Physiological and molecular changes induced in Catharanthus roseus in response to phytoplasma infection

Phytoplasmas are specialised plant pathogenic bacteria, transmitted by sap sucking insect vectors and responsible are for a number of economically important crop diseases. Typical disease symptoms include phyllody, virescence, little leaf, increased branching, stunting and yellowing. Catharanthus roseus, or Madagascar periwinkle, is often used as an experimental host for phytoplasma disease due to the fact that infection produces a good range of representative symptoms. Very little is known about the mechanisms of phytoplasma pathogenicity, and the main aim of this study was to learn more about changes induced in C. rose us in response to phytoplasma infection. Sweet potato little leaf (SPLL) infection induces a reduction in leaf size in C. roseus. Leaf size is controlled by two main mechanisms, initially leaves grow via mitotic divisions but when these cease a period of cell expansion occurs. Upper epidermal cells of healthy and SPLL infected plants were investigated for histological changes at the 2nd and 5th leaf stage. At all stages there was a reduction in cell size, though this was not always enough to account for total reduction in leaf size suggesting some inhibition of cell proliferation occurs. The leaf ratio was affected positively in SPLL infected plants, suggesting disease has a greater affect on growth in the width direction than the length. Phyllody is defined as the replacement of flowers with leaf like structures, but very little work has been done on investigating the actual properties of phyllodous leaves. The upper epidermis of flowers from soybean phyllody (SP) infected plants was investigated. In general three cell types were found: conical cells, cells that resembled closely those from the upper epidermis of leaves and cells that appeared to be a hybrid between the two, with a flattened conical cell appearance. Though C. roseus provides a useful host for phytoplasma disease, it lacks the genomic resources available to other species. The Xspecies microarray technique allows the use of a microarray for a species it was not originally designed for. In this instance transcripts from SPLL infected C. rose us were run on the ATH1 Arabidopsis GeneChip®. A hybridisation efficiency of 150 was chosen. The micro array indicated up-regulation of ARF7 and PIN6, genes involved in auxin response and transport, as well as two miRNA processing genes, HASTY and XRN3. Down-regulation of genes involved in photosynthesis was observed. The expression of a number of transcripts was then monitored with quantitative PCR. In general, the qPCR results were not in agreement with the array data. This could be a result of the probes actually binding to other, closely related transcripts within the C. rose us genome. Changes in phytohormones occur in many plant infections, and have been proposed as an explanation for the symptoms seen in phytoplasma infected plants. The levels of cytokinins were measured in the stem, root, leaf and floral tissues of healthy and infected plants, but no significant changes in cytokinin content were seen. A semi-quantitative technique was used to study changes in gene expression in auxin and cytokinin responsive genes. Down-regulation of the cytokinin receptor was seen in all tissues except the leaf. An increase in expression of the auxin influx carrier AUX1 occurred within infected root material, but the transcript was dramatically down regulated in other tissues. It is evident that changes in gene expression occur in infected plants. Gene expression is repressed by DNA methylation. Changes in DNA methylation were monitored using Methylation Sensitive AFLP. Changes in CpNpG methylation were detected in plants infected with Vinca coconut phyllody and Rhemannia glutinosa. Some isolated sequences returned showed homology to the chloroplast and mitochondrial genomes. These organelle genomes were thought to be free of cytosine methylation, raising questions about whether this hypothesis is correct.EThOS - Electronic Theses Online ServiceGBUnited Kingdo