Molecular Biotechnology. Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Plant Genetic Engineering: Methodology
Plant transformation with the Ti plasmid from Agrobacterium tumefaciens

The Gram-negative soil bacterium Agrobacterium tumefaciens is a phytopathogen that transforms plant Cells during its life cycle. This transformation leads to The formation of crown gall, a tumor that disrupts normal plant growth (Fig. 17.1). This disease, which has serious agronomic consequences, affects only dicotyledonous plants, particularly grapes, stone fruit trees, and roses. Crown gall formation begins with the entry, integration into The plant Cell genome, and expression of a specific segment of bacterial plasmid DNA, known as T-DNA (transferred DNA). T-DNA is a portion of the tumor-inducing plasmid (Ti plasmid) carried by most strains of A. tumefaciens.

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Fig. 17.1. Infection of plants by A. tumefaciens and crown gall formation.

Fig. 17.2. Chemical structures of acetosyringone and hydroxyacetosyringone. These compounds are released by the plant in response to wounding and activate the vir GENES OF THE Ti plasmid.

The length of the T-DNA varies from 12 to 24 kb depending on the strain. Strains of A. tumefaciens that do not contain the Ti plasmid are unable to induce crown gall development.

The infection process begins with the attachment of A. tumefaciens to plant cells at the wound site, often at the Base of the stem (the ROOT crown). It was previously assumed that A. tumefaciens infects wounded plants simply because The Cell wall is disrupted, removing the physical barrier that prevents Bacteria from entering The Cell. However, it is now believed to be due to specific Phenolic Compounds, acetosyringone and hydroxyacetosyringone (Fig. 17.2), which are released by the wounded plant. These compounds are similar to certain products of the main plant biosynthetic pathway for secondary metabolites, such as lignins and Flavonoids. Acetosyringone and hydroxyacetosyringone activate virulence (vir) genes, which are located in a 35-kb region of the Ti plasmid outside the T-DNA. The products of the vir genes are required for the transport and Integration of the T-DNA (Fig. 17.3) into the plant cell genome. There are at least seven different vir genes.

Fig. 17.3. Genetic Map of the Ti plasmid (not to scale). The T-DNA contains auxin, cytokinin, and opine genes, which are transcribed and translated only in plant cells. Outside the T-DNA lie the vir Gene cluster, the gene(s) encoding opine Catabolism enzyme(s), and THE ORIGIN OF Replication (ori), which ensures stable plasmid maintenance in A. tumefaciens. L and R represent the left and right flanking sequences, respectively.

Fig. 17.4. Biosynthesis of auxin and cytokinin mediated by Enzymes encoded by the T-DNA genes of the A. tumefaciens Ti plasmid. A. Auxin synthesis begins with the Conversion of Tryptophan to indole-3-acetamide, catalyzed by tryptophan monooxygenase. This is followed by The conversion of indole-3-acetamide to indole-3-acetic acid by the enzyme indole-3-acetamide hydrolase. B. Cytokinin synthesis involves the attachment of the isopentenyl group of isopentenyl diphosphate to 5'-AMP, catalyzed by the enzyme isopentenyltransferase, to form isopentenyladenosine monophosphate.

Following the attachment of Ti-plasmid-bearing A. tumefaciens to the PLANT CELL AND the activation of the vir genes, the T-DNA is transported into the cell, apparently via a mechanism analogous to The transfer of plasmid DNA from a donor to a recipient cell during conjugation. During this process, the T-DNA is in a single-stranded form, and it is in this form that it integrates into the plant chromosomal DNA.

The transition of T-DNA into a single-stranded form begins with the generation of nicks at both of its flanking sequences. Consequently, the right flanking sequence ends up at the 5' end of the single-stranded T-DNA, and the left at the 3' end. It is assumed that the integration of T-DNA into the plant genome depends on specific sequences located within the right flanking sequence, which contains a 25-bp repeat. A similar repeat is present in the left sequence, but, as shown by deletion mutagenesis, it does not participate in integration.

Most T-DNA genes are activated only after integration into the plant genome. Their products induce the formation of crown gall. The iaaM and iaaH genes, also known as tms1 and tms2, respectively, encode enzymes involved in the Synthesis of the plant hormone auxin (indole-3-acetic acid). The iaaM gene encodes the enzyme tryptophan 2-monooxygenase, which catalyzes the conversion of tryptophan to indole-3-acetamide, while the iaaH gene encodes indole-3-acetamide hydrolase, which catalyzes the formation of indole-3-acetic acid from indole-3-acetamide (Fig. 17.4, A). In addition, the T-DNA carries the tmr gene (also known as the itp gene), which encodes isopentenyltransferase, an enzyme that catalyzes the attachment of an isoprenoid side chain to 5'-AMP to form the Cytokinins isopentenyladenine and isopentenyladenosine monophosphate (Fig. 17.4, B). Hydroxylation of these compounds by plant enzymes yields the cytokinins trans-zeatin and trans-ribosylzeatin, respectively. Both auxin and cytokinins regulate plant cell GROWTH AND DEVELOPMENT, but when present in excess, they can induce tumor formation, such as crown gall, in plants. In addition to the auxin and cytokinin genes, the T-DNA of each specific Ti plasmid contains a gene determining the synthesis of a compound from the opine class. Opines are unique Condensation products of amino and keto acids, or Amino Acids and sugars. For example, the condensation of Arginine and pyruvic acid yields octopine; arginine and alpha-ketoglutarate yields nopaline; and a bicyclic derivative of glutamic acid and sugar yields agropine (Fig. 17.5). Opines are synthesized within the crown gall and then secreted. They can be utilized as a carbon source (and sometimes as a nitrogen source) by any A. tumefaciens strain that carries the gene(s) for the catabolism of the corresponding opine on its Ti plasmid (Fig. 17.3), located outside the T-DNA. Most other soil microorganisms studied are unable to utilize opines as a carbon source. Thus, evolution has produced a unique set of mechanisms through which each strain of A. tumefaciens genetically transforms plant cells into "biological factories" for The production of carbon compounds that only these bacteria themselves can utilize.

Fig. 17.5. Chemical structures of three opines: octopine, nopaline, and agropine.