Apr 23, 2010

Research Projects

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Transgenic fungus-resistant barley – effects on gene expression and plant substances

(2005 – 2008) Friedrich-Alexander University Erlangen-Nuremberg, Chair of Biochemistry

Topic

Two lines of genetically modified barley are to be investigated for their effects on parasitic and beneficial fungi (that live in symbiosis with the plant). One of the lines produces a chitinase, a protein that breaks down the cell walls of pathogenic fungi. The other line produces a glucanase, an enzyme which improves the barley’s quality for brewing. Glucanase can also break down glucanes, which are parts of fungal cell walls.

These questions were examined in detail:

• How and to what extent are transgenic plants infested with fungal pathogens? Is the interaction between plant and beneficial fungi in the field affected? (Project partner: University of Giessen)
• How does the production of the recombinant proteins affect the expression of other genes and the content and quality of the corn? (Project partner: University of Erlangen)

The following description relates to the research area of the University of Erlangen.

Summary

The differences in the gene activity are greater within the conventional barley strains than when compared to the transgenic lines. The insertion of an additional gene into the barley lines did not have an effect on either the gene activity of the other genes or on the composition of the metabolites – regardless of whether this involved a transgenic or a non-transgenic barley strain. Mycorrhizal fungi, however, show no significant influence on the gene activity.

Experiment description

Plant material

Plant material was available from two transgenic barley lines and their non-transgenic parent lines. The transgenic lines were developed and supplied by Washington State University in the US. The plants’ increased resistance potential to the important fungal pest Rhizoctonia solani has already been demonstrated in laboratory experiments over there.

Used Genes

• Chitinase: A gene was inserted into the plants of the first barley strain which codes for the enzyme chitinase. It is produced in all parts of the plant. Chitinases cause the specific breakdown of chitin, the key component of fungal hyphal walls. The gene comes from a beneficial soil fungus found throughout the world (Trichoderma harzianum), which parasitizes a range of fungi which cause significant economic damage to crops.
• Glucanase: The second strain of transgenic barley produces the enzyme glucanase. The gene for glucanase was isolated from a soil bacterium (Bacillus amyloliquefaciens), and it improves the quality of the barley for brewing. Grains with glucanase also have nutritional advantages. Glucanases enhance the digestibility of cereals, which historically have had limited use in animal feed.

The gene for glucanase is only active in the endosperm tissue of the grain (the tissue that feeds germinating seeds). Nonetheless, the possibility exists that glucanases could be released into the soil at germination. Because the enzyme can break down the cell walls of certain fungi, the enzyme could influence the ability of both beneficial and harmful fungi to colonise the plant.

Greenhouse and field

Experiments were carried out in the greenhouse and in the field.

For the greenhouse trials, the transgenic barley lines were infected with two parasites (Rhizoctonia solani or Fusarium graminearum) and a beneficial fungus (Piriformospora indica). Plants colonised by P. indica show a range of positive effects, such as increased growth and increased resistance to pathogens or to drought stress.

In order to establish whether there is a correlation between effects on the metabolism and the expression of the transgenes, barley lines that produce three different levels of chitinase are used. In addition, a line that produces glucanase and the parent lines are also used.

The field trials were conducted on the site of the University of Giessen. From 2006 to 2008 the two transgenic barley lines and their non-transgenic parent lines were sown and studied on plots measuring twelve square metres each. The barley lines were cultivated with and without a commercially available mycorrhizal fungus. This was worked into the ground before sowing.

Analysis of metabolites

Biochemical separation methods (HPLC) were used to produce profiles capable of revealing quantitative differences in the build-up of metabolites. Metabolites with quantitative deviations were selected and analysed (spectroscopic techniques).

Producing transcription profiles

The introduction of foreign genes and their products can lead to unexpected changes in the gene expression pattern.

The following questions were of interest here:

• Are there differences between the transcription patterns of transgenic and non-transgenic plants?
• If so, how do the resistance genes influence the transcription of other genes that are linked to plant defence mechanisms?

In order to determine the possible impact of the two resistance genes, transcription profiles were drawn up. DNA microarrays were used to analyse to what extent a gene is transcribed under certain conditions. The transcription strengths of the transgenic and non-transgenic plant cells were to be compared to identify any differences.

To create the transcription profiles, RNA was extracted from tissue samples from the transgenic and non-transgenic plants at three different times during the plant growth period. The barley lines were cultivated with and without a commercially available mycorrhizal fungus, which is incorporated in the soil before sowing.

Quantitative and qualitative measurement of the barley corns

The expression of the resistance genes can also affect the substances in the barley corns, their number and weight. The project therefore recorded this data.

Results

Analysis of metabolites

The first phase of the project developed methods for identifying metabolites in barley leaves. Since almost every class of substance to be measured needs its own preparation method and there is only a limited quantity of sample material available, it was necessary to optimise the preparation methods so that as many classes of substance as possible could be covered by just a few methods. For this reason, a large number of preliminary experiments were carried out with specially planted non-transgenic plant material. The evaluation of these experiments showed that the methods adapted for analysing the metabolites are suitable.

These methods were then used to examine the transgenic and non-transgenic greenhouse samples infected with parasitic fungi or with a beneficial fungus for various metabolites.

An evaluation of the initial data showed that under field conditions different varieties of barley and different levels of fungal infestation result in differences in the metabolites measured and in the transcription profiles. No transgene-linked changes were found. These results were to have been confirmed in a repeat trial.

Since it was not possible to repeat the trials because of crop vandalism in 2006/2007, limited greenhouse trials were set up for the repetition in order to be able to draw statistically relevant conclusions. Evaluation of the repeat trials is not yet complete.

The examination of outdoor and greenhouse trials showed that the influence of the strain or the environment as well as the colonisation by mycorrhizal fungi have a significant effect on the composition of the metabolites. However, the expression of an additional gene had no measurable influence.

Construction of transcription profiles

To investigate the effect of an additional gene in the genome of two different barley lines, transcription profiles of leaf material from outdoor and greenhouse trials were compiled and evaluated.

No statistical differences were found in the gene activity between non-transgenic and transgenic barley plants. A difference in expression of 22 genes was found only in one transgenic line and the conventional reference strain. In addition, the colonisation of the plants through mycorrhizal fungi was only influenced gene expression slightly in one transgenic barley line (Golden Promise).

However, large differences in the gene activity between control lines were measured. More than 1600 genes were differentially expressed to various degrees between the two strains. The function of most of these genes is unknown.