Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

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Abstract

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

Main

The aspergilli are a ubiquitous group of filamentous fungi spanning over 200 million years of evolution. Among the over 185 aspergilli are several that have an impact on human health and society, including 20 human pathogens as well as beneficial species used to produce foodstuffs and industrial enzymes1. Within this genus, A. nidulans has a central role as a model organism. In contrast to most aspergilli, A. nidulans possesses a well-characterized sexual cycle and thus a well-developed genetics system. Half a century of A. nidulans research has advanced the study of eukaryotic cellular physiology, contributing to our understanding of metabolic regulation, development, cell cycle control, chromatin structure, cytoskeletal function, DNA repair, pH control, morphogenesis, mitochondrial DNA structure and human genetic diseases.

We present here the genome sequence for A. nidulans, and a comparative genomics study with two related aspergilli: A. fumigatus2 and A. oryzae3. A. fumigatus is a life-threatening human pathogen, and A. oryzae is used in the production of sake, miso and soy sauce. A. oryzae and A. fumigatus lack known sexual cycles, and their study relies on A. nidulans as a genetic model. Our analysis of these organisms focused on the genomic bases of their differing physiologies, while investigating their common eukaryotic biology. Our results yield new insights into eukaryotic genome evolution, the evolution of mating-type loci, the potential for sexual reproduction in the two asexual species, and the role of conserved sequence elements in gene regulation.

Genome assembly and annotation

The genome sequence of A. nidulans was assembled from deep whole-genome shotgun (WGS) coverage obtained by paired-end sequencing from a variety of clone types (see Methods). An average of 13 × sequence coverage was generated including ×3 coverage produced and provided by Monsanto. The Arachne package (http://www.broad.mit.edu/wga/) was used to assemble the sequence, and the resulting assembly consists of 248 sequence contigs with an N50 length of 282 kilobases (kb) (that is, 50% of all bases are contained in contigs of at least 282 kb). Contigs were assembled into 89 scaffolds with a total length of 30.06 megabases (Mb) (including gaps between contigs) and an N50 length of 2.44 Mb. A total of 28.5 Mb (95%) of the assembly was anchored to the A. nidulans genetic map4,5 through meiotically mapped markers with sequence and markers located by haploidization or hybridization to electrophoretically separated chromosomes (see Supplementary Information). By comparison with previously published pulse-field gel electrophoresis data, we estimate that the assembly comprises 96.3% of the complete genome. The assembly was annotated using the Calhoun system, as described in the Methods and Supplementary Information.

Phylogenetic relationship

Previous work based on large subunit rDNA data has led to a widely accepted phylogeny of the aspergilli in which A. nidulans and A. oryzae are more related to one another than A. fumigatus6. However, single gene phylogenies can contradict organismal phylogenies7. In principle, whole-genome data provide greater resolving power by allowing trees to be constructed based on concatenated sets of genes7. Using this approach to study the relationship of the three aspergilli, we find support for an alternative phylogeny8 (Fig. 1a).

Figure 1: Phylogenetic tree and representative dot plot.
figure1

a, Phylogenetic tree showing the relationship between three Aspergillus species compared using N. crassa and F. graminearum as an outgroup. Branch lengths correspond to substitutions per site calculated using a maximum likelihood approach. An identical topology was predicted using C. immitis as an outgroup. b, Dot plot of A. nidulans (horizontal) and A. fumigatus (vertical) genomes. Axes represent the concatenation of all chromosomes for the corresponding genome. Gridlines indicate the boundaries between chromosomes and axis labels indicate chromosome number. Elements in the dot plot represent protein homology translated to genomic coordinates.

We established this relationship using a set of 3,034 predicted orthologues across the three aspergilli, Neurospora crassa and Fusarium graminearum. We constructed trees for 75 randomly selected sets of 20 concatenated genes7, using the N. crassa and F. graminearum genes to root the trees (see Methods). All 75 cases produced the phylogeny shown in Fig. 1a in which A. fumigatus and A. oryzae are sister taxa and A. nidulans branches earlier. This phylogeny is further supported by 86% of trees built for each of the 3,034 orthologues individually. Consistent with this phylogeny, A. fumigatus has over twice as many genes with top Blast hits to A. oryzae than to A. nidulans, and A. oryzae has almost twice as many genes closer to A. fumigatus than A. nidulans. A. nidulans has roughly a similar number of top hits to A. fumigatus and A. oryzae. To confirm further the rooting of the tree, we repeated the analysis using predicted gene fragments (see Methods) from the genome sequence of Coccidioides immitis as an outgroup (which is closer to the aspergilli than N. crassa and F. graminearum). Ninety-four per cent (34 out of 36) of 50-gene phylogenies with C. immitis as the outgroup support the relationship of Fig. 1a, as do 60.8% (93 out of 153) of single gene phylogenies (only 21% support the rDNA phylogeny).

Overall genome and proteome comparison

Although in the same genus, the three aspergilli differ considerably in their genome sequences. Predicted orthologues shared by all three species (three-way orthologues) display an average of only 68% amino acid identity. A. fumigatus and A. oryzae share 70% identity, and each has 66–67% identity with A. nidulans. This protein identity is comparable to that between mammals and fish9, which diverged 450 million years ago. The three species also differ considerably in genome size (Table 1). The largest, A. oryzae (36 Mb), is 31% bigger than the smallest, A. fumigatus (28 Mb), and 24% bigger than A. nidulans (30 Mb). This difference seems to be due to an acquisition of sequence in A. oryzae3 rather than loss in both A. nidulans and A. fumigatus. Finally, the genomes show extensive structural reorganization (Fig. 1b).

Table 1 Comparison of genome characteristics

Conserved synteny and genome evolution

These three aspergilli provide an opportunity to study eukaryotic genome evolution over a divergence approaching the limit of conserved long-range synteny. To characterize pairwise conserved synteny, we used an algorithm based on hierarchical clustering that delineates regions of conserved synteny while also retaining information about internal micro-rearrangements (see Methods). Using this method, the majority (77–79%) of each genome assembly could be mapped to conserved syntenic blocks with at least one other genome (Table 2). Figure 2 shows a projection of the homologous blocks onto the chromosomes of A. nidulans and, contrasted with Fig. 1b, illustrates the considerable extent of conserved synteny despite extensive rearrangement.

Table 2 Characteristics of pairwise conserved synteny
Figure 2: Aspergillus comparative map.
figure2

Conserved synteny between A. nidulans and A. oryzae and A. fumigatus. Syntenic regions are represented by two vertical columns of coloured blocks. The left and right columns represent syntenic blocks to A. fumigatus and A. oryzae, respectively, coloured by chromosome as indicated by the key. Nested blocks show synteny at finer resolutions. Blocks outlined in black are in opposite orientations in A. nidulans relative to those in red. Red blocks in black blocks (and vice versa) represent inversions. The green and purple lines display repeat density (Rep) and G + C content (G + C) in A. nidulans, both in 5-kb windows with increasing values to the left. Black circles represent centromeres.

The results of this analysis reveal two notable trends. First, large regions lacking detectable long-range synteny are readily apparent. As has been observed for mammals, nematodes and yeasts10, repeats and subtelomeric sequences are associated with these heavily rearranged regions. This may have specific implications for fungi, as subtelomeric regions in the aspergilli are enriched for secondary metabolite genes thought to have a role in niche adaptation and virulence. The rapid rearrangement of subtelomeric regions may facilitate the species-specific evolution of these genes (Supplementary Information).

The second notable trend is the distribution of lengths of unbroken regions between micro-rearrangements within pairwise syntenic blocks. The random breakage model of genome evolution predicts that such lengths should be exponentially distributed. Although the mean breakpoint lengths differ, in all three pairwise comparisons the distribution of lengths shows close agreement with the model prediction (Supplementary Information). It thus seems that syntenic blocks, comprising the majority of the Aspergillus chromosomes, are evolving in a manner consistent with random breakage.

For each pairwise comparison, the third Aspergillus genome allows the determination of rearrangements specific to each branch of the unrooted tree (see Methods). The results of this analysis provide a quantitative estimate of the different rearrangements contributing to long-term eukaryotic genome evolution (Fig. 3).

Figure 3: Rates of branch-specific rearrangements.
figure3

a, The rates of different breaks broken down by break type for each branch. Bars represent minimum and maximum values obtained using either of the two non-target genomes as reference (see Methods). b, A stacked plot of the same data showing the relative contribution of break types within each branch for all three branches. See text and Methods for more details.

Structural evolution not correlated with molecular evolution

In vertebrates, nematodes and arthropods, it has been reported that the rates of structural evolution and nucleotide evolution are correlated11,12,13. However, our analysis of the Aspergillus genomes suggests that this expected correlation does not always hold for eukaryotes.

The data in Fig. 3 reveal a considerably higher overall rate of genome reorganization in the lineage of A. oryzae compared to A. fumigatus. Nearly all categories of disruption are at least twofold greater in A. oryzae relative to A. fumigatus. For example, A. oryzae displays a more than twofold greater rate of insertion than A. fumigatus. This is consistent with the larger genome size of A. oryzae3. Surprisingly, our analysis also indicates that chromosomal breaks are more common in A. oryzae than A. fumigatus. Although apparent intrachromosomal rearrangements could arise from successive inversion events, this cannot explain interchromosomal rearrangements. These interchromosomal breaks are also not the result of assembly error, as confirmed by optical mapping3 and polymerase chain reaction (PCR) validation of eight predicted interchromosomal breaks.

In contrast, several measures indicate that the rates of amino acid evolution in predicted orthologues are similar between these two species. An examination of predicted three-way orthologues shows that the distribution of amino acid identity is roughly similar for both A. oryzae and A. fumigatus compared to A. nidulans, as are non-synonymous divergences (Supplementary Information). In addition, branch lengths predicted from phylogenetic trees (see above) indicate a comparable rate of substitution for both A. oryzae and A. fumigatus. Taken together, these data lead to the conclusion that structural and molecular evolution in the aspergilli is not correlated. A similar conclusion has been reached in the analysis of two microsporidian genomes, although in this case gene evolution seems to be accelerated relative to genome rearrangement14. Thus, large-scale and small-scale evolutionary processes in eukaryotes can operate at different relative rates in a species-specific manner.

Sex and the evolution of the mating-type loci

Unlike A. nidulans, which has a known sexual cycle, A. fumigatus and A. oryzae are only known to reproduce through asexual mitotic spores. We sought insight into the evolution of this apparent difference by comparing the three genomes. Our results, in conjunction with an accompanying paper and another study2,15, suggest that both A. fumigatus and A. oryzae may be capable of sexual reproduction.

Sexual reproduction in ascomycete filamentous fungi is governed, in part, by two different mating-type genes that establish sexual compatibility: one gene encodes a protein with a high mobility group (HMG) domain, whereas the other encodes a protein with an alpha box domain. We refer to these genes here as the HMG and alpha mating-type genes, and to their chromosomal locations as MAT loci. Homothallic fungi typically possess both mating-type genes and are self-fertile. Heterothallic fungi possess only one mating-type gene and require a partner with a different mating-type gene. In heterothallics, the two mating-type genes typically occupy the same chromosomal location in different haploid genomes but lack sequence similarity, and are thus termed idiomorphs rather than alleles16.

A. nidulans is known to be homothallic, and both HMG and alpha mating-type genes have been identified17,18. Our analysis confirmed that the HMG and alpha loci are unlinked, which is unusual although not unprecedented in homothallic fungi19. We identified a single HMG mating-type gene in A. fumigatus, as previously reported20, and a single alpha mating-type gene in A. oryzae.

A comparison of all four MAT loci revealed extensive conserved synteny (Fig. 4a). The A. oryzae alpha locus and the A. fumigatus HMG locus display conserved synteny over 1.7 Mb on either side of the mating-type genes. Within this region of conserved synteny, the two mating-type genes occupy nearly identical positions, although offset with different orientations. Furthermore, one flank of both the A. fumigatus and A. oryzae loci is syntenic with 409 kb of the A. nidulans HMG locus downstream region, whereas the other flank is syntenic with 34 kb of the A. nidulans alpha locus downstream region (Fig. 4a). The four loci also show conservation of a number of genes associated with MAT loci in other species including N. crassa or one of nine yeast species previously analysed21.

Figure 4: Comparison and evolutionary model of Aspergillus MAT loci.
figure4

a, Conserved synteny between loci. Grey lines indicate predicted orthologues. Red genes indicate orthologues from the left flank (as drawn) of the A. nidulans alpha locus with the left flanks of the A. fumigatus and A. oryzae loci. Cyan genes indicate orthologues with the right flank of the A. nidulans HMG locus. The bottom panel shows the region near mating-type genes. Genes labelled and outlined in black are associated with MAT loci in other fungi. Only partial accession numbers (suffixes) are shown in the figure. For full accession numbers, the numbers shown in the panel should replace the asterisks in the following examples: A. nidulans (AN****.1); A. fumigatus (59.m0****); A. oryzae (AO0703270000**). b, Model of structural evolution of the MAT loci. Braces represent multiple haplotypes at the same genomic locus. The experimental identification of other isolates (indicated by an asterisk) was reported in another study15. The light blue arrow indicates a 360-bp HMG gene fragment. AF, A. fumigatus; AN, A. nidulans; AO, A. oryzae.

Extending the analysis to 215 genes implicated in the fungal mating process, pheromone response, meiosis and fruiting body development revealed that every gene (except for the mating-type genes) that can be identified in A. nidulans is also present in both A. fumigatus and A. oryzae (Supplementary Information), including several genes for which the only known function is related to sexual reproduction.

Although sexual reproduction may have been lost very recently in both A. fumigatus and A. oryzae, providing one explanation for the residual presence of mating process genes, these data suggested the possibility that both A. fumigatus and A. oryzae may be capable of sexual reproduction. Moreover, the pattern of synteny among the four MAT loci leads to an evolutionary scenario for this hypothesis, as shown in Fig. 4b. According to this model, it is predicted that A. oryzae and A. fumigatus isolates exist with the opposite mating-type genes to those present in the strains that were sequenced. In addition, these opposite mating-type genes should be present at the identical locus, consistent with a heterothallic idiomorphic configuration.

As reported in detail in another study15, these predictions have been experimentally verified. Using a PCR-based multiplex mating-type assay, isolates of both mating types of A. fumigatus and A. oryzae were identified. For both species, the opposite MAT locus from the complete genome was sequenced and demonstrated to have the idiomorphic organization predicted. Within the idiomorphic region the opposite mating-type genes appear to be offset with respect to one another, as predicted by our model. In addition, the A. fumigatus alpha MAT locus was found to contain a 360-base pair (bp) fragment of an HMG gene15 neighbouring the idiomorphic region, suggesting that the transition from homothallism to heterothallism in the A. oryzae and A. fumigatus ancestor occurred by gene loss (Fig. 4b).

Although the model of Fig. 4b predicts a homothallic ancestor for all three species, it is possible that heterothallism was ancestral and a transition to homothallism occurred in the A. nidulans lineage. This would be consistent with data from Cochliobolus species for which heterothallism appears to be ancestral, and conversions to homothallism have been described19. However, two factors conflict with this scenario for the aspergilli. First, the offset positions of the mating-type genes within the idiomorphic regions of the A. fumigatus and A. oryzae MAT loci, and the apparent fragment of the HMG gene neighbouring the A. fumigatus alpha locus, are consistent with gene loss from a homothallic ancestor. Second, heterothallism in the aspergilli is rare22,23. Only three heterothallic aspergilli have been previously characterized, of which one, A. heterothallicus, groups in phylogenies with known homothallic species, suggesting a conversion to heterothallism in this case as well22. Mitotic, homothallic and heterothallic species are observed intermixed in several fungal lineages, leading to debates about the fungal ancestral state19,24,25,26. Taken together, our results provide evidence that conversion from homothallism to heterothallism is possible, and suggest that the predominance of a particular sexual strategy may vary within different clades.

Although the finding of MAT genes in supposedly asexual fungi has been previously reported15,27,28,29,30,31,32 and genes related to sexual reproduction have been found in the ‘asexual’ yeast Candida albicans, this report is the first comprehensive survey of sexual reproduction genes in two different filamentous fungi thought to be asexual. In addition, our results provide an experimentally supported evolutionary model associating large-scale synteny and genome rearrangement with a specific and significant difference in biology between these aspergilli. These results for A. fumigatus and A. oryzae have important and specific potential implications for health and industry. The lack of a sexual cycle in A. fumigatus and A. oryzae has precluded classical genetic analysis, impeding efforts to study these organisms and necessitating the use of the relatively distant A. nidulans as a genetic model. The possibility for mating—still speculative at this stage—raises the medically and industrially important potential for developing genetic tools for these fungi.

Conserved non-coding sequences

Detecting and characterizing conservation of sequences outside of protein coding regions is a promising method for identifying potential functional elements. Regulation in yeast has been extensively studied; however, in the aspergilli few transcription factor binding sites have been experimentally verified. Comparing the three aspergilli provides an opportunity to identify the most constrained functional elements.

To do so we aligned three-way orthologous genes including 1 kb of sequence upstream and downstream using Mlagan33. Strict filters were then applied to delineate unambiguous orthologous intergenic regions (see Methods). Given the divergence of the aspergilli, it is expected that intergenic regions would not show significant conservation, and frequently this was found to be the case. However, in many instances, blocks of nearly perfect three-way conservation are observed. An example for one intergenic region is shown in Fig. 5a.

Figure 5: Active conservation of non-coding regions.
figure5

a, Example region between a conserved pair of orthologous histone H2A and H2B genes (left and right blue arrows). The three lines from top to bottom correspond to the sequences of A. nidulans, A. fumigatus and A. oryzae aligned using Mlagan. Letters on the red background indicate 100% conserved bases. b, Conservation scores of maximal subsequences for observed intergenic alignments (red), and models of neutral and random sequence alignment (both fixed and variable length).

To assess which regions were conserved owing to purifying selection rather than neutral mutation or chance, we developed models for alignments of neutral and random sequence. Unlike mammals, where ancient conserved repeats provide a natural model for neutral evolution, few such repeats exist in the aspergilli (see below). Instead, we synthesized alignments of neutral sequence by concatenating randomly selected aligned columns of fourfold degenerate sites. We also controlled for chance alignment introduced by potential aligner bias by aligning randomly selected intergenic regions. Using a simple conservation scoring function we calculated maximal scoring subsequences and compared results between orthologous regions and the control models (Fig. 5b). A noteworthy aspect of the data in Fig. 5b is the similarity between the neutral and random models. According to our models, neutral sequence is effectively saturated for mutation, confirmed by an independent analysis of synonymous sites in protein coding sequences.

Comparing results between real and control alignments, we selected a minimum score of 22 for regions unlikely (P < 0.015) to be conserved by neutral evolution or chance. We denote a subsequence scoring above this threshold as a high-scoring conserved sequence (HCS). On the basis of this analysis, we predict 5,801 HCSs corresponding to 2% of alignable orthologous intergenic regions.

Prediction of functional motifs

We expect HCSs to be enriched for functional elements. The challenge is to discover these functional elements and make testable predictions about their biological functions. In preliminary analyses, several conserved regions could be identified as known functional elements. For example, we observed conservation delimiting a known 3′ untranslated region (UTR) element of the A. nidulans areA gene that regulates messenger RNA stability in response to cellular nitrogen levels34. We also identified three TPP binding riboswitches, one of which has not been described in Aspergillus (Supplementary Information).

To enrich computationally HCSs for sequences corresponding to functional elements, and to derive clues about their biological functions, we modified the approach used by ref. 35 (see Methods). Briefly, we identified common subsequences (or ‘patterns’) that appeared in at least four HCSs across all three Aspergillus genomes. These patterns were searched in three-way conserved orthologues to identify genes in which the subsequence occurred in the 500-bp upstream or downstream regions (a ‘co-occurrence’). A number of conservation criteria were then applied (see Methods). We identified a total of 69 conserved patterns (‘conpats’), occurring in at least four HCSs, that showed enrichment for co-occurrences and exhibited a bias for occurring 500 bp upstream or downstream of genes.

The results of this analysis for the 35 most common patterns are shown in Fig. 6 (all 69 patterns available in Supplementary Information). These include motifs that match known or predicted Aspergillus or other fungal functional sequences. For example, CPCA/GCN4, the master regulator of the cross pathway control system in fungi, is known to bind to the palindromic site TGASTCA36. In yeast, microarray studies have identified 539 genes probably regulated by GCN4 that show a preference for amino acid biosynthetic genes and several ribosomal proteins and translation factors37. One of the patterns identified by our analysis (ID 2483) matched the CPCA binding site, co-occurred preferentially upstream of genes, and was enriched in genes associated with amino acid transport and metabolism (COG category E), and translation, ribosomal structure and biogenesis (category J). Furthermore, the 19 genes with co-occurrences of this pattern include 7 (37%) predicted orthologues to the 539 known yeast regulated genes, representing a sixfold enrichment (P-value < 1 × 10-5). This includes two known A. nidulans CPCA regulated genes (trpB38 and hisHF39). This pattern probably corresponds to the known Aspergillus CPCA binding site.

Figure 6: Selected conserved patterns.
figure6

Column one shows the conpat unique ID. Column two shows the sequence logo representation of conpat weight matrix. Column three shows fungal binding factors with sequence similarity to the conpat. Columns four and five show the number of genes with a co-occurrence of conpat upstream and downstream. Column six shows the preference for co-occurring preferentially 5′ or 3′ of the gene. Column seven shows the fraction of co-occurrences overlapping three-way conserved regions. Column eight shows the preference for co-occurring on a particular strand relative to the gene. Column nine shows COG categories showing significant enrichment (the number of genes with co-occurring conpats in the category is indicated in parentheses). Enrichment results for yeast orthologue cellular location are available in the Supplementary Information. Single asterisk, P < 1 × 10-3; double asterisk, P < 1 × 10-4; triple asterisk, P < 1 × 10-5. bHLH, basic helix-loop-helix.

A second pattern shows strong correspondence with the binding site for Puf family genes40. Puf proteins regulate mRNA translation and mRNA decay through interactions with 3′ UTR sequences. In Saccharomyces cerevisiae, which has five Puf genes, Puf3p has been shown to bind specifically mRNAs encoding mitochondrial proteins40 and requires a 3′ UTR motif with consensus UGUANAUA40. Four different patterns identified by our analysis (ID 1710, 2077, 1144 and 2378; see the full table in Supplementary Information) match or include the Puf binding motif and display a strong downstream bias. Three also show enrichment for predicted orthologues in S. cerevisiae that localize to mitochondria. Taking all four patterns together, we find a 6.8-fold enrichment (P < 4.3 × 10-11) for genes with orthologues to yeast mitochondrial genes. In addition, we find a threefold enrichment (P < 0.0003) for genes with yeast orthologues predicted to be bound by Puf genes in a genome-wide affinity tag analysis40. Although a functional role for the Puf family has not been experimentally demonstrated in Aspergillus, all three genomes possess five loci with 5–8 Puf domains, as predicted by HMMER and PFAM (including one with a predicted RNA binding domain, as with Puf1p and Puf2p in yeast). Together these data suggest that, as in yeast, Puf genes may bind to and regulate mitochondrial mRNAs in the aspergilli.

Only a small number of transcription factor binding sites and control elements are known for filamentous fungi in general, including Aspergillus. These predicted patterns are thus promising targets for future experimental validation.

Regulatory upstream open reading frames

A significant proportion (32%) of HCSs are conservatively predicted to lie within transcribed but untranslated regions of genes (UTRs), consistent with the known role of UTRs in regulating gene expression, particularly mRNA translation (for example, Puf binding domains). One important class of translational control elements is short upstream open reading frames (uORFs) in 5′ UTRs41, which can regulate the expression of downstream protein-coding genes in several ways. First, they can modulate the efficiency of ribosome re-initiation at downstream start codons in a manner dependent on cellular state. Second, uORFs can produce cis-acting peptides that stall ribosomes. Finally, the presence of uORFs can affect mRNA stability. Functional uORFs can be as short as three amino acids and occur at varying distances and multiplicity upstream of the protein-coding gene, occasionally overlapping the downstream start codon. Functional uORFs have been reported in a range of species including plants, animals and fungi41. In Aspergillus, a small number of genes with validated uORFs have been reported (Supplementary Information). To determine the full extent with which they may regulate gene expression, we analysed the three Aspergillus genomes for uORFs.

We identified UTR sequences using expressed sequence tag (EST) alignments for A. nidulans genes, and examined them for open reading frames (see Methods). Of 1,606 genes with identified UTRs, 21% (358) have upstream ORFs. A similar proportion was found (18% or 82 out of 463) when we restricted our analysis to three-way orthologues for which the start codons align exactly within multiple alignments, suggesting that these uORFs are not due to mis-annotation. A corresponding analysis of genes with ESTs in the N. crassa, F. graminearum and Magnaporthe grisea genomes found uORFs associated with 22%, 10% and 16% of genes, respectively. We further extended the analysis in A. nidulans using a conservative estimate of 5′ UTR length (see Methods), and identified an additional 958 genes with potential uORFs of which 165 genes have three-way orthologues. In total, 1,316 genes in A. nidulans are predicted to possess uORFs.

Not all identified uORFs have a detectable impact on gene expression41. To enrich the set of predicted uORFs for those likely to be functional, we looked for those conserved in all three aspergilli. On the basis of a strict criterion requiring alignment of the uORF start and stop codons, we find 38 conserved uORFs (13% of 331 genes with uORFs and predicted orthologues) (Supplementary Information). Of these corresponding Aspergillus genes, 14 have predicted uORFs upstream of orthologues in N. crassa, F. graminearum or M. grisea. Additionally, three also have predicted orthologues in S. cerevisiae with uORFs conserved across four related yeast species.

These 38 conserved uORFs represent strong candidates for experimental investigation. As a preliminary validation, we tested two novel conserved uORFs for their ability to modulate protein synthesis in vitro (Fig. 7). Briefly, oligonucleotides containing each uORF were fused to a luciferase reporter gene, and controls were constructed with disabled uORF and/or reporter gene start codons. Differential expression in a cell-free translation system between intact and control constructs measures the impact of the uORF on translation. This system can detect small (twofold) changes in translation, and can discriminate uORFs that do not reduce translation in vivo from those that do (see Methods). As can be seen in Fig. 7, both uORFs tested display a 5–10-fold repressive effect on the translation of the downstream reporter gene.

Figure 7: Prediction and validation of conserved uORFs.
figure7

Conserved uORFs show a 5–10 times repressive effect on reporter gene translation. a, Alignments of two tested uORFs. uORFs are shown in purple boxes, and protein-coding genes in a blue background. Conserved bases are in upper case and start/stop codons are highlighted. b, Experimental design. The A. nidulans sequence from 26 nucleotides upstream of each uORF to four codons in the protein-coding gene was fused with a firefly luciferase gene. Controls were generated with start codons for both the uORF and the luciferase gene (+/+), the luciferase gene only (-/+), and the uORF only (+/-). Starts were deleted by alteration to ATT. mRNA from each construct was used to programme a cell-free translation system. c, Results of translation assays. The luciferase activity of all constructs (normalized to the -/+ construct) is shown on the y axis. Error bars show the average of the absolute deviation from the mean. The autoradiogram shows 35S-Met-labelled firefly luciferase obtained by in vitro translation of the same mRNAs.

These results provide the first genome-wide list of predicted conserved uORFs for any organism, and suggest that uORFs could have a substantial role in regulating gene expression in Aspergillus. Previous reports estimate that 2–4% of genes in S. cerevisiae contain uORFs42, whereas a review of sequences in UTRdb predicted that 5–10% of eukaryotic UTRs contain ORFs43. Our results predict that in filamentous fungi the proportion may be twice as high.

Aspergillus physiology

Peroxisomes are organelles containing enzymes for the breakdown of fatty acids (β-oxidation), removal of hydrogen peroxide and synthesis of cholesterol and bile acids. Peroxisomes have critical roles in fungi where they are involved in growth, secondary metabolism and pathogenesis. In mammals, defects can lead to developmental and neurological disorders. Proteins are targeted to the peroxisome either by a carboxy-terminal tripeptide sequence or an amino-terminal nine-amino-acid sequence. Using these signals we predicted peroxisomal proteins in Aspergillus (Supplementary Information). Our analysis reveals peroxisomes in Aspergillus to be more similar to mammals than yeasts in two respects. First, our data suggest that the aspergilli, like mammalian cells, perform β-oxidation in both peroxisomes and mitochondria and possess two sets of genes for all β-oxidation enzymes targeted to both the mitochondria and the peroxisome, as supported by recent experimental results44. In contrast, S. cerevisiae metabolizes fatty acids fully to acetyl-CoA only in peroxisomes45. Second, Aspergillus peroxisomes are more similar to those of mammals than those of yeasts in that they possess putative peroxisomal acyl-CoA dehydrogenases. In addition, all three aspergilli appear to encode both mitochondrial and peroxisome forms of an ATP-dependent protease of the LON (La domain) family associated with peroxisomes in mammals46. S. cerevisiae has a single copy of this protease (Pim1/Lon1) targeted to mitochondria47.

One of the hallmarks of the filamentous fungi is their ability to undergo polarized hyphal growth. This requires positional cues that mark polarized growth sites, locally activating Rho-related GTPase signalling modules that promote cytoskeletal reorganization48. The three aspergilli possess the expected genes involved in signalling and cytoskeletal organization for polarized growth, but there is a marked lack of known positional markers (such as the yeast bud site markers Bud3p, Bud8p and Bud9p; see Supplementary Information). Proteins implicated in the transport or modification of bud markers, including Axl1p, Rax1p and Bud7p, were predicted, however. This suggests that filamentous fungi mark polarized growth sites with positional cues, but that the markers themselves may consist of novel cell wall proteins.

Most of the interspersed repeats in all three genome sequences correspond to relics of transposable elements (see Supplementary Information). Surprisingly, only 1.3% of the largest genome assembly of A. oryzae consists of transposable elements, compared to 3% of A. nidulans and A. fumigatus. All three genomes contain essentially all major classes of eukaryotic transposable elements, although the overall variety is relatively low. A number of unusual features were also observed in Aspergillus transposable elements (Supplementary Information). Copy numbers per family range from 1 to 100; there are also many fragments and ‘footprints’ (evidence of recombinational excision). In addition, some members of all transposable element families longer than 400 nucleotides are characterized by numerous C to T transitions. In A. nidulans and A. fumigatus, these predominantly affect cytosines in CpG and CpA doublets (no preference is apparent in A. oryzae). Moreover, repeat density is correlated with A + T richness in all three species (Fig. 2). The predominance of transition mutations is consistent with the operation of RIP (repeat-induced point mutation)49, and all three aspergilli have a single predicted homologue (called DmtA) to the DNA methyltransferase rid that is essential for RIP in N. crassa49. Apart from the putative rid homologue, no additional DNA methyltransferase genes were identified, consistent with failures to demonstrate methylation in these fungi. Although RIP has not been demonstrated in any Aspergillus species, if active it may be more similar to the mild form in M. grisea49, as many transposable elements in these species are mutation-free.

Conclusion and perspective

The A. nidulans genome sequence and our comparative analysis with the genome sequences of A. fumigatus and A. oryzae have shed new light on the physiology of these fungi, as well as insight into aspects of genome evolution and gene regulation likely to be common to all eukaryotes. These results represent the initial step in realizing the full potential of these genomes. As a result of the genome analysis, efforts are underway to cross different isolates of A. fumigatus and A. oryzae. The identified conserved sequences also represent a rich set of targets for further experimental investigation. These efforts and ongoing sequencing projects for additional aspergilli promise to change fundamentally our understanding of this important group of medically, industrially and scientifically relevant fungi.

Methods

Complete details of the methods used are available in Supplementary Information.

A. nidulans sequencing, assembly and analysis

The A. nidulans genome strain FGSC A4 was sequenced by the WGS method to a depth of 10 × . An additional 3 × sequence coverage was provided by Monsanto (http://www.monsanto.com/). All sequence was assembled using Arachne. The A. nidulans genome was annotated as described in Supplementary Information. A. fumigatus and A. oryzae were assembled and annotated as described separately2,3.

Phylogenetic analysis

A total of 3,034 predicted orthologues among A. nidulans, A. oryzae, A. fumigatus, N. crassa and F. graminearum were aligned at the protein level, back translated to DNA codons, and large gaps (> 9 bp) were removed. Random sets of 20 DNA alignments were concatenated and passed to Phylip to generate 100 bootstrap replicates and a consensus maximum parsimony tree. Maximum likelihood trees were calculated on each replicate and a consensus tree was produced. Repeating with 1,000 bootstrap samples led to essentially identical results. For rooting with C. immitis, C. immitis orthologous CDS regions based on TBlastN were retrieved, translated and aligned at the protein level with the aligned portions of the Aspergillus genes. Maximum parsimony trees were then generated and filtered for those with 100% bootstrap values at all nodes. The C. immitis sequence is available at http://www.broad.mit.edu/annotation/fungi/coccidioides_immitis/.

Hierarchical synteny mapping and branch-specific rearrangements

Protein homology anchors were detected using BlastP and filtered to retain only hits scoring >80% of the score of the best hit to each query protein. Contiguous sets of homologous proteins with conserved order and orientation were grouped into clusters. Pairs of clusters were then merged into successively larger clusters by tolerating successively larger breaks between clusters. Branch-specific breaks were determined by identifying regions without breaks between a reference species and query species, and then identifying breaks in that region between the reference and the third (target) species. Such breaks were considered specific to the third species. Breaks were classified according to the pattern of apparent rearrangement.

Identification of non-coding conserved sequences

Genomic sequence for predicted three-way orthologues, including 1 kb upstream and downstream, were multiply aligned using Mlagan33. An additive scoring function was used to identify maximal scoring subsequences. A cutoff of 22 was used to define HCSs unlikely to occur by chance (P < 0.015) according to models of neutral sequence and random sequence alignment. To model alignments of neutrally evolving sequence, aligned columns of fourfold degenerate sites were selected randomly and concatenated. To model alignments of random sequence, randomly selected intergenic regions from each genome were aligned. Fixed and variable length alignments were generated for both models. For each model, 1,000 simulated alignments were generated and maximal scoring subsequences were identified. The number of subsequences for each score was normalized by the number of aligned nucleotides. These rates were used to determine the score cutoff above.

Prediction of functional motifs

Each HCS was represented as a position-specific probability matrix (PSPM) derived from the three-way alignment. Each PSPM was compared to each other PSPM and matching PSPMs were clustered. Local multiple alignments for each cluster were generated and the resulting multiple alignments and corresponding weight matrices were termed conpats. For each conpat, we used the corresponding weight matrix with MAST to identify instances where the conpat co-occurred upstream or downstream of orthologous genes in all three of the aspergilli. A series of conservation tests was then applied to the set of predicted co-occurrences for each conpat as described in Supplementary Information.

Prediction and validation of upstream open reading frames

Genome sequences for three-way orthologues, including 1,000 bp upstream and downstream, were multiply aligned using Mlagan. For 25% of A. nidulans genes 5′ UTR sequences were predicted from EST alignments. On the basis of the length distribution of these EST-predicted UTRs, we used 60 bp upstream of predicted AUG codons as a conservative estimate of 5′ UTRs for genes lacking ESTs. When no ESTs were available, UTRs for orthologues where considered when all three annotated start codons aligned within 40 bp. We identified uORFs ≥12 bp, with a maximum 1-bp overlap with the protein-coding gene's ATG. Conserved uORFs were identified as those for which the start and stop codons were exactly aligned within the multiple alignments. To experimentally validate uORFs, synthetic oligonucleotides containing each uORF, 26 nucleotides upstream of the uORF, the region between the uORF and the protein coding AUG, and the first four codons of the protein coding gene were fused in frame with a firefly luciferase gene. Three different control constructs were also generated. Capped and polyadenylated synthetic mRNA were prepared from each construct and equal amounts were used to programme cell-free extracts from N. crassa. Differential translation between the intact construct and the controls was measured using a luciferase activity assay as well as through the production of 35S-Met-labelled firefly luciferase obtained by in vitro translation of the same mRNAs.

The A. nidulans genome sequence is available at http://www.broad.mit.edu/ and has been deposited at DDBJ/EMBL/GenBank under the project accession AACD00000000.

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Acknowledgements

The authors would like to thank M. Zody, X. Xie, M. Kellis, M. Kamal, J. Taylor, G. Turgeon and E. Lander for numerous helpful conversations, comments and critical readings of the paper. We also thank all members of the sequencing platform at the Broad Institute. We thank R. Dean for providing the BAC library used in sequencing. We especially thank M. Brudno for his help in using Mlagan to align the three genomes, and R. Morris for his many contributions to the A. nidulans sequencing project and subsequent analyses. This work was supported in part by grants from the NIH, as well as the NIH Research Supplement for Underrepresented Minorities, the DFG, the CMPB and the BBSRC. Author Contributions B.W.B. developed and led the A. nidulans sequencing project. J.B. performed the assembly of A. nidulans. C.C. and J.E.G. analysed the phylogenic relationship of the three organisms. J.E.G. performed the analysis of conserved synteny and genome evolution. R.F., B.M. and P.D. performed the comparative analysis of sexual reproduction genes. J.E.G., M.P., P.D. and B.M. analysed the mating-type loci. S.E.C. and J.E.G. analysed conserved non-coding sequences and computationally analysed uORFs. S. Batzoglou and S.-I.L. assisted with the use of Mlagan. M.B., C.C.S. and M.S.S. performed the experimental uORF validation. M.C., M.H., G.H.B., O.D. and C.D. analysed transcription, known transcription factors and binding sites. A.P. and S.G.J. predicted non-coding RNAs using PFAM and RFAM. J.C., V.K., J.J., S.P. and J.E.G. analysed repeat sequences and RIP. M. Farman analysed telomeres and subtelomeric gene content. M.H. analysed the Aspergillus peroxisomes. S.H. and M. Momany analysed hyphal growth and RhoGTPases. J.R.W. and W.C.N. provided the sequence and annotation for A. fumigatus. T.T., T.K., K.A. and M. Machida provided the sequence and annotation for A. oryzae. D.W.D. co-ordinated interactions between the different sequencing centres and scientific communities. J.E.G. and S.A.O. coordinated the comparative analyses. J.E.G. wrote and edited the paper, and produced the figures.

Author information

Correspondence to James E. Galagan.

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The A. nidulans genome sequence has been deposited at DDBJ/EMBL/GenBank under the project accession AACD00000000. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Detailed descriptions of methods and materials (DOC 101 kb)

Supplementary Data

The data and description of methods used to derive the phylogenetic relationship of the the three Aspergilli (XLS 1353 kb)

Supplementary Data

Description of Hierarchical Synteny Clustering method, detailed discussion of sytenic breaks near to repeats and telomeres, description of fit of genome breakage to random break model, data supporting lack of decelerated evolution in A. oryzae, and data supporting similar rates of protein evolution in A. oryzae and A. fumigatus (DOC 537 kb)

Supplementary Data

Description of methods for uORF comparative analysis. Raw data with all uORFs and conserved uORFs. (XLS 7577 kb)

Supplementary Data

Description of methods used for prediction of Non-coding conserved patterns. List of all patterns, locations of patterns, and known Aspergillus and yeast transcription factor binding sites. (XLS 1619 kb)

Supplementary Data

Description and table summarizing transposable elements in the three Aspergilli (DOC 39 kb)

Supplementary Data

Description of A. nidulans assembly and integration with the genetic map. (DOC 305 kb)

Supplementary Data

Comparative analysis of Aspergillus peroxisomes (DOC 40 kb)

Supplementary Data

Comparative analysis of hyphal growth (DOC 86 kb)

Supplementary Data

Description of A. nidulans gene prediction methodology and gene accuracy statistics. (DOC 49 kb)

Supplementary Data

215 genes implicated in sex in fungi and their presence in all three Aspergilli (XLS 54 kb)

Supplementary Data

Description of predicted TPP Riboswitches (DOC 48 kb)

Supplementary Figure

High resolution version of Figure 2 (PDF 497 kb)

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