Development of an Agrobacterium vitis transformation system for grapevine

Joubert, Dirk Albert, 1973- (2000-03)

Thesis (MSc)--University of Stellenbosch, 2000.

Thesis

ENGLISH ABSTRACT: Agrobacterium tumefaciens-mediated transformation technology has been used in a variety of applications throughout the fields of cellular and molecular plant biology as well as plant physiology. Research is conducted in order to extend this application range and overcome some of the intrinsic limitations of the Agrobacterium transformation system. Predominantly, these limitations can be attributed to the host range specificity of A. tumefaciens, as well as adverse effects induced on explant tissue by active plant defence mechanisms, triggered by the plant-pathogen-interaction. Typically, this active defence mechanism culminates in the hypersensitive response (HR), characterised by localised cell death and necrosis. Not all Agrobacterium species, however, share the same host range and some have evolved the ability to infect plant species not normally considered hosts of A. tumefaciens. This host range specificity can be exploited to extend the application of existing Agrobacterium transformation systems. In an attempt to establish an efficient transformation system for Vitis vinifera which, has proven very difficult to transform with A. tumefaciens, indigenous A. vitis strains have been evaluated as possible host-specific transformation agents. Strains of Agrobacterium vitis should be suitable for this type of endeavour, since they have evolved several unique characteristics directly linked to the infection of their hosts. These include the ability to utilise, tartrate, a host abundant carbon source, as well as the production of an acid polygalacturonase that could play a role during the infection process. The proposition that the evolution of A. vitis is a fairly recent event is also confirmed by the relatively little divergence observed between A. tumefaciens and A. vitis. In this study, a selection of A. vitis strains were evaluated in screenings designed to accentuate desirable traits in strains such as good infectivity of grapevine material (presumably an indicator of an efficient mechanism of gene transfer to be exploited in an engineered transformation system) as well as a favourable reaction (causing no necrosis) on grapevine somatic embryos. Two strains produced large tumours on grapevine cuttings and caused little necrosis on the somatic embryos. Significant variation in infectivity as well as callus necrosis was observed between the strains as well as in a genotype-specific manner on the host material. This genotypic-specific effect of either host or pathogen could be an indication of the degree of specialisation developed by plant pathogens to infect specific hosts. On the basis of these results, it was possible to select an A. vitis strain for further biochemical and genetic characterisation. Simple biochemical analysis classified the strain as an octopine strain. DNA-DNA hybridisation techniques combined with a plasmid walking technique resulted in the partial characterisation of the T-DNA of the selected A. vitis strain. A partial restriction enzyme map of the T-DNA was constructed and the T-DNA and flanking areas were cloned. Significant differences, most notably, the absence of a TB-area as well as the absence of the agrocinopine (aes) gene from the 5' area of the T-DNA, were observed. Partial sequencing data indicated the presence of at least four conserved T-DNA genes located on the TA-DNA, as well as the presence of three bacterial insertion (IS-)elements flanking the region. Two of these IS elements, both related to the IS 110 family of IS elements have not yet been reported in A. vitis. In fact, these two elements seem to be the 5' and 3' ends of a disrupted element and could therefore have played an evolutionary role in the development of this strain. This study provides fundamental background for the development of a more efficient transformation system specific for grapevine, exploiting same of-the unique characteristics of one of its pathogens, A. vitis.

AFRIKAANSE OPSOMMING: Agrobacterium tumefaciens-gebaseerde transformasiesisteme word in "n wye reeks van toepassings in die velde van sellulêre- en molekulêre plantbiologie asook plantfisiologie aangewend. Navorsing word voortdurend onderneem om die inherente beperkinge van die Agrobacterium-transformasiesisteem te oorkom en sodoende die toepassingsveld van die sisteem verder te verbreed. Die beperkinge tipies aan dié sisteem kan hoofsaaklik toegeskryf word aan die gasheerspesifisteit van A. tumeteciens, asook die negatiewe reaksies op eksplantmateriaal wat deur die plant se aktiewe verdedigingsmeganisme, soos ontlok deur die plant-patogeen interaksie, veroorsaak word. Hierdie aktiewe verdedigingsmeganisme lei gewoonlik tot In hipersensitiewe respons (HR) in die plant, wat deur gelokaliseerde selafsterwing en nekrose gekenmerk word. Alle Agrobacterium-spesies het egter nie almal dieselfde gasheerreeks nie en sommige rasse het as gevolg van evolusionêre ontwikkelings die vermoë verkry om plantspesies wat normaalweg buite die gasheerreeks van A. tumefaciens val, te infekteer. Hierdie tipe gasheerspesifisiteit kan uitgebuit word om die toepassingsmoontlikhede van bestaande Agrobacterium-transformasiesisteme te verbreed. In In poging om In effektiewe transformasiesisteem vir Vitis vinifera, In moeilik transformeerbare gewas, te ontwikkel, is inheemse rasse van Agrobacterium vitis ondersoek as moontlike gasheerspesifieke transformasie-agente. Rasse van A. vitis behoort uiters geskik te wees vir so "n toepassing, aangesien hulle verskeie unieke eienskappe, wat direk aan die infeksie van die gasheer gekoppel is, vertoon. Van hierdie eienskappe is onder meer die vermoë om tartraat, In koolstofbron volop in druifplante, te benut. A. vitis produseer verder ook In suur poligalaktorunase wat vermoedelik In rol in die infeksieproses speel. Die voorstel dat die evolusionêre ontwikkeling van A. vitis In redelike onlangse gebeurtenis is, word onderskryf deur die betreklike homogenisiteit met A. tumefaciens. In hierdie studie is "n groep A. vitis-rasse met behulp van siftingsprosedures wat daarop gemik is om gesogte eienskappe in rasse uit te wys, beoordeel. Die vermoë om druifplantmateriaal te infekteer (wat vermoedelik "n aanwyser van "n effektiewe meganisme van geenoordraging is wat in "n gemanipuleerde transformasiesisteem benut kan word), sowel as 'n gunstige reaksie (d.w.s geen nekrose) op druifplant somatiese embrio's is van die gesogte eienskappe waarvoor gesoek word. Twee rasse het groot tumors op druifplant-stingelsegmente veroorsaak terwyl hulle bykans geen weefselskade op somatiese embrio's geïnduseer het nie. Betekenisvolle verskille in infektiwiteit en in kallusnekrose is tussen die rasse sowel as in 'n genotipe-spesifieke-verhouding waargeneem. Hierdie genotipe-spesifieke effek, kenmerkend van óf die gasheer óf die patogeen, kan aanduidend wees van die vlak van spesialisasie wat heers by die infeksie van spesifieke gashere. Na aanleiding van bogenoemde resultate was dit moontlik om 'n A. vitis-ras te selekteer wat verder aan biochemiese en genetiese analises onderwerp kon word. Eenvoudige biochemiese analises het dit moontlik gemaak om die ras as oktopien te klassifiseer. DNA-DNA hibridisasietegnieke gekombineerd met 'n unieke plasmiedwandeltegniek het gelei tot die gedeeltelike karakterisering van die geselekteerde A. vitisras. In Gedeeltelike restriksie-ensiem (RE) kaart van die T-DNA kon gevolglik opgestel word. Die T-DNA en die aangrensende gedeeltes is boonop gekloneer. Betekenisvolle verskille, spesifiek die afwesigheid van In TB area, sowel as die afwesigheid van die agrosinopien-sintasegeen (acs) aan die 51-kant van die T-DNA, is waargeneem. Gedeeltelike basispaaropeenvolgingsdata het egter die teenwoordigheid van minstens vier gekonserveerde T-DNA-gene, asook die teenwoordigheid van drie bakteriese invoegingselemente (IS) aan weerskante van die area, geïdentifiseer. Twee van hierdie elemente, wat beide homologie vertoon met die IS110 familie van IS elemente, is nog nie vantevore in A. vitis aangetref nie. Dit wil boonop blyk of dié twee elemente die 51 - en 31 - areas van In onderbroke element vorm, wat dus In moontlike aanduiding is van hul potensiële rol in die evolusionêre ontwikkeling van die ras. Hierdie studie verskaf basiese inligting wat daartoe kan lei dat 'n doeltreffender transformasiesisteem spesifiek vir druifplante ontwikkel word deur van die unieke kenmerke van een van sy patogene, A. vitis, uit te buit.

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