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Functional
Organization
of
T-DNA -
To study the detailed functional organization of both the octopine and the nopaline T-region, transposons Tn3 and Tn5 were introduced into isolated DNA fragments derived from the T-region and substituted in the Ti plasmid by in vivo recombination.
Specific deletions, removing one or more T-DNA transcripts, have been constructed for both octopine and nopaline T-DNA. These specific mutations and deletions can therefore be used to study the functions of some transcripts.
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Several mutant Ti plasmids are apparently avirulent on some plants, but on Kalanchoe and tobacco the mutations mainly change tumor morphology. Other mutations result in the loss of opine synthesis. Some mutations resulting in the inactivation of the mapped transcripts have no obvious phenotype.
Remarkably, all T -DNA functions shown to affect the tumor phenotype (and also host range) are located in the "common" T-DNA segment. Instead of undifferentiated octopine tumors, the mutant octopine tumors show root or shoot proliferation from the callus. Six different well defined transcripts have been derived from this common region of octopine and nopaline T ON As (viz., 5, 2, 1, 4, 6a, and 6b).
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Inactivation of transcript 4 results in root formation from the tumor on Kalanchoe stems but renders the plasmid avirulent on Kalanchoe leaves. The product of this gene must therefore prevent root formation and can thus be compared to the effect obtained with normal plant tissue by increasing cytokinin concentrations or lowering the auxin content.
The two transcripts 1 and 2 prevent shoot formation by both normal and transformed plant cells. The effect of these genes might be analogous to raising auxin or lowering cytokinin levels. Transcript 5 might inhibit the organization of transformed cells in teratoma. Transcripts 6a and 6b mainly affect tumor size.
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Agropine type tumors do not contain the homology to transcripts 6a and 6b but based on the presence of homologous DNA, should have the transcripts 5, 2, 1, and 4 in common with the octopine and nopaline tumors.
The rhizogenes root cultures contain only homology between the gene for transcript 1 and the octopine T-DNA, Octopine type II plasmids have very little homology with these common genes, although homologous DNA was detected at low stringency in the region of the transcript.
It has been inferred that none of the T -DNA transcripts are essential for the transfer of T-DNA segments into the plant genome. Opines were synthesized at the wounds infected with any of the Ti plasmid mutants tested, including those unable to induce a tumorous phenotype. This indicates that all the T-region mutants can still transfer the T-DNA genes.
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No single gene is absolutely necessary for tumor formation, perhaps because the common genes work cooperatively and synergistically. This may be illustrated best by the observation that mutant containing deletions of the genes for either transcripts 5, 7, 2, and 1 or 4: are able to induce tumors while deletions of all transcripts are not.
The T-DNA may playa role in the production of phytohormones by the transformed plant cells, either directly, i.e., through the synthesis of auxins and/or cytokinins, or indirectly, i.e., through interaction with the plant biosynthetic pathways.
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Root-forming tumors, brought about by inactivation of transcript 4 in both octopine and nopaline Ti plasmids, no longer' produced roots when complemented by the external addition of cytokinins. This could be explained if the cytokinin content were lower in these tumors compared to wild type tumors; moreover, extra cytokinins stimulate cell growth in tissue culture.
These mutant Ti plasmids resemble in many ways the Ri plasmids, which either induce unorganized tumors or proliferating roots, depending on the host plant. Hairy-root tissue contains agropine, indicating stable transformation of the differentiated tissue.
Notwithstanding the remarkable correlations between the effects pf genes 1 and 2 and the similar effects of exogenous auxins on the one hand, and of gene 4 and the similar effect of exogenous cytokinins on the other', it should be pointed out that there is as yet no formal proof that these genes are directly involved in the formation of these plant growth factors.
As described above, the T-DNA-containing tumor lines have different morphogenic phenotypes, either producing defective shoots (teratomas) or roots (hairy-root) or undifferentiated tumors, depending on the type of inciting Ti plasmid and host plant cell.
Experiments have shown that the expression of T-DNA functions can be controlled either at the gene transcription level or controlled epigenetically.
T-DNA linked genes go through meiosis as a Mendelian genetic trait. It was found by genomic hybridizations that the shoot derived from transformed plants retained only the T -DNA segment corresponding to the transcript, i.e., octopine synthase, while the tumors at least the majority of the cells contained the whole T-DNA stretch with the inserted Tn.
It still has to be demonstrated whether those T-DNA deletions occur during the induction phase or subsequent to T-DNA integration in the plant genome. Either case may have implications for plant genetic engineering.
If deletion occurs during the induction phase of transformation, this means that the octopine synthase gene can be transferred by itself and be stably incorporated into the plant genome.
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