AbstractWe know little about the intracellular, phytopathogenic mollicutes, the phytoplasmas. Phytoplasmas cause disease in several hundred plant species worldwide; however without an understanding of their biology and pathogenicity the ability to avert phytoplasma disease is low. Insights into the genetic architecture of phytoplasmas are increasing through the employment of molecular techniques. Previously, much of this research has focused on the analysis of the 16SrRNA and other ribosomal genes. While this facilitated the definition of major phytoplasma groups, the capacity to distinguish between closely related phytoplasmas is limited, as is the biological information provided by such analysis. As more non-ribosomal phytoplasma genes are identified, greater insights will be obtained into many aspects of their biology, pathogenicity, and genetic diversity.
This study aimed to contribute to phytoplasma research by analysing a region of the sweet potato little leaf strain-V4 (SPLL-V4) phytoplasma genome. Physical mapping indicated the region was in proximity to the putative origin of replication, and thus it was expected to contain integral phytoplasma genes. The specific objectives of this project were the identification of genes in the pH80 random clone, and the analysis of the glucose inhibited division protein A (gidA) gene. This gene has been previously identified in the SPLL-V4 and tomato big bud (TBB) phytoplasmas, but in TBB, the gene was identified in three separate random clones. We wanted explore this to determine if there were multiple copies of the gene in phytoplasmas, which may have significant implications for its function.
The pH80 random clone was sequenced and submitted to various database search programs. The genes putatively identified were examined via phylogenetic and protein analysis programs to gain insights into their phylogeny and function. Fragments of the genes identified were amplified from the TBB phytoplasma and subjected to RFLP and sequence analysis to compare the genes between these two closely related phytoplasma isolates. The copy number of the gidA gene in TBB and SPLL-V4 was determined using southern hybridisation, and the arrangement of the TBB gidA random clones investigated using PCR.
Three genes were identified within the 2.7 kb insert of the pH80 clone. These genes encoded the proteins PotB. PotC, and PotD, which form part of ATP-binding-cassette transport system responsible for polyamine uptake. This result was further supported by further structural analysis, which indicated that PotB and PotC are integral membrane proteins, and PotD is a polyamine binding, surface exposed membrane protein. Phylogenetic analysis of the proteins supported phytoplasma classification in the class mollicutes. Comparison of fragments of the TBB and SPLL-V4 polyamine transport genes indicated little diversity and supported the close relationship between these phytoplasmas. Finally, one copy of the gidA gene was shown to be present in the phytoplasmas, with two of the TBB gidA random clones adjacent on the chromosome.
The identification and characterisation of this region of the SPLL-V4 genome enhanced our understanding of phytoplasma genomics. The identification of a surface-exposed membrane protein was of particular significance to phytoplasma research, with emerging studies involving the expression of recombinant antibodies against membrane proteins, which eventually may present a pathway for engineering phytoplasma resistance in plants.
|Date of Award||Nov 2002|
|Supervisor||Karen Gibb (Supervisor), Claire Streten (Supervisor) & Lucy Thanh Thuy Tran-Nguyen (Supervisor)|