Genomic characterization of the conditionally dispensable chromosome in Alternaria arborescens provides evidence for horizontal gene transfer
© Hu et al.; licensee BioMed Central Ltd. 2012
Received: 20 September 2011
Accepted: 8 March 2012
Published: 6 May 2012
Fungal plant pathogens cause serious agricultural losses worldwide. Alternaria arborescens is a major pathogen of tomato, with its virulence determined by the presence of a conditionally dispensable chromosome (CDC) carrying host-specific toxin genes. Genes encoding these toxins are well-studied, however the genomic content and organization of the CDC is not known.
To gain a richer understanding of the molecular determinants of virulence and the evolution of pathogenicity, we performed whole genome sequencing of A. arborescens. Here we present the de-novo assembly of the CDC and its predicted gene content. Also presented is hybridization data validating the CDC assembly. Predicted genes were functionally annotated through BLAST. Gene ontology terms were assigned, and conserved domains were identified. Differences in nucleotide usage were found between CDC genes and those on the essential chromosome (EC), including GC3-content, codon usage bias, and repeat region load. Genes carrying PKS and NRPS domains were identified in clusters on the CDC and evidence supporting the origin of the CDC through horizontal transfer from an unrelated fungus was found.
We provide evidence supporting the hypothesis that the CDC in A. arborescens was acquired through horizontal transfer, likely from an unrelated fungus. We also identified several predicted CDC genes under positive selection that may serve as candidate virulence factors.
KeywordsAlternaria arborescens Illumina sequencing Conditionally dispensable chromosome Horizontal gene transfer Polyketide synthase Host specific toxins
The rapid development of next-generation sequencing technologies over the past decade has led to a flood of both de-novo sequencing and re-sequencing projects in almost every branch of the tree of life. Within the fungal kingdom, comparative genome studies have led to the unexpected finding that large genomic regions may be variable among isolates of a given species. One category of these variable regions are unique chromosomes referred to as supernumerary or conditionally dispensable because they are not typically required for saprophytic growth [1–3]. These chromosomes have been identified in many fungi including Magnaporthe oryzae [4–6], Fusarium oxysporum , Nectria haematococca [8, 9], Mycosphaerella graminicola , Cochliobolus heterostrophus , Leptosphaeria maculans , and Alternaria alternata [13, 14].
Plant pathogenic fungi in the genus Alternaria infect a remarkable range of host plants and are major causes of agricultural yield losses . Conditionally dispensable chromosomes (CDCs) are carried by several of the small-spored, plant-pathogenic Alternaria species [13, 14, 16]. These chromosomes are generally less than 2.0MB in size, and may be transmitted horizontally between isolates in a population, potentially conferring new pathogenic attributes to the receiving isolate [17–20]. Loss of the CDC can also occur during repeated sub-culturing, resulting in the transition from a pathogenic to saprophytic form of the fungus . Several genes coding host specific toxins (HSTs) have been located to gene clusters on CDCs, including those producing AF-toxin from the strawberry pathotype , AK-toxin from the Japanese pear pathotype , and ACT-toxin from the tangerine pathotype . These toxins share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid structural moiety, with the genes encoding each toxin sharing a high degree of homology [21–25]. In addition, the AMT gene from the apple pathotype, a gene involved in host-specific AM-toxin cyclic peptide biosynthesis, is located on a small chromosome of 1.1 to 1.7 Mb [13, 26], with at least four copies involved in AM-toxin biosynthesis . The only other gene sequences identified to date on CDCs are extended families of transposon-like sequences (TLSs) .
Horizontal gene transfer (HGT) is the movement, without recombination, of stable genetic material between two individuals . HGT may not only occur between different individuals of the same species, but also between species or even between bacteria and fungi or between fungi and oomycetes [29, 30]. In fungi, the movement of plasmids, mycoviruses, transposable elements, gene clusters, and whole chromosomes have been demonstrated from one individual to another . The first theory to explain gain and loss of HSTs was proposed in 1983 . It has then been hypothesized that the genome content of CDCs in Alternaria species were acquired through HGT events . The most well studied example of HGT in fungi is the movement of the ToxA gene from the wheat blotch pathogen Stagonospora nodorum to Pyrenophora tritici-repentis, the causal agent of tan spot of wheat [33, 34]. This horizontal transfer event was identified by nucleotide sequence similarity and structural comparisons between genes from both species. The direction of transfer was inferred by the fact that the ToxA gene consisted of a single haplotype in P. tritici-repentis but 11 haplotypes in S. nodorum isolates.
Alternaria arborescens (synonym A. alternata f. sp. lycopersici), the fungus that produces host-specific AAL toxin, is the causal agent of stem canker of tomato [35, 36]. It has been observed in pulsed field gel electrophoresis (PFGE) studies that A. arborescens carries one CDC of 1.0-Mb [16, 37]. To date, only two genes have been reported to be carried on this CDC including ALT1, which is a PKS gene involved in AAL toxin biosynthesis [38, 39], and AaMSAS, also a PKS gene [40, 41]. A CDC deletion mutant of A. arborescens generated through restriction enzyme mediated integration (REMI) showed a toxin and pathogenicity minus phenotype . In addition, in protoplast fusion experiments, a CDC from A. arborescens was observed to transfer into the strawberry pathotype, and subsequently introduced new tomato pathogenicity to the fusant .
In this study, we used a next generation sequencing approach to produce a draft sequence of the A. arborescens genome and used a novel bioinformatics approach to separate CDC contigs from the essential chromosome (EC) contigs. The gene content of the CDC was analyzed to answer the following questions: (1) What is the difference between the CDC and EC genome content at the nucleotide level? (2) Are CDC genes under positive selection and could they represent additional virulence factors in addition to the known toxin encoding genes? (3) Is the evolutionary history of the CDC the same as that of the ECs, and is there any evidence of a HGT event? In answering these questions, we confirmed a different genome content pattern of the A. arborescens CDC and found evidence for HGT.
Sequencing & assembly
A. arborescens strain EGS 39–128 (CBS 102605)  was sequenced by a whole genome shotgun approach using the Illumina Genome Analyzer II, which resulted in ~50 million paired-end short reads of 75 bp representing 90X average coverage of the predicted genome content. De-novo assembly was performed using Velvet  (version 0.7), and confirmed by Edena  and Minimus2 . The assembly resulted in 1,332 contigs with a N50 of 624KB and total size of 34.0MB ( Additional file 1: Table S1; Assembly has been deposited at DDBJ/EMBL/GenBank under the accession AIIC00000000. The version described in this paper is the first version, AIIC01000000.) One hundred thirty-seven large contigs with lengths greater than 10KB and representing 98% of the genome assembly content were chosen for further analysis.
Marker-assisted identification of contigs carrying toxin biosynthetic genes
Identification of the remaining CDC contigs and validation by Southern hybridization
Alignment of A. arborescens marker gene contigs and A. brassicicola contigs
Gene prediction, length, GC3-content, and repeat identification
Nine thousand, one hundred sixty-seven genes were predicted by FGENESH  using pre-trained Alternaria parameters, of which 209 genes were assigned to CDC contigs and 8958 to EC contigs. The average length of each predicted gene was 1.8 KB, and the gene density was 3.7KB per gene. Compared to gene predictions for A. brassicicola (average gene length = 1.3KB, gene density = 3.0KB per gene), A. arborescens genes were longer and present in lower density. To evaluate the origin of the CDC, the predicted genes residing on the CDC and EC contigs were compared at the nucleotide level, including gene length, GC3-content , repeat load, and codon usage bias. This analysis showed that CDC genes are about 200bp shorter on average than EC genes (P = 2.36E-09) and have significantly lower GC3-content ( P = 0.028). Repeat regions composed 5.3% of CDC contigs while only 0.6% of EC contigs (Additional file 1: Table S2). It should be noted that some repeat regions could be lost in short read sequences de-novo assemblies, however, even with possible suppressed numbers, this result indicates approximately 10X repeat enrichment in the CDC compared to the EC.
Codon usage analysis
Annotation of EC genes
The assembly results showed the size of essential chromosomes region collectively to be 33.0 MB with 8958 predicted genes. RepeatMasker identied only 0.12% of the EC region as simple repeats (about 50bp in length) and 0.08% as low complexity, indicating that short repeats may be lost during de novo assembly of Illumina sequencing reads. For secreted protein identification, 1099 (12.2%) of the EC proteins were predicted to contain signal peptides, and were functionally annotated using BLAST to the NCBI database with more than 98% of the genes returning at least one hit with an E-value < 1.0E-3. From the BLAST results, we identified 212 transcription factors, 98 oxidase proteins, 202 kinase proteins, 279 transporters, 81 Cytochrome P450s, and 45 different proteases.
Annotation of CDC genes
Several host-specific toxin genes and transposon-like sequences have been reported to be carried by CDCs in Alternaria . We used two methods to annotate the functions of resident CDC genes: (1) they were blasted against the NCBI non-redundant database as well as Pfam  and NCBI CDD  to search for functional domains; (2) they were scanned to identify transcription factors, PKS genes, NRPS genes, P450s, transporters, and pathogenicity related genes.
Gene ontology terms were assigned to CDC genes based on BLAST matches with sequences whose function was previously characterized . Ninety CDC genes were assigned to a biological process, 51 for molecular function, and 15 for cellular component (Additional file 2: Figure S2). Among the biological process assignments, 54% of genes were assigned to “metabolic process”, and 10% to “biosynthetic process”. Enrichment of metabolic and biosynthetic process in CDC genes as compared to EC genes supported the observation that Alternaria CDC genes were enriched for polyketide synthases (PKS) and toxin synthases. Molecular function terms showed a significant percentage (39%) to “nucleotide/nucleic acid binding”, which showed an enrichment of transcription factors and gene regulation elements.
To provide a more detailed characterization of putative CDC genes, each was translated to identify protein families. Among the 209 predicted CDC proteins, 31 were identified as carrying PKS domains. Two proteins were found to carry highly modular domains: KS-AT-KR-ACP on CDC_141 and KS-AT-DH-ER-KR-ACP on CDC_165. The remaining 29 PKS proteins each carried 1 or 2 ACPs (Acyl carrier protein) domains. Seven proteins were found to carry NRPS domains: 3 Enterobactin domains, 2 Bacitrancin domains, 1 Pyochelin domain, and 1 CDA1 domain. Two proteins were identified as hybrid PKS-NRPS. Seven proteins were identified as P450 monooxygenase proteins. For transcription factors, 24 proteins were characterized to contain TF domains, in which Zn2Cys6 was the prominent group. Multiple ADP/ATP transporters, ABC transporters, ion transporters and major facilitator superfamily (MFS) transporters were also found in CDC protein group. Additionally, it was found that multiple proteins carrying FAD binding domains and oxidoreductases. Finally, 37 proteins were identified as putative pathogenicity related genes through scanning CDC genes in the pathogen-host interactions database (PHI-base)  (E-value < 0.05). See Additional file 3 for a complete CDC gene annotation list.
Secondary metabolite biosynthetic gene clusters
Evolutionary selection of CDC genes and domains
Origin of CDC
Comparison to other CDC containing fungi
Compared to other recently published assemblies of CDCs in filamentous fungi, A. arborescens has a relatively small number of CDCs (one) and the size (1.0Mb) is small. M. graminicola was reported to have the highest number of dispensable chromosomes with upwards of 8 ranging in size from 0.39 to 0.77MB . Three CDCs in N. haematococca [9, 60], and 4 complete CDCs and partial region of another 2 in F. oxysporum were identified. In other Alternaria species, identified CDCs are relatively larger such as 1.05Mb in the strawberry pathotype , 1.1 to 1.7 Mb (depending on strains) in the apple pathotype , and 4.1 Mb in the Japanese pear pathotype [61, 62]. In A. arborescens, only 1 dispensable chromosome is present, representing only 3% of the genome content, which is significantly smaller than other cases and may suggest a more recent acquisition or different origin.
PKS and NRPS clusters
Phytopathogenic fungi produce a diverse array of secondary metabolites, including host-selective toxins conferring pathogenicity . It was reported in two basidiomycete maize pathogens candidate effector genes were located in small clusters that were dispersed throughout (both "are" change to "were") the genome . However, in some other fungi, especially ascomycetes, genes coding for toxins can co-locate in clusters consisting of more than 10 contiguous genes. A well-known example is the trichothecene biosynthetic gene cluster in F. graminearum which contains 10–12 genes including a terpene synthase gene, P450 monooxygenase genes, acyl transferase genes, regulatory genes, and transporter genes . While in A. fumigatus, 26 SMB clusters were identified, each containing 5–48 genes . In our study, 29 PKS, 5 NRPS, and 2 hybrid PKS-NRPS genes were found on the CDC, with larger density compared to other fungi. However, among 10 predicted SMB clusters, most were relatively small and only carried 3–8 genes, which may not represent the true cluster size due to short contigs length that may divide one large cluster into two or more. One example of an identified cluster was located on contig Node_309 which consists of 5 genes, including 2 PKSs, 1 NRPS putatively coding for enterobactin, a phosphate transferase gene, and a MFS transporter. It lacks regulators, P450s, and transporters compared to other typical clusters. However, this cluster locates at the edge of the contig. Only 5 genes away from this cluster, another small cluster containing PKS, NRPS, P450s and an ABC transporter was identified, suggesting these two could be part of a larger cluster (see Additional file 3: Supplementary CDC annotation list). In this study, PKS genes were identified by screening the PKS sequence database, especially the domain database, which include: ketoacyl synthase (KS), acyl transferase (AT), ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), and acyl carrier protein (ACP, also known as PP domain). The KS, AT, and ACP domains are essential for PKS genes . Two PKS genes were identified to have multiple domains above: KS-AT-KR-ACP in CDC_141, and KS-AT-DH-ER-KR-ACP in CDC_165. The remaining 29 PKS genes each carries 1 or 2 ACP domains. Despite these conserved domains, other domains carried by these genes were divergent, indicating variance and multifunction of each PKS genes (Additional file 1: Table S6). However, at least 3 domain families were found to be enriched in the indentified PKS genes: ABC_membrane (4 identified), NADB_Rossmann (7 identified), and P-loop NTPase (6 identified), suggesting these proteins are transmembrane and catalyzing enzymatic reactions. In the NRPS and hybrid PKS-NRPS gene group, enterobactin, bacitracin, pyoverdine, syringomycin, and CDA1 domains were identified, 4 of which were reported from to bacteria [67–70]. We eliminated the possibility of these genes originating from bacterial sequencing contamination by BLAST comparing all assembly contig sequences against the NCBI All Bacterial database with 2017 genome sequences, and found that the species with most hits was Streptomyces coelicolor with > 80% identity. However, only 0.7% of the entire S. coelicolor genome was covered. Indicating that either these genes have an origin from bacteria or their product proteins interact with each other and require a highly conserved structure that was retained during evolution.
Horizontal gene transfer
According to the horizontal gene transfer hypothesis, A. arborescens may have acquired its CDC from another Alternaria species, from a fungus other than Alternaria, or possibly from a bacterium or virus . There are at least two other possible explanations for its origin: (1) CDCs were present in an Alternaria ancestor, but were independently lost during vertical transmission in other non-pathogenic Alternaria species. (2) CDCs arose from essential chromosome as a copy first but then went under divergence so no obvious orthology could be detected. To test which of the three models fits this case best, we built a complete EC protein library and blasted all CDC proteins against it to detect any possible orthology. Out of 209 CDC proteins, we found 12 (5.7%) showing orthology to EC proteins. Although the low orthology percentage alone could not exclude the “duplication and divergence” model, taken together with differences on GC3-content and codon usage bias, the possibility that this model fits is minimal.
To distinguish between HGT and vertical transmission hypothesis, we identified differences between A. arborescens CDC and EC genes in length, GC3-content, and codon usage bias. There was limited orthology detected between two groups; CDC genes showed discordant phylogenetic relation with EC, and had higher similarity to other fungi than A. brassicicola. From previous phylogenetic analysis of 13 A. alternata isolates collected worldwide, CDC genes from different isolates were almost identical despite diverse EC background . Taken these results together, we concluded that the HGT model may serve as the best fit model in this case. Additionally, these data support the theory proposed in 1983 by Nishimura that Alternaria species acquired HSTs by HGT .
In this study, we identified evidence for the possibility of HGT event occurred in A. arborescens. For Alternaria, this strategy has its advantages. First, as a pathogen with a wide host range, as observed in nature, transportable pathogenicity chromosome may increase pathogen's adaptation to environment. Second, loss of a CDC when there's no host may reduce the cost of carrying extra genome content. Third, as asexual fungi, horizontal transfer may compensate the lack of genetic recombination.
In this study, we identified A. arborescens CDC sequences through a whole genome sequencing and de-novo assembly process. By comparing nucleotide usage between CDC and EC contigs, we found evidence supporting HGT in A. arborescens. We also identified some predicted CDC genes under positive selection that may serve as virulence factors. However, questions still remain, such as the similarity and difference among CDCs from different A. arborescens isolates. To better understand CDC characteristics and mechanisms of HGT, other Alternaria isolates need to be sequenced.
Materials and methods
Sequencing, assembly & alignment
A. arborescens DNA was extracted following a protocol described  and the sequencing library was prepared using the Illumina Paired-End DNA Sample Prep Kit. Sequencing was performed using Illumina Genome Analyzer II. Short reads were assembled de-novo using Velvet, and assembly quality was improved by a pipeline including two alternate assemblers: Edena , and Minimus2 . Parameters including k-mer length for Velvet and hash length for Edena were optimized by sequential step changes. The alignment between A.arborescens and A.brassicicola was conducted using the Nucmer program in the MUMmer suite , with parameter c = 15, l = 10. Alignments with identities lower than 90% or lengths shorter than 100bp were removed.
On the CHEF gel membrane presented in Figure 2, lane 1 contains size markers, lane 2 contains A. arborescens chromosomes that had degraded, and lane 3 contains intact A. arborescens chromosomes. Southern hybridization was conducted using the GE health CDP-Star kit with 5 gene probes, including 1 CDC marker gene ALT1, 3 predicted CDC genes, and 1 predicted EC gene. Primers (Additional file 1: Table S7) were designed using Primer3  (v0.4.0). Blots were stripped between hybridizations to ensure no probes from previous hybridization remained. Film was exposed for 48 hours.
Gene prediction, codon usage analysis & repetitive DNA identification
Gene prediction was conducted using FGENESH , an ab initio gene predictor provided in the Softberry website. A pre-trained Alternaria matrix was used to optimize predictions. Both CDSs and protein sequences were generated and converted into fasta format files. ACUA  was used for calculating CAI and RSUC for each gene, and CAI distribution curves from the CDC group and EC group were compared to each other. Student’s F-test was used to test statistical significance. RepeatScout  was used for de-novo identification of repeat sequences in both CDC and EC sequences. The repeat libraries were then aligned back to CDC and EC contigs using Nucmer to calculate the repeat percentage for each group.
Blast2go  was used to annotate genes by “BLASTX” to the NCBI non-redundant protein database and then GO term assignment from the gene ontology database. Annotation of conserved domains was identified by scanning proteins through Pfam and NCBI CDD. PKS and NRPS genes were identified through scanning an online database SBSPKS . The Fungal transcription factor database (FTFD)  was used to identify transcription factors. Transporters, P450s, and oxidoreducatases were identified based on BLAST and domain inspection. Potential secreted proteins were predicted using Signal 3.0 . Pathogenicity and virulence factors were identified through scanning CDC genes in the pathogen-host interactions database (PHI-base) .
Estimating Ka/Ks Ratios
A. arborescens proteins were blasted against A. brassicicola proteins to generate a match list between the two groups with a bits score cut-off at 300. The gene sequences coding for aligned proteins were extracted by an in-house PERL script. Prank  was used to conduct codon alignment, in which two protein sequences were aligned first and then DNA sequences were aligned based on the corresponding protein alignments. The codon alignment result was then entered into “Codem” in PAML  (v4.0) for Ka and Ks calculation with model M0. In calculating, the Nei and Gojobori  method and Yang and Nielsen  method were used.
Conditionally dispensable chromosome
Nonribosomal peptide synthetase
Host specific toxin
Horizontal gene transfer
Pulsed field gel electrophoresis
Restriction enzyme mediated integration
Clamped homogenous electric fields
Codon adaptation index
Relative synonymous codon usage
Conserved domains database
Secondary metabolite biosynthesis
Blast score ratio.
We are grateful to Dr. R.C. Venu at The Ohio State University (OSU) for preparing the sequencing library and the OSU Molecular Cellular Imaging Center (MCIC) for performing Illumina sequencing. We thank Drs. Kun Huang and Hideaki Kikuchi at the OSU Department of Biomedical Informatics for providing access to the high performance computing cluster. This work was supported by grant 2009–012 from the Ohio Agricultural Research and Development Center’s Research Enhancement Competitive Grants Program (SEED).
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