Mutation of the genes virA, virB, virC, and virG of the Agrobacterium tumefaciens octopine-type Ti plasmid pTiRlO was found to cause a 100-to 10,000-fold decrease in the frequency of conjugal transfer of this plasmid between Agrobacterium cells. This effect was not absolute, however, in that it occurred only during early times (18 to 24 h) of induction of the conjugal transfer apparatus by octopine. Induction of these mutant Agrobacterium strains by octopine for longer periods (48 to 72 h) resulted in a normal conjugal transfer frequency. The effect of these vir gene mutations upon conjugation could be restored by the introduction of cosmids harboring wild-type copies of the corresponding disrupted vir genes into the mutant Agrobacterium strains. In addition, transfer of the self-mobilizable plasmid pPHlJI was not impaired in any of the mutant Agrobacterium strains tested. (15,17,20,33,34), agrocinopines A and B induce the transfer of nopaline-type Ti plasmids (9), and agrocinopines C and D direct the conjugation of agropine-type Ti plasmids (9). The conjugal transfer of Ti plasmids is normally repressed, although transfer-constitutive mutations have been isolated (10,21,33). In octopine-type Agrobacterium strains, these mutations map to the region of the Ti plasmid near the genes responsible for the catabolism of octopine (7). Octopine catabolism is also inducible by octopine, and it has been hypothesized that octopine catabolism and conjugal Ti plasmid transfer are under the control of a common repressor (17,21,33 plasmid at similar 24-base-pair directly repeated sequences that flank the T-DNA (1,18,40,48,51,54,55 (31,32,47). Single-stranded T-DNA molecules (T-strands) that accumulate in the bacterium have been hypothesized to be the form of the T-DNA that is transferred to the plant in a process resembling the conjugal transfer of some plasmids between bacteria (39, 41). A single-stranded-DNA-binding protein, encoded by the virE operon, binds to these T-strands (4-6, 16) and presumably serves to protect the T-DNA intermediate as it exits the bacterium and enters the plant cell, possibly using a membrane apparatus encoded by the virB operon (11,45,52).We recently discovered that certain opines, including octopine, nopaline, leucinopine, and succinamopine, can augment the activity of acetosyringone in the induction of the vir genes and the generation of T-strands in induced Agrobacterium cells (49). Because opines had an effect on vir gene activity, we speculated that perhaps vir genes could affect the activity of processes, such as conjugal Ti plasmid transfer, regulated by opines. We show here that mutations in certain vir genes can markedly influence the conjugal transfer frequency of the Ti plasmid under some opine induction conditions. These data suggest an interaction between the processes controlling the transfer of the Ti plasmid between bacteria and those regulating the transfer of the T-DNA to plants.
Previous work has shown that the octopine synthase (ocs) gene encoded by the Agrobacterium tumefaciens Ti-plasmid contains an upstream activating sequence necessary for its expression in plant cells. This sequence is composed of an essential 16-bp palindrome and flanking sequences that modulate the level of expression of the ocs promoter in transgenic tobacco calli. In this study, we have used RNA gel blot analysis of RNA extracted from transgenic tobacco plants to show that the octopine synthase gene is not constitutively expressed in all plant tissues and organs. This tissue-specific pattern of expression is determined, to a large extent, by the 16-bp palindrome. Histochemical analysis, using an ocs-lacZ fusion gene, has indicated that the 16-bp palindrome directs the expression of the ocs promoter in specific cell types in the leaves, stems, and roots of transgenic tobacco plants. This expression is especially strong in the vascular tissue of the leaves, leaf mesophyll cells, leaf and stem guard cells, and the meristematic regions of the shoots and roots. Sequences surrounding the palindrome in the upstream activating sequence restrict the expression of the ocs promoter to fewer cell types, resulting in a reduced level of expression of beta-galactosidase activity in the central vascular tissue of leaves, certain types of leaf trichomes, and the leaf primordia.
Previous work has shown that the octopine synthase (ocs) gene encoded by the Agrobacterium tumefaciens Ti-plasmid contains an upstream activating sequence necessary for its expression in plant cells. This sequence is composed of an essential 16-bp palindrome and flanking sequences that modulate the level of expression of the ocs promoter in transgenic tobacco calli. In this study, we have used RNA gel blot analysis of RNA extracted from transgenic tobacco plants to show that the octopine synthase gene is not constitutively expressed in all plant tissues and organs. This tissue-specific pattern of expression is determined, to a large extent, by the 16-bp palindrome. Histochemical analysis, using an ocs-lacZ fusion gene, has indicated that the 16-bp palindrome directs the expression of the ocs promoter in specific cell types in the leaves, stems, and roots of transgenic tobacco plants. This expression is especially strong in the vascular tissue of the leaves, leaf mesophyll cells, leaf and stem guard cells, and the meristematic regions of the shoots and roots. Sequences surrounding the palindrome in the upstream activating sequence restrict the expression of the ocs promoter to fewer cell types, resulting in a reduced level of expression of beta-galactosidase activity in the central vascular tissue of leaves, certain types of leaf trichomes, and the leaf primordia.
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