T4 lysozyme potatoes: Effect of T4 lysozyme in the soil, release of recombinant DNA from the plant and horizontal gene transfer in the soil

Topic

Transgenic potato plants were developed to control phytopathogenic bacteria which cause serious damage during cultivation and storage of potatoes.

Resistance to Erwinia carotovora, the pathogen that causes soft rot and blackleg, was produced by transferring the gene for a lysozyme from the T4 bacteriophage into the plant genome of the potato. Lysozymes attack the bacterial cell wall and cause it to dissolve (lysis).

The formation of T4 lysozyme as a defence against pathogens in plants represents a new quality for the environment. The project focuses on two possible environmental effects. For instance, lysozyme could have detrimental impacts on the soil ecosystem if it is released from the plants and retains its activity in the soil. The project will also check whether functional transgene DNA is present in the rhizosphere and whether horizontal gene transfer is possible.

Summary

It was possible to demonstrate that the T4 lysozyme potato shows increased lysozyme activity in the tubers, which can explain the increased resistance to Erwinia carotovora.

A novel test showed that active lysozyme is released from the roots into the surrounding area. T4 lysozyme possesses bactericidal activity against the pathogen Erwinia carotovora. In addition, a large proportion of the tested soil bacteria species, including various plant pathogens, proved sensitive to it. However, T4 lysozyme quickly lost its effectiveness in arable soil.

Functional transgene DNA was found in rhizosphere extracts from the release experiments.

The possibility of horizontal gene transfer to bacteria was tested using a newly developed monitoring procedure. In laboratory experiments it was possible to demonstrate the transfer of plant DNA into the genome of artificially prepared recipient bacteria – but not to naturally occurring bacteria.

Experiment description

The impacts of transgenic T4 lysozyme potato strains were assessed in the laboratory, greenhouse and in the field and compared with one transgenic control strain without the T4 lysozyme gene and with the non-transgenic parent variety Désirée. Highly sensitive measuring methods were developed for this purpose.

Setting up tests to check for T4 lysozyme activity against bacteria

Turbidity measurement. Plant-pathogenic Erwinia carotovora strains and other plant-pathogenic bacteria and soil bacteria were tested for their sensitivity to lysozyme using photometric tests. These tests are based on the reduction in turbidity of bacterial solutions to which defined quantities of purified T4 lysozyme are added. A reduction in turbidity occurs when the bacteria are sensitive to lysozyme. The test can also be used on soil and plant extracts.

Survival test. Plating was used to determine the survival of bacterial cells that had been exposed to defined lysozyme concentrations.

Release of T4 lysozyme from the roots

Nutrient solutions, into which roots of transgenic T4 lysozyme potatoes had grown, were tested for the presence of T4 lysozyme using the Western Blot method.

The presence of active lysozyme on the root surfaces of transgenic lysozyme potatoes was detected using a newly developed test procedure. The procedure involves fixing indicator bacteria on the hair-like roots, where they are exposed to the released lysozyme for a defined period. The proportions of living and dead bacterial cells are then measured by colouring them with fluorescent dyes and evaluating them using fluorescence microscopy.

Monitoring transgenic plant DNA in the soil

By using special DNA probes it is possible to “fish” recombinant DNA out of soil samples and to purify it efficiently, so that it can then be amplified using PCR (magnetic capture hybridization).

Evidence of possible gene transfer to soil bacteria

In order to check whether transgenic DNA in the soil is functional, and that bacteria could therefore use its information, a new test system was developed. Bacterial strains of Acinetobacter were used as recipient bacteria. They have an incomplete resistance gene. To repair this gene they need a particular gene segment from the transgenic potato plants. If the required transgenic DNA is present in the soil sample, Acinetobacter uses it to recreate an intact resistance gene. Successful reproduction of the bacterium then serves as evidence of the presence in the sample of the transgene under investigation.

Results

The aim of this project was to investigate the effect of released lysozyme on the microbial ecosystem in the soil and trace the transgenic DNA after the plant material rots.

Effect of T4 lysozyme

It was demonstrated using various novel methods that potatoes which produce transgenic lysozyme have high activity against bacteria. The lysozyme fights the bacteria which cause soft rot and black leg in potatoes. Small amounts of T4 lysozyme are released from the roots in active form. In the laboratory experiment this led to the death of bacteria on the root surface. In arable soil, however, the lysozyme very quickly loses its effect.

Monitoring the transgenic plant DNA

A highly sensitive monitoring test was developed for the T4 lysozyme gene. Using magnetic probes, recombinant DNA was detected in almost all transgenic test variants.

Evidence of horizontal gene transfer

The rhizosphere of transgenic potato plants contains intact transgene DNA. This was demonstrated through the fact that specially constructed recipient bacteria can take up and integrate the DNA. Naturally occurring bacteria, however, were not able to use the transgene DNA. Horizontal gene transfer did not take place at this level.