Cloning and identification of genes involved in the interaction between the bacterial stone fruit pathogen Pseudomonas syringae pv. syringae strain NV and plum trees

Appel, Maryke (2001-03)

Thesis (PhD)--Stellenbosch University, 2001.

Thesis

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.

AFRIKAANSE OPSOMMING: Bakteriese kanker van steenvrugte, wat deur Pseudomonas syringae pv. syringae veroorsaak word, is een van die mees verwoestende siektes van landbougewasse in Suid-Afrika. Chemiese beheermaatreëls het geheel en al misluk en effektiewe langtermyn beheerstrategieë sal op die teling van weerstandbiedende gasheerbome moet staatmaak. Ondersoeke na die molekulêre basis van die interaksie tussen P. s. pv. syringae en steenvrugbome is in samewerking met die LNR-Vrugte-, Wyn- en Wingerdnavorsingsinstituut in Stellenbosch van stapel gestuur om tot sulke telingsprogramme by te dra. Die doelwit van hierdie proefskrif was om gene wat betrokke is in die interaksie tussen die bakteriese kanker patogeen en steenvrugbome te kloneer en te identifiseer. In die eerste gedeelte van die studie is die harpien-koderende geen van 'n plaaslike ras van die patogeen, P. s. pv. syringae NV, geamplifiseer in 'n polimerase kettingreaksie (PKR)-strategie met peilers wat op die hrpAZB-geenopeenvolgings van die boontjiepatogeen, P. s. pv. syringae 61, gebaseer is. Volgordebepaling van hierdie hrpZpssNv-geen het 'n hoë vlak van homologie (96%) tussen die harpien-koderende gene en harpien proteïene van die twee rasse getoon. Die hrpZpssNv-geen is vervolgens in die uitdrukkingsvektor pMAL-c2 gekloneer en uitgedruk in Escherichia coli. Hierdie sisteem is vir die produksie van suiwer, biologies-aktiewe, rekombinante HrpZpssNv-proteingebruik. In die tweede gedeelte van die studie is die differensiaalvertoon (DD) tegniek gebruik om gene te identifiseer wat deur P. s. pv. syringae en/of sy harpien elisitar in steenvrugbome geïnduseer word. Vir hierdie doel is aktief-groeiende lote van twee Prunus sa/icina kultivars, die matig weerstandbiedende kv. 'Laetitia' en die hoogs vatbare kv. 'Songold', met rekombinante harpienpssNvproteïen of lewende P. s. pv. syringae NV bakterieë behandel. 'n Onbehandelde- en verwondingskontrole is in die eksperiment ingesluit. Totale RNA is 24 uur na behandeling vir vergelykende mRNA-analise geïsoleer. DD-profiele is met vyftien peilerkombinasies gegenereer. Agt kandidaatbande is geheramplifiseer en gekloneer, waarna hul DNA-opeenvolgings bepaal is. Trutranskriptase-PKR is gebruik om die ekspressiepatrone van die gekloneerde bande in die oorspronklike RNA monsters na te gaan. Daar is vasgestel dat twee van die bande, DDc en DD4, differensieel tussen kultivars en/of behandelings uitgedruk is, terwyl geen verskille in die ekspressievlakke van die oorblywende ses bande (DDa, DOe, 003, DOS, 006 en DO7) waargeneem is nie. BLAST-soektogte het betekenisvolle ooreenkomste vir DDe, DD4 en DD7 met plant weerstandsgeassosieerde gene opgelewer.

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