A nopaline-type overdrive element, and its influence upon Agrobacterium-mediated transformation frequency and T-DNA copy number in Nicotiana tabacum : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand/Aotearoa

Abstract

Overdrive is an enhancer element located outside and adjacent to the right border of the T-DNA in Agrobacterium tumefaciens octopine-type tumour-inducing (Ti) plasmids. This element is necessary for maximal enhancement of T-strand production and subsequent A. tumefaciens-mediated plant transformation frequency, and only the octopine-type overdrive had been characterised in any detail. A putative overdrive has been identified in the nopaline-type Ti-plasmid pTiT37 on the basis of its homology with known octopine-type overdrive sequences, particularly the eight base-pair so-called overdrive consensus core. The putative nopaline-type overdrive core, however, is only 75% homologous to that of all known overdrive core regions. Furthermore, as there are other sequences throughout the nopaline-type T-region that share 75% homology with the overdrive consensus core, the precise location of the nopaline-type overdrive is undetermined, although all nopaline-type T-region fragments exhibiting overdrive-like activity contained the putative overdrive core adjacent to the right border. The role of this particular putative core in T-DNA transfer has never been established. Deletions were made in the putative nopaline-type overdrive consensus core adjacent to the right border of a binary plant transformation vector derived from pTiT37. This was to establish whether this putative overdrive core does have a role as a transmission enhancer as proposed (Peralta et al., 1986; Van Haaren et al., 1988; Culianez-Macia and Hepburn, 1988). Two deletions were selected for the full study. The first encompassed the putative nopaline-type overdrive core flanked by 3 bp (5') upstream, and 4 bp (3') downstream, and was located in pANDY9. The other, located in pANDY10, encompassed the putative consensus core plus the entire region sharing homology with the octopine-type overdrive. This second deletion was to determine whether the core alone could account for overdrive-like activity, or whether further sequences are necessary to produce the effect. The vector with no deletions in the putative nopaline-type overdrive region was pANDY8. As determined by quantitative Nicotiana tabacum transformation assays, both deletions of the putative nopaline-type overdrive core (pANDY9, pANDY10) equally decreased the rate at which calli appeared, and equally decreased transformation frequency by 47% compared with that of pANDY8. That deletion of the putative core influenced plant transformation frequency provided strong evidence that it was indeed an overdrive-like core. Furthermore, in a virC2 mutant environment, the plant transformation frequency was reduced markedly for all three plasmids (approximately 90% reduction compared to when in the wild-type vir environment). However, there was no difference in the plant transformation frequencies of the pANDY8-10 series in a virC2 environment. This indicated that the mechanism by which the deletions influenced plant transformation frequency did not act independently of the virC operon, which is further evidence of overdrive-like activity. The type of vir regulon influenced the effect of the deletions in the putative overdrive. The transformation frequency of the plasmid with the intact putative overdrive region (pANDY8) was very similar in both an octopine-type vir environment (21.7 organogenic calli per 10 leaf discs in LBA4404) and a nopaline-type vir environment (18.7 organogenic calli per 10 leaf discs). However, in an octopine-type vir environment, deletions in the putative core resulted in a 47% decrease in transformation frequency, whereas in a nopaline-type vir environment the deletions had no effect upon transformation frequency. This may be due to a higher level of vir gene products (a feature associated with nopaline-type vir regulons), particularly VirDl and VirD2 compensating for the lack of a fully active putative overdrive. Southern analysis of plants arising from the transformation experiments (in an octopine-type vir environment) revealed that removal of the putative nopaline-type overdrive core halved the incidence of multiple T-DNA insertion events from 34.7% (pANDY8, intact nopaline-type overdrive) to 12.2% (pANDY9) and 14.3% (pANDY10). Deletion of the nopaline-type overdrive core also restricted the insert number to a maximum of two, rather than four or more. This is the first time that deletions in the regions outside the T-DNA have been shown to influence T-DNA copy number.