Scientific topics covered under the projec

The goal of MOBITAG, i.e. the build-up of infrastructure for the development and testing of novel agricultural biotechnologies, can be reached only through practical training. Investigations targetted to several topics specified below are consistent with the aims of European research declared in Decision No. 1982/2006/EC of the European Parliament and the Council (7th Framework Program). They are in the focus of contemporary biology, require international collaboration, and are of pivotal importance for the advance of agricultural biotechnologies. Following paragraphs describe briefly specific research tasks.

1. Genome analysis

Genome composition is a precise characteristic of each individual. Various methods of DNA analysis allow a rapid identification of species and their genotypes, as well as of introduced transgenes. Research conducted in the frame of MOBITAG will focus on the detection and identification of the viral and viroid infections in plants and on the identification of insect pests and their genotypes. Genome analysis of plants will also be performed as part of the transgenosis research (along with the distinction of various transgenes).

1.1. Plant viroids (small pathogenic RNAs) show fast evolutionary adaptations to new conditions and crops. They represent an expanding group of plant pathogens. In some species viroids propagate at ultra low levels and their detection based on the plan phenotype is impossible. Viroid diagnosis based on sensitive and specific methods like real-time PCR, RT PCR in combinations with molecular hybridization methods must be developed and certified for diagnosis. The team has already experience in this type of work (Matoušek et al. 2007b). Proposed research will focus on the monitoring of viroids that cause great economic losses in Central Europe: potato viroids in the potato, tomato, and ornamentals, hop viroids, grapevine viroids, and fruit trees viroids. Thermodynamic methods will be exploited to monitore viroid populations. Research is supported by project 1QS500510558 of the Academy of Sciences.

1.2. Plant viruses and other pathogens are currently diagnosed serologically or by PCR. Neither of these methods is suitable for the simultaneous detection of a large number of pathogens (molecules). We propose to implement the strategy of oriented suspension array technology that had been developed for medical purposes. This method possesses the advantage of a liquid-type kinetics and allows simultaneous detection of various molecules. Up to 100 different detection micro beads can hybridize simultaneously and detect up to 100 different molecules. The method can be based both on nucleic acid hybridization and on antigen-antiserum reaction. The complexed nucleic acids and proteins, respectively, are subsequently sorted in a flow cytometer and analysed. We already have certain experience in the detection of pathogens by either PCR or the microarray technique. These skills will be enhanced through the training of new staff. Research is supported by grant 522/07/P338 from the Czech Science Foundation.

1.4. Molecular cytogenetic analysis of sex chromosomes in the lepidopteran pests. There is a high demand to develop efficient and species-specific methods for the control of certain moths. The sterile insect technique (SIT), which proved efficient in several species, relies on the rearing and release of large numbers of genetically sterile insects that hamper the reproductive potential of the wild population. Current SIT programmes for the management of lepidopteran pests are based on bisexual releases. Male-only releases, which would be more efficient and better acceptable by farmers, have never been tested, due to the lack of efficient ways of female elimination. We propose to develop genetic sexing strains in several lepidopteran pests that are suitable for SIT (reviewed by Marec et al., 2005). Sex chromosomes of the selected species will be examined with the methods of molecular genetics and cytogenetics to identify regions involved in the sex determination. Laser microdissection of these regions will be used to develop sex-chromosome painting probes, establish sex-chromosome libraries, identify the sex-linked genes and design molecular markers for the identification of sex-specific bacterial artificial chromosomes (BACs) in available BAC libraries. Further work will include synteny mapping of the sex-linked genes. Most of the research will be done on the codling moth, Cydia pomonella, which is the key pest of most pome fruit and some walnut orchards in temperate regions around the world (Fuková et al. 2007). Several other important pests such as the flour moth Ephestia kuehniella, the cabbage moth Mamestra brassicae, and the armyworms Spodoptera sp., will also be examined. Research is funded by grant 206/06/1860 from the Czech Science Foundation and through the Research Contract 12055/R of the International Atomic Energy Agency, Vienna, Austria.

2. Exploitation of natural compounds

Natural compounds have been widely exploited in plant protection for centuries. Crude extracts of the tobacco, the neem tree, and other plants represent the simplest use of natural products. Synthetic derivatives of natural compounds, such as pyrethroids, are another class of plant protectants. More sofisticated is the application of either agonists or antagonists of natural compounds, such as insect hormones (juvenoids and ecdysteroid agonists). The GMOs represent another step in the exploitation of natural compounds. Typical example is the transfer of Cry genes from Bacillus thuringiensis to plants that then produce the corresponding insect toxin and become pest resistant. We plan to promote research focused on the identification of natural products that could be applied in plant protection by means of the GM techniques.

2.1. Proteinase inhibitors for plant protection. The production of proteinase inhibitors is one of the mechanisms used by plants for their protection against herbivores. The inhibitors curb digestive proteinases and thereby reduce insect growth and reproduction. However, during the million years of their co-evolution with plants many insects found a counter-defence, most often in the production of proteinases that are insensitive to the inhibitors from plants. However, insects are unlikely to possess defence against their own proteinase inhibitors. We have identified such inhibitors in insect silk and demonstrated that one of them has a great potential for biotechnologies (Kludkiewicz et al. 2005). Different type of both proteinase and amylase inhibitors have been found in the digestive system of cockroaches (Vinokurov et al. 2007). In frame of MOBITAG we want to identify genes of these inhibitors and probe their potential in the plant protection. Support is currently provided by project IAA5007402 from the Grant Agency of the Academy of Sciences.

2.2. Insect neurohormones. Insects contains a variety of regulatory neuropeptides that possess vital functions. Their presence in excessive amounts or at wrong times causes lethal disorders in insect performance but we do not know yet how to achieve such a hormonal disbalance. Insertion of neuropeptide genes into plants can be considered but the hormones delivered to the insects via the food are likely to be destroyed by their digestive enzymes. Topical application has been considered impossible because insect integument is not permeable to hydrophilic compounds. However, our recent study showed that adipokinetic hormones (AKHs) can penetrate through the cuticle (Kodrík et al., 2002) and affect energy metabolism as well as behaviour of several insects, including serious crop pests. AKHs also interact with other insect hormones and modulate insect sensitivity to the insecticides and other stressors. We intend to explore the action of topically administered AKHs on the African migratory locust Locusta migratoria, the Colorado potato beetle Leptinotarsa decemlineata and several aphid species. The work will include AKH quantification by ELISA and investigations on the mode of AKH interaction with the juvenile hormones and insecticides. Research is supported by grant 522/7/0788 from the Czech Science Foundation.

2.3. Insect growth factors. Two families of Drosophila growth-promoting proteins are implicated in regulating cell growth and proliferation. The family of the Imaginal Disc Growth Factors (IDGFs) has six members related to chitinases. The family of the Adenosine Deaminase Growth Factors (ADGFs) has also 6 members, which are related to adenosine deaminases and have intrinsic enzymatic activity required for their mitogenic function (Žurovec et al. 2002). The physiological roles of ADGFs and IDGFs are not clear. ADGFs are involved in the decrease of extracellular adenosine and the genetic analysis showed that their presence is vital. ADGF-A,-C and-D mutants display multiple phenotypes including mortality and defects in various tissues. IDGFs are lectins that bind certain sugar moieties on the cell surfaces. Using the microarray RNA-expression analysis we found that IDGFs are important regulators of antibacterial and antifungal response. Preliminary data with IDGF gene disruption using RNA interference suggest that metamorphosis is a critical period for their function. Since insect homologs of IDGFs and ADGFs differ from the known vertebrate growth factors, they have a potential to be used in insect pest control as a new type of IGRs (insect growth regulators). We intend to explore this possibility using recombinant growth factors derived from a baculoviral or a bacterial expression system. Research is funded by the Grant Agency of the Czech Academy of Sciences (project IAA500070601).

3. Transgenosis and transgene expression

Crop production depends on the plant genotype, environmental conditions (soil quality, nutrient supply, irrigation, etc.), agrotechnical measures, and crop protection against diseases and herbivores. The genotype has been traditionally modified by lengthy breeding procedures but modern science provides tools for direct and targeted changes of the plant genotype. Such genetic modifications (GM) have opponents but rapid spreading of the GM crops (grown on more than 100 million ha in 2007) testifies to their advantages. GM maize, which expresses Cry1Ab gene derived from the soil bacterium Bacillus thuringiensis and is thereby resistant to the stem borers, has been approved for cultivation in Europe and is grown commercially in the Czech Republic. It is likely that new genes will be used for the production of insect-resistant GM crops. MOBITAG includes preparation of new GM plants and development of novel techniques of plant transgenosis. At a later stage, GM plants will be prepared with trangenes identified in the research tasks listed under B.1.1.5.2.

3.1. Spruce resistant to the bark beetle. The Norwegian spruce bark beetle, Ips typographus, is the most serious forest pest in large areas of the Euroasia. Partial control is achieved with the pheromone traps and insecticide application but an outbreak is imminent after any substantial damage to the spruce stands. A permanent spruce protection could be achieved through transgenosis. We plan to prepare GM spruce that will express a Cry toxin lethal for the bark beetle. This work requires a reconstruction of the natural Cry3A gene (De Maagd et al. 2003). Substantial modification of domain II, loop 1, is planned to increase toxin effectivity on the bark beetles. Recombinant toxins will first be produced in an expression system based on Escherichia coli to verify their action on I. typographus. Transgenic spruce embryogenic lines expressing the modified cry3A gene will then be developed and characterized. Research is funded by grant QH71290 from the National Agency for Agricultural Research.

3.2. MYB and TF biotechnology of plants. Transcription factor (TF)-mediated biotechnology will be developed with the specific Myb, bHLH amd bZip regulatory factors. This approach has a potential to regulate plant morphogenesis and metabolome (e.g. Broun 2004). The main goal of TF biotechnology research is to prepare systems for plant transgenosis that would modify desired parameters, for example the production of drugs such as prenylflavonoids or to increase the plant yield. Regulatory factors Myb, bZIP and bHLH will be isolated and analyzed on heterologous transgenotes (Matoušek et al. 2007a). The newly isolated genes will be employed for metabolome modifications in the hop (Humulus lupulus, management of the lupulin composition and content) and medicinal herbs. Agricultural traits will be affected via the morphogenetic action of TFs. TF biotechnology is a way to prepare new valuable materials for plant breeders. Research will be performed in collaboration with the partners in Belgium (group of Prof. D. DeKeuleleire) and Germany (Prof. G. Weber). Research is supported by grant 521/08/0740 from the Czech Science Foundation

3.3. Plant production of nucleolytic enzymes. The analysis of plant nucleolytic enzymes and their antitumorogenic potential is a new and promising research topic. Development of a large scale production of the plant-derived apoptotic nucleases in plants will be mediated via the system of Agrobacterium tumefaciens infiltrations. Since the nucleases possess wide antitumorogenic activities without unwanted side effects (Souček, 2006), the work will continue with a study of the mechanism of their cytostatic action. This research is conducted in collaboration with several institutions in the Czech Republic as well as with Prof. D. Riesner of the Düsseldorf University. Research is supported by grant 521/06/1149 from the Czech Science Foundation.

4. Safety aspects of GM crops

Chemical insecticides have played crucial role in the rise of agricultural production and the combat of diseases transmitted by the insects and ticks. However, they have also caused unwanted side effects and inflicted environmental damage persisting for decades. This experience calls for caution when a new technology is introduced. The deployment of transgenic plants is quite responsible step that should be based on the precautionary principle. Scientists must evaluate the risks and benefits of the use of transgenic plants, compare them with the risks and benefits of the standard techniques, and eventually propose, case by case, either acceptance or a refusal of the GM plant under investigation. Since all commercial insect-resistant GM crops carry a Cry gene from the bacterium Bacillus thuringiensis, we shall concentrate on the environmental safety of such Bt crops. Specific action of Cry toxins on a narrow range of the target species is generally considered to be one of the major advantages of the Bt crops (and of the sprays of B. thuringiensis) in comparison with the chemical pesticides. Uniform concentration of the toxin in major plant parts such as the leaves and stems, is regarded as another major advantage because it reduces the risk of resistance development. However, great variations in the toxin content have been reported. We shall address validity of both premises: Cry specificity and the uniformity of transgene expression in Bt crops.

4.1. The mode of Cry action. We do not know exactly why the Cry toxins act very specifically on certain insects or nematodes. For example, Cry1Ab is highly active on the European corn borer, Ostrinia nubilalis, but inactive on most other moths, all beetles, flies, and other insects. We chose to examine the cause of target specificity using Cry3Aa and beetles of the Tenebrionidae family. Some species of the family, such as Tenebrio molitor, are highly sensitive, but their close relatives, for example Tribolium confussum, are resistant. We shall search for the cause of this difference at the level of digestive proteinases that may destroy the toxin, and at the level of toxin receptors in the gut wall and other tissues. Current research is supported by grant 522/06/1591 from the Czech Science Foundation.

4.2. Measurements of transgene products. Trangenes are easy to detect but the proteins they encode are difficult to quantify. This is exemplified by the reports on unexplained fluctuations of the Cry toxins by more than one order of magnitude in the commercial Bt crops. The scatter and the random distribution of the established values indicate that part of the problem lays in the reliability of the quantification methods. Cry toxins are routinely quantified with the immunological kits of US provenience, which are sold in Europe by the French firm Agdia. Rough and chiefly qualitative measurements are done with paper stripes coated with the antibody linked to a dye, why quantitative data can be obtained with the ELISA kits. We know from experience that different kit batches yield different results, which makes the situation even more confusing: measured differences in the Cry content may reflect genotypic differences among the plants, effects of environmental factors on transgene expression or the stability of the transgene product, but they may also be due to faulty measurements. There is clearly a need for standardization that we plan to achieve by comparing the results of ELISA and those of a bioassay. Research is supported by grant 1M06030 from the Czech Ministry of Education, Youth and Sports.

Cited references