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Extrachromosomal and chromosomal DNA analysis of 'Candidatus phytoplasma Australiense'

Tran-Nguyen, Lucy (2007). Extrachromosomal and chromosomal DNA analysis of 'Candidatus phytoplasma Australiense'. PhD Thesis, Charles Darwin University.

Document type: Thesis
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Author Tran-Nguyen, Lucy
Title Extrachromosomal and chromosomal DNA analysis of 'Candidatus phytoplasma Australiense'
Institution Charles Darwin University
Publication Date 2007
Thesis Type PhD
Subjects 0607 - Plant Biology
Abstract The aim of this study was to increase our understanding of phytoplasma genomics by sequencing and analysing extrachromosomal and chromosomal phytoplasma DNA. Extrachromosomal DNA (eDNA) is believed to play an important role in pathogenicity, host specificity and virulence in plant pathogenic bacteria. In this study, eDNA was characterised for the two most significant phytoplasmas in Australia - tomato big bud (TBB) phytoplasma and ‘Ca. P. australiense’. Using the eDNA sequence data, replication strategies were proposed for both phytoplasmas. Based on an analysis of the TBB eDNA encoded rep gene from multiple isolates, a unique mechanism for replication was proposed that involves the dnaG gene. The ‘Ca. P. australiense’ eDNA rep gene contained conserved domains for both bacterial plasmids and geminiviruses. Similarity matches between the closely related Australian and New Zealand ‘Ca. P. australiense’ eDNAs were found to be only 41- 61%. This level of difference suggests that the eDNA is not under the same evolutionary pressure as is chromosomal DNA, and/or a range of different mechanisms leading to genetic diversity are operational. Phytoplasmas cannot be cultured so the difficulty in obtaining sufficient quantities of high quality phytoplasma DNA hinders whole genome analysis. Despite this, the desire to sequence phytoplasmas is fuelled by the current paucity of knowledge about phytoplasma pathogenicity. In this study, conventional and modern techniques were optimised to obtain sufficient quantities of genomic DNA to sequence the entire ‘Ca. P. australiense’ genome. Although phytoplasma DNA could be extracted from pulsed field gels, quantities were too low for subsequent manipulation. DNA extracted from cesium chloride gradients was successfully used to enrich phytoplasma DNA suitable for sequencing. The ‘Ca. P. australiense’ chromosome was 879 324 bp and is among the largest phytoplasma genomes to be sequenced. It contained potential mobile units (PMUs) that may account for the fact that it is smaller than the closely related New Zealand ‘Ca. P. australiense’ strain. The mechanism by which ‘Ca. P. australiense’ PMUs may have been laterally transferred is unknown, because inverted repeats characteristic of composite transposons were missing. It is possible that ‘Ca. P. australiense’ PMU mobility relies on helper elements or by association with phage particles. The genomes of ‘Ca. P. australiense’ and the previously published ‘Ca. P. asteris’ (OY-M and AY-WB) were compared with a particular focus on metabolic processes. Many essential metabolic pathways were completely missing or greatly reduced in all phytoplasmas, but all contained numerous genes for membrane transporters. This observation is not surprising for a pathogen that occupies the nutrient rich phloem of its host plant. There is still much to learn about the pathogenicity and minimal metabolic capability of ‘Ca. P. australiense’, but with close to 50% ORFs not assigned to any known function, there exists a rich archive of information for further discovery.


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