Oct 30, 2002

Research Projects

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Investigating the influence of transgenic virus-resistant sugar beet on other viruses

(1999 – 2002) Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Virology, Microbiology and Biosafety; Braunschweig / Institute of Sugar Beet Research (IfZ); Göttingen

Topic

By transferring certain virus genes, it is possible to produce plants with a high level of virus resistance. It is, however, conceivable that recombination may take place between the viral RNA segments that are formed in the transgenic plant and the RNA in more or less distantly related viral strains that attack the transgenic plants. This could result in viruses with a modified pathogenicity. Certain viruses are clearly more prone to recombination than others.

The following questions were investigated using sugar beet with a genetically engineered resistance to the A-type rizomania virus (BNYVV – beet necrotic yellow vein virus) as a model:

• Do recombinations take place between the introduced transgene in the sugar beet and other strains of the virus or other viruses?
• Do recombinations also occur in non-transgenic plants that are infected with various virus strains (mixed infection)?
• If modified viruses do develop, are they affected by infection-stimulating conditions (higher temperature, damp soil and light supply)?
• If such viruses do develop, do they display a higher pathogenicity than the existing viruses?

The project also aimed to investigate whether new viruses have already formed on fields in which transgenic virus-resistant sugar beet has been released in previous years.

Summary

• No viral recombination was found, either in the transgenic plants or in the plants with mixed infection.
• In a model experiment in the greenhouse, the transgenic virus-resistant beet plants were usually free of infection at low temperatures. At higher temperatures infection often appeared in the transgenic plants only after four months.
• Since no new viruses have been found to date, the question concerning higher pathogenicity could not be investigated.
• Neither were any new viruses resulting from recombination found in the soil of former release fields.

Experiment description

Green house experiments. The experiments were carried out under conditions that were presumed to be more conducive to the development of new viruses by recombination than conditions in the field: the soil in the greenhouse was more heavily infected with viruses. Infected lateral roots remained in the soil after the beets were removed, thereby increasing the virus content. In addition, three sugar beet generations were planted per year in the experiment, whereas normally, they would be planted on the same field only every third year.

The soil used in the greenhouse contained B-type BNYV viruses and BSB viruses (beet soil-borne virus, which is widespread in European sugar beet areas). Two different transgenic beet lines were studied on this soil. They displayed a higher susceptibility to BNYVV than other transgenic lines:

• transgenic sugar beet with virus resistance produced by transferring the coat protein gene of the BNYVV A-type,
• transgenic sugar beet with virus resistance based on the suppression of certain virus genes (co-suppression); the plants also contain the coat protein gene of the A-type BNYVV.

By way of comparison, a non-transgenic beet variety with mixed infection and a high susceptibility to viruses was also investigated.

Detecting recombination. The root samples were tested for virus content and recombination using ELISA and PCR. To do this, the samples were examined for a specific genome segment, one end of which can come only from the virus, the other end of which may also come from the transgene. Special methods were used to test the PCR products for changes that can be traced back to recombination.

Influence of infection-stimulating conditions. Two different growing conditions were tested:

• with day temperatures of 24°C
• with day temperatures of 18°C

New viruses. Soils of release fields infected by viruses were investigated for new viruses resulting from recombination. In addition, non-transgenic sugar beet plants were planted in the greenhouse in soil samples in which transgenic, virus-resistant sugar beet had been planted in 1995, 1996 or 1997. Their roots were examined. The samples taken after harvest were analysed using molecular biological methods (ELISA and PCR).

Results

Enough BNYV viruses were present in the plants of both transgenic lines under the chosen conditions in the greenhouse for recombinations to be expected in theory.

Recombination. In plants with mixed infection results were often obtained in the first instance that appeared to point to recombination. However, these methods often produce PCR products that simulate recombinations where none are present (artefacts). These artefacts were also obtained when sap samples were mixed from plants that were infected only with A-type BNYVV or only with B-type BNYVV. The recombinations in these mixed saps could not have developed in the plants infected with only one virus; they must have developed during the PCR process. In order to rule out such “false positive” results, a special PCR method was developed and all previously tested samples were re-tested. This time, no recombination was detected in any of the trial plants; not even in those with mixed infection.

Overall, the experiments show that the detection of recombinations must be carried out with extreme care in order to rule out artefacts.

Effect of infection-stimulating conditions

• When plants were infected with BSBV, the different cultivation conditions had no effect.
• With BNYVV infection, most of the transgenic beet plants were not infected at low temperatures.
• At higher temperatures, infections in the transgenic plants could often be reliably detected only after four months.
• The non-transgenic plants were all infected.

New viruses. Even in the sugar beet plants cultivated in the soil of former release fields (infected with BNYVV B-type) no recombination has so far been found. The A-type coat protein gene formed there between 1995 and 1997 by the transgenic plants could no longer be detected in the soil.