TiPlasmids are Transferred from Bacteria to Plants

Although the F-plasmid of E. coli is limited in its host range to a few enteric bacteria, it can actually promote DNA transfer between E. coli and yeast! Similarly, broad host range plasmids of the IncP, IncQ and IncW incompatibility groups can mobilize DNA from gram-negative bacteria into both gram-positive bacteria and yeast. In both cases, the range of species in which the plasmid can survive and replicate is much smaller than the range of species to which DNA may be transferred. Therefore, many plasmids are degraded or destroyed after they are transferred to an incompatible cell. Some DNA mobilized in this manner may survive if it is recombined with the host chromosome or resident plasmids.

The greatest versatility in plasmid transfer is shown by the highly specialized Ti-plasmids (Ti = tumor-inducing) that allow certain bacteria to insert DNA into the nucleus of plant cells. The Ti-plasmid is carried by soil bacteria of the Agrobacterium group, in particular A. tumefaciens, and confers the ability to infect plants and produce tumors, inside which the bacteria grow and divide happily. This results in tumor-like swellings on the stems of infected plants, a condition known as "crown gall disease". The related Ri-plasmid is carried by Agrobacterium rhizogenes, which infects roots and causes hairy root disease.

Agrobacterium is attracted by chemicals, such as acetosyringone, which are released by wounded plants. It then enters via the wound and transfers a portion of crown gall Type of tumor formed on plants due to infection by Agrobacterium carrying a Ti-plasmid

Ti-plasmid Tumor-inducing plasmid. Plasmid that is carried by soil bacteria of the Agrobacterium group and confers the ability to infect plants and produce tumors

FIGURE 16.21 Structure of the Ti-Plasmid

The Ti plasmid of Agrobacterium has several regions. The T-DNA region is flanked by two inverted repeats and contains genes for auxin and cytokinin, which induce the plant cells to grow, and genes for opine synthesis, a carbon source for Agrobacterium. This region is transferred into the plant cell by the expression of the transfer genes found on the other part of the Ti plasmid. The Ti plasmid also has an origin of replication and genes for opine breakdown.

the Ti plasmid into the plant cell by a mechanism similar to bacterial conjugation. A slight abrasion that is trivial to the health of the plant is of course sufficient for the entry of a microorganism. The result is a crown gall tumor that provides a home for the Agrobacterium at the expense of the plant.

The Ti-plasmid consists of several regions (Fig. 16.21), but only one segment, the T-DNA (tumor-DNA), is actually transferred into the plant cell, where it enters the nucleus. The T-DNA is flanked by 25 bp inverted repeats. Any DNA included within these repeats will be transferred into the plant cell. Consequently, Ti-plasmids have been widely used in the genetic engineering of plants. The virulence genes on the plasmid are responsible for cell-to-cell contact and transfer of the T-DNA but do not themselves enter the plant cells.

Acetosyringone, which attracts the bacteria to the wounded plant, also induces the virulence genes, thus facilitating the transfer of the T-DNA region (Fig. 16.22). Ace-tosyringone binds to VirA protein in the Agrobacterium membrane. This activates VirG, which in turn switches on the other vir genes, including virD and virE. VirD makes a single-stranded nick in the Ti-plasmid at the left border of the T-DNA and the T-DNA unwinds from the cut site. The single-stranded T-DNA is bound by VirE protein and unwinding stops at the right border. The Ti-plasmid then replicates by a rolling circle mechanism as the single stranded T-DNA region enters the plant cell. Overall this results in DNA transfer from the bacteria into the plant cells. The mechanism resembles bacterial conjugation and the "virulence" genes of the Ti-plasmid are equivalent to the tra genes of other plasmids. The T-DNA then integrates at random into a chromosome in the plant cell nucleus.

Once inserted, the genes in the T-DNA are switched on. The enzymes they encode synthesize two plant hormones, auxin and cytokinin. Auxin makes plant cells grow bigger and cytokinin makes them divide. When this happens rapidly in the absence of normal cell differentiation, the result is a tumor (Fig. 16.23 and Fig. 16.24).

auxin Plant hormone that induces plant cells to grow bigger cytokinin Plant hormone that induces plant cells to divide

T-DNA (tumor-DNA) Region of the Ti-plasmid that is transferred into the plant cell nucleus

Cytokinin Opine genes synthesis genes

Left border

(inverted repeat)

Right border (inverted repeat)

Cytokinin Opine genes synthesis genes

Right border (inverted repeat)

Left border

(inverted repeat)

Opine breakdown

Virulence genes (i.e. tranfer genes)

Origin of replication

Opine breakdown

Virulence genes (i.e. tranfer genes)

Origin of replication

Bacteria carrying Ti-plasmids infect plants and cause the formation of tumors.

Only part of the Ti-plasmid enters the plant cell, where it integrates into the plant chromosomes.

Injured plant

Bacteria incorporated

Tumor forms

FIGURE 16.22 Formation of Tumor by Agrobacterium

Agrobacterium are attracted to an injured region of a plant by sensing molecules of acetyosyrigone. The bacteria enter the plant through the open wound, and begin colonizing the area. The plant cells are stimulated to divide and a tumor forms around the bacteria.

Molecules of acetosyrigone are sensed by the Agrobacterium

Molecules of acetosyrigone are sensed by the Agrobacterium

FIGURE 16.22 Formation of Tumor by Agrobacterium

Agrobacterium are attracted to an injured region of a plant by sensing molecules of acetyosyrigone. The bacteria enter the plant through the open wound, and begin colonizing the area. The plant cells are stimulated to divide and a tumor forms around the bacteria.

Bacteria invade

Agrobacterium

Bacteria invade

Plant cells divide

Plant cells divide

Agrobacterium

FIGURE 16.23 Crown Gall Tumor Caused by Agrobacterium

A crown gall tumor formed by Agrobacterium is shown on a tree trunk. © E. R. Degginger, Photo Researchers Inc.

FIGURE 16.23 Crown Gall Tumor Caused by Agrobacterium

A crown gall tumor formed by Agrobacterium is shown on a tree trunk. © E. R. Degginger, Photo Researchers Inc.

Modified Ti-plasmids are widely used in genetic engineering of plants.

The T-DNA also carries genes that subvert the plant cell into making opines. These are unusual nutrient molecules that are made at the expense of the plant cell but can only be used by bacteria that carry special genes for opine breakdown. The genes for opine degradation are found on the part of the Ti-plasmid that does not enter the plant cell. So the Agrobacterium can grow by using the opines but the plant cannot use them. Other bacteria that might infect the plant are also excluded as they do not have the opine breakdown genes either.

Modified Ti-plasmids are widely used in the genetic engineering of plants. The genes for plant hormones and opine synthesis are removed and the genes to be trans-

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