Department of Biochemistry

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Browsing Department of Biochemistry by Author "Appel, Maryke"

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    Cloning and identification of genes involved in the interaction between the bacterial stone fruit pathogen Pseudomonas syringae pv. syringae strain NV and plum trees
    (Stellenbosch : Stellenbosch University, 2001-03) Appel, Maryke; Bellstedt, D. U.; Mansvelt, E. L.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Bacterial canker of stone fruit, caused by Pseudomonas syringae pv. syringae, is one of the most destructive crop diseases in South Africa. Chemical control has failed completely and effective long-term management strategies will have to rely on the breeding of resistant host trees. To assist in such breeding programmes, investigations into the molecular basis of the interaction between P. s. pv. syringae and stone fruit trees have been undertaken in collaboration with the ARC-Fruit, Wine and Vine Research Institute in Stellenbosch. The aim of this dissertation was to clone and identify genes that are involved in interaction between the bacterial canker pathogen and stone fruit trees. In the first part of the study, the harpin encoding gene of a local strain of the pathogen, P. s. pv. syringae NV, was amplified in a polymerase chain reaction (PCR) strategy with primers based on the hrpAZB sequences of the bean pathogen, P. s. pv. syringae 61. Sequencing of this hrpZpssNvgene revealed a high degree of homology (96%) between the harpin encoding genes and harpin proteins of the two strains. The hrpZpssNvgene was subsequently cloned into the pMAL-c2 expression vector and expressed in Escherichia co/i. This system was used for the production of purified, biologically active, recombinant HrpZpSSNV protein. In the second part of the study, differential display (DD) technology was used to identify genes that are induced in stone fruit trees in response to P. s. pv. syringae and/or its harpin elicitor. For this purpose, actively growing shoots of two Prunus sa/icina cultivars, the moderately resistant cv. 'Laetitia' and the highly susceptible cv. 'Songold' were treated with recombinant harpinpssNvprotein or live P. s. pv. syringae NV bacteria. An untreated control and wounding control was included in the experiment. Total RNA was isolated for comparative mRNA analysis 24 hours after treatment. DD profiles were generated with fifteen primer combinations. Eight candidate bands were re-amplified, cloned and sequenced. Reverse transcription PCR was employed to verify the expression patterns of the cloned bands in the original RNA sample set. Two bands, DDc and DD4 were shown to be differentially expressed between treatments and/or cultivars, while no differences in the expression levels of the remaining six bands (DDa, DDe, DD3, DD5, DD6 and DD7) were observed. BLAST similarity searches yielded significant matches for DDe, DD4 and DD7 with plant defense-related genes.
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    Investigations into the production of a harpin elicitor by Pseudomonas syringae pv. syringae isolated from a nectarine tree
    (Stellenbosch : Stellenbosch University, 1996) Appel, Maryke; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Department of Biochemistry.
    ENGLISH ABSTRACT: Bacterial canker of stone fruit trees, caused by Pseudomonas syringae pv. syringae, has become one of the most destructive crop diseases in South Africa Failure of chemical control of the disease has rendered the selection and breeding of resistant host trees an important aspect of future control strategies. To assist in such breeding programmes, investigations into the molecular basis of the host-pathogen interaction were initiated The fundamental ability of phytopathogenic pseudomonads, xanthomonads and non-soft rot erwinias to cause necrotic diseases in their hosts and hypersensitivity in non-host plants is controlled by their widely conserved hrp gene clusters. The only known secreted hrp gene product, dubbed "harpin", has been identified as the molecule ("elicitor"') both responsible and required for eliciting hypersensitivity or disease symptoms. In this study, the production of a harpin elicitor by a strain of Pseudomonas syringae pv. syringae, isolated locally from nectarine tree (P. s. pv. syringae NV) was investigated. The HR test in tobacco was used to assess the elicitor activity of bacterial fractions. It was established that the bacterium produces an extracellular protein elicitor similar to harpin Pss the harpin elicitor of the wheat and bean pathogen, Pseudomonas syringae pv. syringae 61. Antibodies were raised against harpin Pss and used to confirm homology between the elicitors of the two strains, using Western blot analysis. Homology between the two proteins was exploited on the gene level in the design of a polymerase chain reaction strategy for the amplification of the harpin encoding gene of P. s. pv. syringae NV from its genomic DNA. Partial sequencing of the single PCR product and Southern blot hybridization with a probe based on the P. s. pv. syringae 61 harpin encoding gene, confirmed its identity as the harpin encoding gene of P. s pv syringae NV.