EAS Cluster Evolution

In the study of the taxonomic distribution of the eas genes in epichloë strains, the cluster was found to be either completely absent or present. This pattern was also observed over a broad selection of strains tested for the presence of lpsB and lpsA, where neither gene was found without the other (Johnson et al., 2007b). The presence or absence of the cluster is strain- rather than species-dependent, indicating that the cluster has been frequently lost during evolution. This result was not expected as a similar, larger analysis with ltm genes showed that some strains contained different mixtures of genes, as well as several in which the whole cluster was present or absent (Young, 2005). The LTM cluster 3 in particular (ltmE and ltmJ) was frequently absent with the other two clusters remaining. Similarly, in fusaria different species contain different tri gene sets for tricothecene synthesis (Kimura et al., 1998a; Quarta et al., 2006). With both the ltm and tri genes the deletion of different genes can lead to a different chemotype, possibly with different bioactivity, for the producing organism, which may retain or give a new selective advantage in a given niche. Different ergot alkaloids have been shown to have different biological effects, albeit in a laboratory setting. Ergopeptines were shown to be active against black beetle (Ball et al., 1997), fall armyworm (Clay and Cheplick, 1989), mammals (Panaccione et al., 2006) and against the nematode Pratylenchus scribneri (Panaccione, 2005), while clavines are more cytostatic and active against bacteria (Panaccione, 2005) but also with some effects on mammals (Panaccione et al., 2006). If activity against insects and nematodes were most important for plant-epichloë symbiota, as intuitively may be expected, there would be selection pressure to keep the entire gene cluster to enable the production of ergovaline. In ecological niches where insect or nematode pressure is low, or where there is redundancy from other alkaloids for protection against a given herbivore, as work in this study may indicate (discussed in section 7.1), selection would be lost for the entire cluster, perhaps explaining its frequent loss. Further analysis of epichloë strains deleted in different eas genes is required to determine potential ecological roles for the various alkaloids which would shed further light on this question.

At the family level, the known ability to produce ergot alkaloids is restricted to some members of the Clavicipitaceae in the order Hypocreales and to some Penicillium species and A. fumigatus from the quite distant order Eurotiales (Schardl et al., 2006). Why are genes for ergot alkaloid synthesis found so discontinuously in the fungal tree of life? There are two possibilities. One is that the gene cluster was present in a very early shared ancestor of Hypocreales and Eurotiales and has been frequently lost throughout ascomycete evolution. The second is that the cluster was horizontally transferred from one order to the other. Panaccione (2005) states that the codon usage and GC content of the C. purpurea and A. fumigatus EAS clusters are not consistent with a horizontal transfer event. The former thus seems more likely, although the conservation of gene order between the shared genes in the A. fumigatus cluster and the N. lolii EAS cluster 1 may provide some support for a transfer event, given both clusters are found in what are likely to be dynamic regions of the genome.

Both the N. lolii and A. fumigatus gene clusters are located in regions linked to the telomere – a common location for biosynthetic gene clusters as detected by fungal genome sequencing projects (Galagan et al., 2005; Nierman et al., 2005; Rehmeyer et al., 2006). Other genes with “disposable” functions are often found subtelomerically, including gene families involved in antigenic variation in Plasmodium (Gardner et al., 2002) and Trypanosoma spp. (Horn and Barry, 2005), and adhesin variability in C. glabrata (Castano et al., 2005) and S. cerevisiae (Halme et al., 2004). Subtelomeric regions of the genome evolve more rapidly than other regions of the chromosome, show high rates of interchromosomal recombination and gene duplication, and are frequently sites for relocation of one copy of a sequence duplicated elsewhere in the genome (Kellis et al., 2003). The abundance of transposon relic sequences adds to the evolutionary potential of the eas cluster. In Magnaporthe oryzae transposable element clusters correlate with increased recombination rate, loss of synteny, gene duplication and sequence divergence from orthologous genes (Thon et al., 2006). Adaptive gene relocation to a

Although it is not known whether this is a general mode of gene cluster evolution, the subtelomeric location of the EAS cluster and many other fungal secondary metabolite biosynthetic gene clusters supports this hypothesis. Adaptive gene relocation is an attractive mechanism to explain different biosynthetic capabilities associated with the evolution of the ergopeptine-producing (contain lpsA, lpsB and cloA), and clavine-producing (contain easI – easN) fungi, subsequent to either the divergence of these lineages or transfer of the cluster.

Analysis of the EAS gene homologues in sequenced aspergillus genomes revealed that the cluster was possibly subject to an ancient duplication. Within the A. fumigatus genome, the gene cluster for fumitremorgen synthesis contains four genes homologous to genes in the EAS cluster. The similarity of the two clusters is highlighted by the FTM cluster being mistakenly labelled as an ergot alkaloid biosynthetic cluster previous to functional analysis (Sheppard et al., 2005). Both of the clusters are found very near to an identical LINE element and to telomeres, perhaps indicating that the clusters arose from telomere recombination. If the EAS cluster is found in distant families due to gene loss in other organisms then it seems likely that it was an EAS cluster that was duplicated and that the duplicated cluster was neo-functionalised in A. fumigatus to synthesise fumitremorgens. In A. nidulans both clusters appear to have diverged and functional analysis is required to determine the product of these clusters. It seems likely that the progenitor EAS cluster contained an NRPS gene as each of the EAS-like clusters, barring the EAS cluster itself, have an associated NRPS; as of course do the clavicipitacean EAS clusters. Further support for this is that of all apergillus NRPS modules, the C. purpurea LpsB enzyme (and by extension the N. lolii enzyme) is most closely related to the first module of the FtmA protein found in the fumitremorgen cluster (Cramer et al., 2006). A full analysis of the EAS genes within the aspergilli and other sequenced fungi was outside the scope of this thesis. Future analysis though seems likely to yield further interesting insights into EAS cluster evolution.