Investigations into the production of a harpin elicitor by Pseudomonas syringae pv. syringae isolated from a nectarine tree

Appel, Maryke (1996)

Thesis (M. Sc.) -- University of Stellenbosch, 1996.

Thesis

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.

AFRIKAANSE OPSOMMING: Bakteriese kanker van steenvrugte, wat deur Pseudomonas syringae pv. syringae veroorsaak word, is tans een van die mees verwoestende siektes van landbougewasse in Suid-Afrika. Die mislukking van chemiese beheermaatreëls het die seleksie en teling van weerstandbiedende gasheerbome 'n belangrike aspek in toekomstige beheerstrategieë gemaak. Studies wat die molekulêre basis van die gasheer-pathogeen intereksie ondersoek, is van stapel gestuur om tot sulke teelprogramme by te dra. Die fundametele vermoë van sekere bakterieë om nekrotiese siektes in gasheerplante en hipersensitiwiteit in nie-gasheerplante te verooraaak, word deur hul wyd-gekonserveerde hrp gene beheer. Die enigste bekende uitgeskeide hrp geenproduk, wat "harpin" genoem word, is geïdentifiseer as die molekuul ("elisitor'') wat verantwoordahk is en vareis word vir die ontlokking van hipersensitiwiteit of siektesimptome. In hierdie studie is die produksie van 'n "harpin" elisitor deur 'n ras van Pseudomonas syringae pv. syringae, wat plaaslik vanaf 'n nektarienboom geïsoleer is (P. s. pv syringae NV), ondersoek. Die HR-toets in tabak is gebruik om die elisitor aktiwiteit van bakteriële fraksies te bepaal. Daar is vasgestel dat die bakterium 'n akstrasellulêre proteïen elisitor produseer wat soortgelyk is aan harpin Pss die ''harpin" elisitor van die toring- en boontjiepatogeen, Pseudomonas syringae pv. syringae 61. Antiliggame is teen harpin Pss opgewek om homologie tussen die elisitors van die twee rasse te bevestig, deur van die Western-klad tegniek gebruik te maak. Homologie tussen die twee proteïene is op geen-vlak uitgebuit om 'n polimerase kettingreaksie-strategie te ontwerp waardeur die ''harpin" koderende geen van P. s. pv. syringae NV vanuit sy genomiese DNA geamplifiseer kon word. Gedeeltelike volgordebepaling van die enkele PKR-produk en Southern-klad hibridisasie met 'n gedeelte van die P. s. pv. syringae 61 "harpin" koderende geen, het bevestig dat die PKR-produk die ''harpin"-koderende geen van P. s. pv. syringae NV is.

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