An analysis of ABC toxin complex genes in fungi and the characterisation of EAS and IDT gene clusters in Metarhizium robertsii.

Abstract

Metarhizium robertsii is a fungus that straddles several ecological roles in the soil. It is a well-characterised generalist entomopathogen, capable of infecting a wide range of insects, but also associates with plant roots in the rhizosphere and as an endophyte. Recent sequencing of the M. robertsii genome revealed a range of uncharacterised genes with potential roles in insect or plant interaction. These include putative genes for an ABC toxin complex and ergot alkaloid (EAS) and indole diterpene (IDT) biosynthesis pathways, which are the subjects of this thesis. Putative ABC toxin complex genes in a range of fungi were identified. These proteins had previously only been characterised in bacteria so to investigate the possibility of horizontal transfer of these genes into fungi, detailed phylogenetic analysis was performed on a set of 69 bacterial and fungal ABC toxin complex amino acid sequences. This showed admixture of the Tc genes between different bacterial phyla, indicating frequent horizontal transfer events (HTEs) between bacteria. Counter to the species phylogeny, the fungal ABC toxin complex sequences clustered together in a clade with bacterial sequences, supporting the hypothesis that fungal ABC complex genes derive from interkingdom horizontal transfer. Specifically, fungal sequences co-clade together with sequences from actinobacteria, cyanobacteria, bacteroidete, and deltaprotoebacteria. Therefore, it is proposed that the fungal ABC toxin complex genes have a non-gammaproteobacterial origin. Many of the fungi possessing these genes associate with plants, suggesting the ABC toxin complex may have been repurposed to deliver plant effectors into plant cells, avoiding the plant pathogen triggered immunity (PTI) response. Initially it was aimed to test this hypothesis by functional analysis of the M. robertsii TcB/C component gene, abcB. However, expression and bioinformatics analysis determined that it was a non-expressed pseudogene. Identification of EAS and IDT genes in M. robertsii showed that it has conserved gene clusters for both pathways. M. robertsii may be capable of producing ergoamide ergot alkaloids and paspaline-like indole-diterpenes, from each respective biosynthesis pathway. This is supported by an ELISA analysis that shows a D-lysergic acid derivative is produced in Mr23 during insect infection. Quantitative PCR shows that the first gene of the EAS pathway, dmaW, is expressed in association with insects and plant roots, as well as in axenic cultures, but its expression may be regulated by light intensity and nutrient status. Overall, this thesis describes an example of an ancient interkingdom horizontal transfer and lays the genetic foundation for understanding the biochemistry and ecological role of ABC complexes, ergot alkaloids, and indole diterpenes in M. robertsii.