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A genomic survey and prediction of the infection strategies employed by Phoma medicaginis var. medicaginis, a fungal pat hogen of alfalfa and the model legume Medicago truncatula

Williams, Angela (2013) A genomic survey and prediction of the infection strategies employed by Phoma medicaginis var. medicaginis, a fungal pat hogen of alfalfa and the model legume Medicago truncatula. PhD thesis, Murdoch University.

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Phoma medicaginis is a fungal pathogen of the order Pleosporales that causes black spot of Medicago sativa (alfalfa, lucerne) and the related legume Medicago truncatula. This study presents a genomic analysis of the isolate OMT5 (PmedOMT5) and the pathogenicity mechanisms it employs during infection of M. truncatula. The genome assembly of ~31Mb covers an estimated 99% of the gene space and analysis outlines the location of repetitive and non-coding RNA, as well as the separately assembled and annotated complete mitochondrial genome. This is the first genome assembly from the genus Phoma, which encodes 11,879 manually-annotated protein-coding genes with biological support from proteogenomic and transcriptomic analysis.

Culture filtrate produced by PmedOMT5 grown in a variety of media, produced a chlorotic and necrotic response when infiltrated into plants from Medicago species and selected other plant species. The phytotoxic component was localised to a fraction containing molecules greater than 10 kDa, indicating that the active molecule is likely to have a proteinaceous component. The active compound or compounds have high thermal and chemical stability, are able to resist heat and pronase degradation and still produce a consistent response in planta after storage for 6 weeks at 4 °C on ice. Unexpectedly, the activity of compounds in the culture filtrate showed little correlation between sensitivity and susceptibility to foliar infection with P. medicaginis. Proteogenomic analysis of the PmedOMT5 culture filtrate yielded a list of bioinformatically predicted effector candidates for future testing.

Transcripts were sequenced via RNA-seq from four life-cycle stages of the pathogen: during in vitro growth, at vegetative (4d) and sporulating (16d) phases, during production of phytotoxic culture filtrate (4w) and during the early stages of plant invasion (1-5 dpi) on M. truncatula leaves. The variation in expression between the conditions was used in a preliminary investigation to identify genes that may play a role in pathogenicity, including prediction of effector candidates. Bioinformatic analysis suggests that PmedOMT5 is able to both detoxify and expel host phytoalexin compounds, which may contribute to its virulence. PmedOMT5 also produces a wide range of cell-wall-degrading enzymes with species-specific expansion of several pectin degrading families when compared to other Pleosporales pathogens.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
Supervisor(s): Oliver, Richard, Lichtenzveig, Judith, Trengove, Robert and Singh, Karam
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