was the first species in the Colletotrichum genus to have its genome completely sequenced (www.broadinstitute.org/annotation/ genome/colletotrichum_group/). Transposable elements are ubiquitous and provide a large amount of genetic variability with recombination and transposition playing important roles in mutation and genomic organization in filamentous fungi (Daboussi and Capy 2003). Transposable elements are divided into two classes based on the presence or absence of an RNA intermediate during transposition. Class I transposable elements, or retrotransposons, transpose via an RNA intermediate. Class II elements (transposons) transpose via a DNA intermediate and are divided into two subclasses that are distinguished by the number of DNA strands that are cleaved during transposition. Subclass I includes transposable elements of the order TIR, which are characterized by terminal inverted repeats (TIRs) of

Marisa Vieira de Queiroz1 Departamento de Microbiologia, Universidade Federal de Vic¸osa, Avenida Peter Henry Rolfs s/n, Campus Universita´rio, Vic¸osa, Brasil. CEP: 36570-000

Abstract: Transposable elements are ubiquitous and constitute an important source of genetic variation in addition to generating deleterious mutations. Several filamentous fungi are able to defend against transposable elements using RIP(repeat-induced point mutation)-like mechanisms, which induce mutations in duplicated sequences. The sequenced Colletotrichum graminicola genome and the availability of transposable element databases provide an efficient approach for identifying and characterizing transposable elements in this fungus, which was the subject of this study. We identified 132 full-sized Tc1-Mariner transposable elements in the sequenced C. graminicola genome, which were divided into six families. Several putative transposases that have been found in these elements have conserved DDE motifs, but all are interrupted by stop codons. An in silico analysis showed evidence for RIP-generated mutations. The TCg1 element, which was cloned from the Brazilian 2908 m isolate, has a putative transposase sequence Submitted 16 Aug 2013; accepted for publication 1 Jan 2014. 1 Corresponding author. E-mail: [email protected]

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various sizes. Transposition is mediated by a transposase that recognizes TIRs and cleaves both strands at each end (Wicker et al. 2007). Subclass I includes several known superfamilies that differ from one another by their TIRs and the size of the target-site duplication (Wicker et al. 2007). The Tc1-Mariner superfamily is one of the most diverse and widespread class II transposable elements. Elements in this superfamily are approximately 1300– 2400 bp long, create TA target site duplications, have a single transposase gene and are flanked by TIRs. Their transposases have characteristic DDE motifs (Plasterk et al. 1999) with an amino-acid triad consisting of two aspartic acid (D) residues and one glutamic acid (E) or a third aspartic acid (D) residue. The three-dimensional structure of this triad creates a catalytic site coordinating two divalent metal ions and aids in nucleophilic reactions to cleave DNA (Yuan and Wessler 2011). These transposases also contain helix-turn-helix (HTH) DNA-binding motifs at the Ntermini to recognize the TIRs (Pietrokovski and Henikoff 1997). The centromeric protein B domain (CENPB), which is found in mammalian centromeric proteins and eukaryotic transposases, also is involved in TIR recognition (Hey et al. 2008). Although they are able to confer advantages to the organism, the presence of transposable elements in the genome may be associated with deleterious mutations. Therefore many organisms have defense mechanisms for the inactivation of transposable elements. Some filamentous fungi use repeat-induced point mutation (RIP) or RIP-like mechanisms that efficiently detect and cause mutations in duplicated sequences. RIP occurs during the sexual life cycle and acts on duplicated sequences that are larger than 400 bp and share more than 80% nucleotide identity. These sequences are inactivated by C:G to T:A mutations (Selker 2002, Galagan and Selker 2004, Clutterbuck 2011). The RID (RIP defective) protein, which is a DNA methyltransferase that has been described in Neurospora crassa, was the first enzyme to be identified as essential to the occurrence of RIP in N. crassa (Freitag et al. 2002). The availability of sequenced genomes and expanding databases has refined transposable element research by making bioinformatics integral to these studies ( Janicki et al. 2011). Dufresne et al. (2011) identified transposable elements in the genomic sequences of four Fusarium species by performing similarity searches with the BLAST algorithm (Altschul et al. 1997) and Fot element as query. Eight new transposon families belonging to the Tc1-Mariner superfamily have been identified in Paracoccidioides Braziliensis (Marini et al. 2010) using a bioinformatics approach together with bench experiments to identify

transposable elements in the sequenced genomes of three phylogenetically distinct lineages. Within the Colletotrichum genus, the Collect1 and Collect2 transposons previously were identified in C. cereale by differential hybridization for identifying repetitive DNA (Crouch et al. 2008), and several retrotransposons, such as CgT1 (He et al. 1996) and Cgret (Zhu and Oudemans 2000) in C. gloeosporioides and RetroCl1 in C. lindemunthianum (dos Santos et al. 2011), were found with the same approach. This study describes the identification and characterization of 6 Tc1-Mariner transposable element families by an in silico analysis of the C. graminicola genome. The TCg1 transposon, which was sequenced from an isolate that was obtained in Brazil, showed potential as a molecular marker in population analyses. All of the identified transposons showed evidence of RIP mutations, demonstrating their importance in inactivating transposons in C. graminicola. MATERIALS AND METHODS Bioinformatic analysis.—A search using keywords that are specific to class II transposable elements (transposase and transposon) was conducted with the C. graminicola sequencing project database (Colletotrichum Sequencing Project, Broad Institute of Harvard and MIT, http://www. broadinstitute.org/). The genome also was analyzed with the RepeatMasker program (version open-3.3, RepBase Update 20110419), which identifies element copies using comparisons between the sequence and a library of known elements (http://www.repeatmasker.org). The identified sequences subsequently were used in an identity search of the genomic sequence via the BLAST algorithm using an Evalue cutoff of 1023 (Altschul et al. 1997). The resulting sequences (together with flanking sequences 1000 bp upstream and downstream) were aligned with the Clustal W multiple sequence alignment program (Thompson et al. 1994) to identify the ends of the transposable elements. To identify the TIRs, the sequences were searched against themselves with the BLAST algorithm (Altschul et al. 1997). Finally, full-sized sequences containing flanking inverted repeats were analyzed with the NCBI protein database (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/) and the Colletotrichum graminicola M1.001 proteins and transcripts database (e-value cutoff of 1e-20). Phylogenetic trees of the amino acid sequences that were aligned with ClustalW were constructed using the neighborjoining method in the MEGA 4 program (Tamura et al. 2007) with bootstrap values for 5000 replicates. The transposase sequences from the other fungi that were used are available at http://www.girinst.org/repbase/. Amino acid sequences for transposases from the OPHIO1, Flipper, Fot5 and tc1 elements also were used (GenBank accession numbers ABG26270.1, AAB63315.1, CAE55867.1, XP_ 002146479.1 respectively). These elements belong to the

BRAGA ET AL.: COLLETOTRICHUM GRAMINICOLA TABLE I.

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Colletotrichum graminicola isolates used in present study

Isolate

Locality

Isolate

Locality

14.04 21.04 37.04 27.05 42.07 91.07 02.08 08.08 23.08 26.08 28.08 29.08 33.08 48.08 21.09 23.09 30.09 32.09 33.09 35.09 37.09 38.09 42.09 43.09

Xanxereˆ, SC, Brazil Campo Moura˜o, PR, Brazil Colombia, SP, Brazil Andradas, MG, Brazil Passo Fundo, RS, Brazil Xapeco´, SC, Brazil Sete Lagoas, MG, Brazil Sete Lagoas, MG, Brazil Sete Lagoas, MG, Brazil Sete Lagoas, MG, Brazil Sete Lagoas, MG, Brazil Sete Lagoas, MG, Brazil Sete Lagoas, MG, Brazil Sete Lagoas, MG, Brazil Campo Moura˜o, PR, Brazil Campo Moura˜o, PR, Brazil Campo Moura˜o, PR, Brazil Campo Moura˜o, PR, Brazil Campo Moura˜o, PR, Brazil Campo Moura˜o, PR, Brazil Campo Moura˜o, PR, Brazil Campo Moura˜o, PR, Brazil Campo Moura˜o, PR, Brazil Campo Moura˜o, PR, Brazil

50.09 53.09 54.09 116.09 117.09 118.09 119.09 121.09 122.09 124.09 125.09 134.09 135.09 150.09 151.09 152.09 153.09 14.09 15.09 12.10 17.10 20.10 21.10 23.10

Cascavel, PR, Brazil Cascavel, PR, Brazil Cascavel, PR, Brazil Irai de Minas, MG, Brazil Irai de Minas, MG, Brazil Irai de Minas, MG, Brazil Irai de Minas, MG, Brazil Irai de Minas, MG, Brazil Irai de Minas, MG, Brazil Irai de Minas, MG, Brazil Irai de Minas, MG, Brazil Jata, GO, Brazil Jataı´, GO, Brazil Jaciara, MT, Brazil Jaciara, MT, Brazil Jaciara, MT, Brazil Jaciara, MT, Brazil Campo Moura˜o, PR, Brazil Campo Moura˜o, PR, Brazil Vilhena, RO, Brazil Vilhena, RO, Brazil Vilhena, RO, Brazil Vilhena, RO, Brazil Vilhena, RO, Brazil

GO, Goia´s; MG, Minas Gerais; MT, Mato Grosso; PR, Parana´; RS, Rio Grande do Sul; RO, Rondo ˆ nia; SC, Santa Catarina; SP, Sa˜o Paulo. fungi Ophiostoma ulmi, Botryotinia fuckeliana, Fusarium oxysporum and Penicillium marneffei respectively. Transposon insertion sites in the sequenced C. graminicola genome were identified by analyzing areas upstream and downstream of the transposon that occupied a total of 3000 bp (target site duplication was excluded) via a similarity search against the NCBI database using the BLASTx algorithm (Altschul et al. 1997). The threshold used was E-value , 10220 and identity . 50%. The RIPCAL program was used to analyze dinucleotides and calculate the RIP indices (Hane and Oliver 2008). The RID protein sequence from Neurospora crassa (GenBank accession number AAM27408.1) was used for BLAST queries against the genome. Isolates and culture conditions.—Forty-nine C. graminicola isolates were provided by the Brazilian Agricultural Research Corporation (Empresa Brazileira de Pesquisa Agropecua´ria – Embrapa) Maize and Sorghum Division (Embrapa Milho e Sorgo), Sete Lagoas, Minas Gerais (TABLE I). Isolates were cultured in potato dextrose agar (PDA) media at 22 C. DNA extraction.—Mycelia from the isolates were ground in liquid nitrogen, and the total DNA was extracted with the method described by Specht et al. (1982). Polymerase chain reaction (PCR) amplification.—Primers (59ACTCCCCCACCCTATATCCG 39) were designed based on the inverted repeats of the most conserved element that

was identified by the bioinformatic analysis. PCR amplification was performed with a PTC-100-MJ Research thermocycler in 50 mL reaction mixtures containing 40 ng DNA, 100 mM of each dNTP, 1 mM primer, 13 thermophilic DNA poly buffer (Promega), 2 mM MgCl2 and one unit of GoTaq DNA polymerase (Promega). The amplification conditions were: 40 cycles consisting of a denaturation step at 95 C for 1 min, an annealing step at 48 C for 1 min and an extension step at 72 C for 1 min. There was also an initial denaturation step at 95 C for 2 min and a final extension at 72 C for 10 min. Cloning and sequencing.—The PCR products were purified, cloned into the pGEMH-T Easy (Promega) vector, transformed into ultracompetent Escherichia coli DH5a cells and sequenced (3730xl DNA Analyzer, Applied Biosystems). The nucleotide sequence of Tcg1 was submitted to GenBank (accession number JX121209). Hybridization analysis.— Total genomic DNA from the isolates was digested by EcoRI and run on an 0.8% agarose electrophoresis gel. The fragments were transferred from the gel to a nylon membrane (Amersham) with standard procedures (Orkin 1990). Staining, hybridization and detection with the probe were performed with the PCR DIG Probe Synthesis Kit and DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) according to the manufacturer’s directions. The 963 bp probe was derived from the internal region of the sequenced element, and high-stringency hybridization conditions were used.

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TABLE II. Proportion of Tc1-Mariner transposons identified in the C. graminicola genome and classified into new families Tc1-Mariner family

No. of elements

TCg1 TCg2 TCg3 TCg4 TCg5 TCg6 Total

45 39 23 16 3 6 132

Dendrograms were constructed with the POPGENE program (Quardokus 2000).

RESULTS Identification of transposons in C. graminicola genome.— A database search using keywords that are related to transposase revealed four open reading frames (ORFs) encoding proteins that are similar to transposases in the Tc1-Mariner superfamily that have been found in other organisms. Two ORFs, encoding proteins of 176 and 242 amino acids in length, resulted in several hits (12 and 67 respectively) in an identity query (with the BLAST algorithm with standard parameters) against the C. graminicola genome. The other two ORFs didn’t result in significant hits. We identified 45 full-sized sequences (flanked by inverted repeats) corresponding to Tc1Mariner elements from these results. Analyses using the RepeatMasker program revealed that 3.06% of the genomic sequence is repetitive but that only 0.5% of these repetitive DNA was homologous to transposable elements from the Repbase database (0.41% retrotransposons and 0.09% transposons). In all, 132 fullsized elements were identified (SUPPLEMENT 1). However, none of these elements contained intact ORFs. All of the putative transposase-encoding sequences were interrupted by stop codons and typically did not have start codons. The elements had an average GC content of 29.5% in contrast with the genomic GC content of 49.1%. The element containing the most highly conserved putative transposase amino acid sequence (fewest number of premature stop codons) also had the highest GC content (48.4%). Elements were divided into six families, which were classified with the definition that was proposed by Wicker et al. (2007) (i.e., two elements belong to the same family if they share at least 80% sequence identity in at least 80% of their coding or internal regions, or terminal repeat regions or both). This approach was hindered by the degenerate element

sequences. Therefore, an element was placed in a family if it shared at least 80% identity with some members. Thus, members from each family shared 65–99% homology overall, and a given element shared at least 80% homology with at least one other element in the family (TABLE II). All elements had a TA duplication target site and TIRs 43–51 bp long (FIG. 1). The TIRs contained direct internal repeats (with the exception of the TCg6 family) 12–17 bp long. Analyses of the internal regions showed homologies to Tc1-Mariner elements from other filamentous fungi, but few contained the characteristic conserved motifs (DDE, HTH, CENPB). Only the TCg1 and TCg3 families had full-sized elements containing conserved DDE motifs (FIG. 2). In the TCg1 family the third residue of the DDE motif was an aspartic acid (D), and in the TCg3 family the third amino acid was substituted by a leucine (L). The TCg1 family was the most abundant (with 45 identified elements) and was subdivided into three subfamilies, TCg1.1, TCg1.2 and TCg1.3 (27, 13 and five elements respectively), based on their TIR sequences, which were clearly distinct between each family member (FIG. 1). The TCg1.1_8 element in this family was 1837 bp long and had perfect 44 bp TIRs, a putative transposase with few premature stop codons (12) and a conserved DDE motif. The internal region of this element shared 77% sequence identity (e value 5 0.0) with the Collect2 transposon from Colletotrichum cereale (SUPPLEMENT 2.1). The TCg2 family was the second most abundant (39 elements) and contained elements with low GC content, with an average of 22.9% (17.1–30.9%). The most conserved element (TCg2_434) in this family was 1862 bp long and had 43 bp TIRs and a putative transposase with 40 premature stop codons. Its internal region shared 64% identity (e value 5 2e-26) with the DAHLIAE1a transposon in Verticillium dahliae, which is another ascomycete phytopathogen (SUPPLEMENT 2.2). The 1888 bp TCg3_64 element belonged to the TCg3 family and was the only one with conserved HTH and CENPB motifs in addition to the DDE motif. The putative TCg3_64 transposase sequence had 28 premature stop codons and shared 51% homology (e value 5 1e-168) with the DAHLIAE2 transposase in Verticillium dahliae (SUPPLEMENT 2.3). The TCg4 family also had a low GC content (av. 23.2%). Its most conserved element (TCg4_31) was 1860 bp long with 45 bp TIRs possessing a 16 bp internal direct repeat and a putative transposase with approximately 50 stop codons and no conserved motifs. The TCg5 family had only three members that shared 81–87% identity. TCg5_1 was 1945 bp long and had 43 bp TIRs with a 17 bp internal direct repeat. Its putative transposase region had 54 premature stop codons

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FIG. 1. Terminal inverted repeats (TIRs) for TCg elements. Comparison of the TIRs of elements from the various Tc1Mariner families that were found in the C. graminicola genome. The arrows below the sequences indicate the internal direct repeats, and the asterisks indicate the mismatches.

and incomplete HTH and DDE motifs. Internal direct repeats were not identified in the TIRs of the TCg6 family. Members of this family were defective and had approximately 50 premature stop codons in the putative transposase region. TCg6_251 was 1717 bp long and had 50 bp TIRs. Phylogenetic analyses using the putative transposase amino acid sequences of the most conserved elements divided the families into three major groups; one contained the TCg1, TCg3 and TCg5 families, and another contained the TCg2 and TCg4 families. The TCg6 family diverged from the other groups (FIG. 3). Transposon insertion sites.—An analysis of the insertion region (1500 bp upstream and downstream of the transposon) showed that some transposons are inserted into regions that are similar to transposase and reverse transcriptase proteins, which are likely indicative of the presence of clusters of transposable elements. In one region of supercontig 136, two full-sized transposon sequences, TCg3_136 and TCg4_136, were separated by a 2041 bp sequence, the corresponding amino acid sequence of which had 40% homology with the reverse transcriptase that was encoded by a retrotransposon in Aspergillus kawachii (GenBank number GAA93096.1). Supercontig 314 contained two transposons, TCg2_314 and TCg5_314, which were separated by just 213 bp, and the region upstream of TCg2_314 contained a sequence that was 339 bp long and coded for an amino acid sequence with 49% homology to the transposase from a Mutator-like element in Metarhizium anisopliae

(GenBank number EFZ03338.1). Two transposons, TCg1.1_98.1 and TCg1.1_98.2, were separated by 2939 bp in supercontig 98. A 1728 bp ORF that was found in this region, which was just 6 bp downstream of TCg1.1_98.1 and coded for a 529 amino acid protein that was classified as a prostacyclin synthase (Supercontig 98: 59657–61384 -, GenBank number EFQ36497.1). In the region 1196 bp upstream of TCg1.1_98.1, there was a 1770 nucleotide ORF that coded for a 589 amino acid protein that shared 27% homology with the transposase in the Crypt1 element of Cryphonectria parasitica (GenBank number AAF97810.1), which belongs to the hAT superfamily of class II DNA transposons. In addition, the TCg1.1_ 98.2 transposon is flanked by regions that are similar to tc1 transposases. We identified several transposons that were inserted close to gene ORFs, these results included (TABLE III, SUPPLEMENT 3). RIP analysis.—Forty-five sequences from elements in the TCg1 family were used in a RIPCAL software analysis. The TCg1.1_8 element was used as a reference because it had the highest GC content. This analysis revealed that the number of transition mutations was higher than the number of transversions and the majority of sequence changes were transitions from C to T and G to A. The C:G transitions did not occur randomly in the mutated sequences; some CpN dinucleotides were preferentially mutated compared with others (Hane and Oliver 2008). In this analysis, the number of occurrences of dinucleotide combinations, which

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FIG. 2. Comparison of the DDE motif of putative TCg element transposases and related fungal transposases. Clustal W alignment of the conserved DDE motifs of TCg1.1_8, TCg1.2_33, TCg1.3_3 (TCg1 subfamilies), TCg3_64 and TCg3_85 (TCg3 family) with the DDE motifs of the OPHIO1 element from Ophiostoma ulmi and Flipper element from Botrytis cinerea (GenBank accession numbers: ABG26270.1 and AAB63315.1 respectively). Conserved amino acid residues are highlighted in black (identical residues) or dark gray (semiconserved substitutions). Asterisks indicate the DDD triad.

are potential RIP targets, were counted. Mutations occurred more often in CpA dinucleotides (CpA to TpA or TpG to TpA mutations), indicating that they were the preferred sites. Two main indices were used to show evidence of RIPs: TpA/ApT and (CpA + TpG)/(ApC + GpT). The first index measures the frequency of RIP products, while the second evaluates their target frequencies. High TpA/ApT and low (CpA + TpG)/(ApC + GpT) values are strong indicators of RIPs (Hane and Oliver 2008). In this

analysis, the TpA/ApT index was 1.64 and the (CpA + TpG)/(ApC + GpT) index was 0.56, which is consistent with the presence of RIP mutations. Reference values of indices for standard RIP are TpA/ApT . 0,89, CpA+TpG/ApC+GpT , 1,03 (Hane and Oliver 2008). A 1253 nucleotide ORF (SUPPLEMENT 4), which codes for a 750 amino acid protein containing a DNA methyltransferase (DMT) domain that is characteristic of RID, was found in the sequenced C. graminicola

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FIG. 3. Phylogeny of TCg elements and related transposases from other fungi. Phylogenetic tree of the putative amino-acid sequences of the TCg elements and of various transposable elements. Amino acid sequences for transposases from the OPHIO1, Flipper, Fot5 and tc1 elements were used (GenBank accession numbers: ABG26270.1, AAB63315.1, CAE55867.1, XP_002146479.1 respectively). The transposase sequences from the other fungi that were used are available at http://www. girinst.org/repbase/.

genome. This domain’s amino acid sequence shares 52% homology with that of the DMT domain of the RID protein in Neurospora crassa (FIG. 4). The identification of this protein, which is involved in the RIP process, supports the possibility that this mechanism may exist in C. graminicola. TCg1 transposon.—The TCg1 transposon was amplified from the C. graminicola 2908 m genomic DNA with primers that were based on TCg1.1_8 TIRs (SUPPLEMENT 5). TCg1 covers 1866 bp and has imperfect 44 bp TIRs with imperfect 12 bp internal direct repeats (FIG. 5). The putative transposase sequence codes for a 532 amino acid protein and has three characteristic conserved motifs that are thought to be essential for transposition, including the HTH, CENPB and DDE motifs (FIG. 6). A DDD

triad also was present. However, the sequence was interrupted by five premature stop codons and the correct start and stop codons were not found. TCg1 shares 78% identity (e value 5 0.0) with Collect2 in C. cereale (SUPPLEMENT 2). Hybridizations using probes that were derived from the internal TCg1 region under stringent conditions revealed that they bound to genomic DNA from various C. graminicola isolates in multiple hybridization patterns, although one of the profiles predominated (FIG. 7). All of the 49 analyzed isolates hybridized with the probe. Four bands showed a strong hybridization signal with the probe, indicating strong identity or an existence of cluster of copies. Other weaker bands were detected, indicating the presence of more divergent sequences that were similar to the probe. A dendrogram was constructed from the four

636 TABLE III.

MYCOLOGIA Partial list of genes identified near the transposons insertion sites Reference

Transposon sequence

Supercontig

Gene

Transposon distance from gene (bp)

GenBank

Colletotrichum database

TCg1.3_23 TCg3_6.2 TCg1.2_56 TCg1.1_45 TCg1.2_142 TCg3_24 TCg1.2_3 TCg1.1_7 TCg1.1_83 TCg2_162 TCg3_46 TC1.1_70

23 6 56 45 142 24 3 7 83 162 46 70

ABC transporter Alpha glucoside transporter Amidase Cytochrome B5-like Cytochrome P450 Cytochrome P450 Epoxide hydrolase GMC Oxidoreductase Hexose transporter KR motif-containing protein MFS transporter Trichodiene oxigenase

D: 62 0* U: 98 D: 192 U: 196 0* U: 86 U: 294 0* D: 213 D: 411 0**

EFQ31083.1 CBV37383.1 EFQ34948.1 EFQ33994.1 EFQ36698.1 EFQ31242.1 EFQ26064.1 EFQ27442.1 CBV37357.1 EFQ36730.1 EFQ34113.1 EFQ35814.1

GLRG_06227.1 GLRG_02169.1 GLRG_10092.1 GLRG_09138.1 GLRG_11846.1 GLRG_06386.1 GLRG_01208.1 GLRG_01937.1 GLRG_11320.1 GLRG_11878.1 GLRG_09257.1 GLRG_11009.1

* Inserted within the gene. ** Part of the transposon (186 bp from the 39 end) was considered to be part of the trichodiene oxigenase gene, likely due to its proximity.

strongest hybridization bands (FIG. 8). The lineages were separated into seven groups, with one group encompassing the majority of the lineages (67%). DISCUSSION The frequency of transposable elements in the C. graminicola genome that was estimated by RepeatMasker (0.5%) is low compared to those that are found in other fungal genomes, where they typically are present at a frequency of 3–20% (Hua-Van et al. 2005). However, more variable rates have been measured in some cases, from 1.1% in Ustilago maydis

(Kamper et al. 2006) up to 58% in Tuber melanosporum (Martin et al. 2010) and 85% in Blumeria graminis (Parlange et al. 2011). Repeat Masker is a homology-based program and relies on sequences present in a previously described transposable elements library (RepBase), thus it can miss elements that are not represented in the library and degenerate copies of elements. It is likely that the C. graminicola genome has a transposable element frequency that is much higher than 0.5%. The degeneration of the elements suggests that the host has a defense mechanism to control transposable element activity, which likely involves RIP. The low

FIG. 4. Comparison of the DMT domain of DNA-methyltransferase from C. graminicola genome with the DMT domain of the RID protein from N. crassa. Clustal W alignment of the amino acid sequences of the DMT domains of the DNAmethyltransferase that was identified in the C. graminicola genome (Supercontig 5:84970–87222, GenBank accession number: EFQ26585.1) with the DMT domain of the RID protein from N. crassa (GenBank accession number: AAM27408.1). Conserved amino acid residues are highlighted in black (identical residues), dark gray (conserved substitutions) or light gray (semiconserved substitutions).

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FIG. 5. Terminal inverted repeat (TIR) from TCg1. Arrows below the sequences indicate internal direct repeats, and asterisks indicate the mismatches.

FIG. 6. Comparison of the DDE motif of putative TCg1 element transposase with related fungal transposases. Clustal W alignment of TCg1 with the DDE motifs in the Flipper element from Botrytis cinerea and the OPHIO1 element from Ophiostoma ulmi (GenBank accession numbers: ABG26270.1, AAB63315.1 respectively). Conserved amino acid residues are highlighted in black (identical residues), dark gray (conserved substitutions) or light gray (semiconserved substitutions). Conserved motifs are: HTH (solid line), CENPB (dashed line) and DDE (dotted line).

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FIG. 7. Hybridization profiles of genomic DNA from various C. graminicola isolates cleaved by EcoRI under high-stringency conditions using a 963 bp probe derived from the internal region of TCg1. Total genomic DNA from 49 Colletotrichum graminicola isolates was digested with the EcoRI restriction enzyme, transferred to a nylon membrane and hybridized with a probe that was derived from the internal region of TCg1. DNA from all isolates was hybridized with the probe. Stronger bands indicate higher identity between the probe and target or perhaps an existence of cluster of copies.

GC contents of the transposable elements compared with that of the whole genome supports the existence of this mechanism. A few elements have perfect TIRs and perfect internal direct repeats, but most have imperfect TIRs. The internal direct repeats in the TIRs are involved in the recognition and binding of the transposase motifs (Bigot et al. 2005). Element degeneration is also reflected by the low frequency of characteristic conserved motifs. Only two families contain elements with conserved motifs. The other families have severely damaged elements with putative transposases that are interrupted by more than 40 stop codons. None of the elements have the characteristics that are necessary to be considered autonomous. Phylogenetic analyses showed that TCg1 and TCg3 are the closest two families, while the TCg6 family probably has a different ancestor or diverged early from the ancestor that gave rise to the other families. RIP analyses revealed that most of the differences from the reference sequence were transitions from C to T and G to A, which is consistent with RIP-induced changes. The TpA/ApT and (CpA + TpG)/(ApC + GpT) index values that were obtained in the present study also are consistent with an RIP-related mechanism.

We observed that the majority of detected transitions were mutations from CpA to TpA. It is believed that the preference for CpA to TpA transitions increases the odds of introducing TAA or TAG stop codons to inactivate the gene (Montiel et al. 2006), which is a possible explanation for the numerous stop codons interrupting the transposases of the identified elements. Evidence for RIP already has been reported in C. graminicola following an analysis of a Gypsy retrotransposon, which has a low GC content and transitions that are characteristic of RIPs (Clutterbuck 2011). Evidence for RIP also was found in C. cereale, which is a related species. Sequences that were derived from the Ccret2 retrotransposon showed TpA/ApT index values of 2.00 and (CpA + TpG)/(ApC + GpT) index values of 0.44 (Crouch et al. 2008). Although C. graminicola rarely has a sexual cycle in nature, these results indicate that RIP is likely rare events or that a RIP-like mechanism occurs in C. graminicola that is independent of the sexual cycle. The TCg1 transposon has three conserved characteristic motifs that are thought to be essential for transposition in addition to the DDD triad that coordinates metallic ions for catalysis. However, this

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element is likely inactive because it has a putative transposase that is interrupted by five premature stop codons. In addition, it has imperfect TIRs with imperfect internal direct repeats that likely impede transposase recognition. TCg1 shares high identity (78%) with Collect2 from C. cereale. Collect2 also has a low GC content and an ORF that is interrupted by stop codons that were probably created by RIP (Crouch et al. 2008). These two species are closely related phylogenetically (Crouch et al. 2006), indicating that TCg1 and Collect2 possibly share a common ancestor. It is also probable that TCg1 is an ancient integration into the C. graminicola genome. A relationship between the original location and the hybridization profile was not observed, indicating that these elements are likely ancient in the analyzed lines. The dendrogram separated the lines into seven groups, revealing that TCg1 potentially may be used in population studies on this phytopathogen. The strategy that was used in the present study efficiently detected transposable elements in C. graminicola and identified six Tc1-Mariner families. Colletotrichum graminicola has multiple degenerate Tc1-Mariner transposable elements with characteristics of RIP-affected sequences, such as those with C to T and G to A transitions showing preferences for CpA to TpA mutations and low GC contents. Sequences possessing all characteristics that are necessary for autonomous elements were not identified; thus it is unlikely that any of the identified elements are autonomous. The TCg1 element is present in several C. graminicola isolates and potentially can be used as a molecular marker for population studies. The presence or absence of TCg1 sequences and hybridization profiles should be analyzed in other phytopathogenic Colletotrichum species. Although there was no evidence that these elements are active in the isolate of C. graminicola sequenced, the possible existence of active copies in field isolates was shown by the difference in the profiles of hybridization. Transposable elements still can generate genetic variability passively by functioning as homologous regions for recombination, possibly resulting in chromosome rearrangements, gene duplications or deletions (Oliver and Greene 2009). Thus these families of inactive elements probably play roles in the genetic variability of C. graminicola. FIG. 8. Dendrogram of hybridization analysis of C. graminicola isolates. Dendrogram constructed from the DNA hybridization results from digested genomic DNA from 49 C. graminicola isolates using the internal region of the TCg1 transposon as a probe.

ACKNOWLEDGMENTS This work was financially supported by the Brazilian Agencies CAPES (Coordenac¸a˜o de Aperfeic¸oamento de

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Pessoal de Nı´vel Superiror) and CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico).

LITERATURE CITED Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402, doi:10.1093/ nar/25.17.3389 Bergstrom GC, Nicholson RL. 1999. The biology of corn anthracnose. Plant Dis 38:596–608, doi:10.1094/ PDIS.1999.83.7.596 Bigot Y, Brillet B, Auge-Gouillou C. 2005. Conservation of palindromic and mirror motifs within inverted terminal repeats of mariner-like elements. J Mol Biol 351: 108–116, doi:10.1016/j.jmb.2005.05.006 Clutterbuck AJ. 2011. Genomic evidence of repeat-induced point mutation (RIP) in filamentous ascomycetes. Fungal Genet Biol 48: 306– 326, doi:10.1016/ j.fgb.2010.09.002 Crouch JA, Beirn LA. 2009. Anthracnose of cereals and grasses. Fungal Divers 39:19–44. ———, Clarke BB, Hillman BI. 2006. Unraveling evolutionary relationships among the divergent lineages of Colletotrichum causing anthracnose disease in turfgrass and corn. Phytopathology 96:46–60, doi:10.1094/PHYTO-96-0046 ———, Glasheen BM, Giunta MA, Clarke BB, Hillman BI. 2008. The evolution of transposon repeat-induced point mutation in the genome of Colletotrichum cereal: reconciling sex, recombination and homoplasy in an ‘‘asexual’’ pathogen. Fungal Genet Biol 45:190– 206, doi:10.1016/j.fgb.2007.08.004 Daboussi MJ, Capy P. 2003. Transposable elements in filamentous fungi. Ann Rev Microbiol 57:275–299, doi:10.1146/annurev.micro.57.030502.091029 dos Santos LV, Queiroz MV, Santana MF, Soares MA, Barros EG, Arau´jo EF, Langin T. 2011. Development of new molecular markers for the Colletotrichum genus using RetroCl1 sequences. World J Microbiol Biotechnol, doi:10.1007/s11274-011-0909-x Dufresne M, Lespinet O, Daboussi MJ, Hua-Van A. 2011. Genomewide comparative analysis of pogo-like transposable elements in different Fusarium species. J Mol Evol 73:230–243, doi:10.1007/s00239-011-9472-1 Freitag M, Williams RL, Kothe GO, Selker EU. 2002. A cytosine methyltransferase homolog is essential for repeat-induced point mutation in Neurospora crassa. Proc Natl Acad Sci USA 99:8802–8807, doi:10.1073/ pnas.132212899 Galagan JE, Selker EU. 2004. RIP: the evolutionary cost of genome defense. Trend Genet 20: 417– 423, doi:10.1016/j.tig.2004.07.007 Hane JK, Oliver RP. 2008. RIPCAL: a tool for alignmentbased analysis of repeat-induced point mutations in fungal genomic sequences. BMC Bioinform 9:478, doi:10.1186/1471-2105-9-478 He CZ, Nourse JP, Kelemu S, Irwin JAG, Manners JM. 1996. CgT1: A non-LTR retrotransposon with restricted

distribution in the fungal phytopathogen Colletotrichum gloeosporioides. Mol Gen Genet 252:320– 331. Hey P, Robson G, Birch M, Bromley M. 2008. Characterization of Aft1 a Fot1/Pogo type transposon of Aspergillus fumigatus. Fungal Genet Biol 45:117–126, doi:10.1016/ j.fgb.2007.10.009 Hua-Van A, le Rouzic A, Maisonhaute C, Capy P. 2005. Abundance, distribution and dynamics of retrotransposable elements and transposons: similarities and differences. Cytogenetic Genome Res 110:426–440, doi:10.1159/000084975 Janicki M, Rooke R, Yang G. 2011. Bioinformatics and genomic analysis of transposable elements in eukaryotic genomes. Chromosome research: an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology 19:787–808, doi:10.1007/s10577-011-9230-7 Kamper J, Kahmann R, Bolker M, Ma LJ, Brefort T, Saville BJ, Banuett F, Kronstad JW, Gold SE, Muller O, Perlin MH, Wosten HA, de Vries R, Ruiz-Herrera J, Reynaga-Pena CG, Snetselaar K, McCann M, Perez-Martin J, Feldbrugge M, Basse CW, Steinberg G, Ibeas JI, Holloman W, Guzman P, Farman M, Stajich JE, Sentandreu R, Gonzalez-Prieto JM, Kennell JC, Molina L, Schirawski J, Mendoza-Mendoza A, Greilinger D, Munch K, Rossel N, Scherer M, Vranes M, Ladendorf O, Vincon V, Fuchs U, Sandrock B, Meng S, Ho EC, Cahill MJ, Boyce KJ, Klose J, Klosterman SJ, Deelstra HJ, Ortiz-Castellanos L, Li W, Sanchez-Alonso P, Schreier PH, Hauser-Hahn I, Vaupel M, Koopmann E, Friedrich G, Voss H, Schluter T, Margolis J, Platt D, Swimmer C, Gnirke A, Chen F, Vysotskaia V, Mannhaupt G, Guldener U, Munsterkotter M, Haase D, Oesterheld M, Mewes HW, Mauceli EW, DeCaprio D, Wade CM, Butler J, Young S, Jaffe DB, Calvo S, Nusbaum C, Galagan J, Birren BW. 2006. Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444: 97– 101, doi:10.1038/nature05248 Marini MM, Zanforlin T, Santos PC, Barros RR, Guerra AC, Puccia R, Felipe MS, Brigido M, Soares CM, Ruiz JC, Silveira JF, Cisalpino PS. 2010. Identification and characterization of Tc1/mariner-like DNA transposons in genomes of the pathogenic fungi of the Paracoccidioides species complex. BMC Genomics 11:130, doi:10.1186/1471-2164-11-130 Martin F, Kohler A, Murat C, Balestrini R, Coutinho PM, Jaillon O, Montanini B, Morin E, Noel B, Percudani R, Porcel B, Rubini A, Amicucci A, Amselem J, Anthouard V, Arcioni S, Artiguenave F, Aury JM, Ballario P, Bolchi A, Brenna A, Brun A, Buee M, Cantarel B, Chevalier G, Couloux A, da Silva C, Denoeud F, Duplessis S, Ghignone S, Hilselberger B, Iotti M, Marcais B, Mello A, Miranda M, Pacioni G, Quesneville H, Riccioni C, Ruotolo R, Splivallo R, Stocchi V, Tisserant E, Viscomi AR, Zambonelli A, Zampieri E, Henrissat B, Lebrun MH, Paolocci F, Bonfante P, Ottonello S, Wincker P. 2010. Perigord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis. Nature 464:1033–1038, doi:10.1038/ nature08867

BRAGA ET AL.: COLLETOTRICHUM GRAMINICOLA Montiel MD, Lee HA, Archer DB. 2006. Evidence of RIP (repeat-induced point mutation) in transposase sequences of Aspergillus oryzae. Fungal genet biol 43: 439–445, doi:10.1016/j.fgb.2006.01.011 Munch S, Lingner U, Floss DS, Ludwig N, Sauer N, Deising HB. 2008. The hemibiotrophic lifestyle of Colletotrichum species. J Plant Physiol 165:41–51, doi:10.1016/ j.jplph.2007.06.008 Oliver KR, Greene WK. 2009. Transposable elements: powerful facilitators of evolution. BioEssays : news and reviews in molecular, cellular and developmental biology 31:703–714, doi:10.1002/bies.200800219 Orkin S. 1990. Molecular cloning—a laboratory manual. 2nd ed. In: Sambrook J, Fritsch Ef, Maniatis T, eds. Nature 343:604–605, doi:10.1038/343604a0 Parlange F, Oberhaensli S, Breen J, Platzer M, Taudien S, Simkova H, Wicker T, Dolezel J, Keller B. 2011. A major invasion of transposable elements accounts for the large size of the Blumeria graminis f.sp. tritici genome. Funct Integr Genomics 11:671–677, doi:10.1007/ s10142-011-0240-5 Pietrokovski S, Henikoff S. 1997. A helix-turn-helix DNAbinding motif predicted for transposases of DNA t r a n s p o s o n s . M o l G e n G e n e t 2 54 : 6 8 9 – 6 95 , doi:10.1007/s004380050467 Plasterk RHA, Izsvak Z, Ivics Z. 1999. Resident aliens—the Tc1/ mariner superfamily of transposable elements. Trends Genet 15:326–332, doi:10.1016/S0168-9525(99)01777-1 Quardokus E. 2000. PopGene. Science 288:458–458, doi:10.1126/science.288.5465.458

641

Selker EU. 2002. Repeat-induced gene silencing in fungi. Adv Genet 46:439–450, doi:10.1016/S0065-2660(02) 46016-6 Specht CA, Dirusso CC, Novotny CP, Ullrich RC. 1982. A method for extracting high molecular weight deoxyribonucleic acid from Fungi. Anal Biochem 119:158–163, doi:10.1016/0003-2697(82)90680-7 Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA 4: Molecular evolutionary genetics analysis software. Mol Biol Evol 24:1596–1599, doi:10.1093/molbev/ msm092 Thompson JD, Higgins DG, Gibson TJ. 1994. Clustal W— improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680, doi:10.1093/nar/ 22.22.4673 Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O, Paux E, SanMiguel P, Schulman AH. 2007. A unified classification system for eukaryotic transposable elements. Nat Rev Genet 8:973–982, doi:10.1038/nrg2165 Yuan YW, Wessler SR. 2011. The catalytic domain of all eukaryotic cut-and-paste transposase superfamilies. Proc Natl Acad Sci USA 108:7884–7889, doi:10.1073/ pnas.1104208108 Zhu PL, Oudemans PV. 2000. A long terminal repeat retrotransposon Cgret from the phytopathogenic fungus Colletotrichum gloeosporioides on cranberry. Curr genet 38:241–247, doi:10.1007/s002940000162

Transposable elements belonging to the Tc1-Mariner superfamily are heavily mutated in Colletotrichum graminicola.

Transposable elements are ubiquitous and constitute an important source of genetic variation in addition to generating deleterious mutations. Several ...
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