Most monocotyledonous crop species have large genomes and genetic transformation tends to be difficult and/or time consuming. Therefore the VIGS technique can be very useful for functional studies of monocot genes.
We are focusing on the use of Barley stripe mosaic virus (BSMV) for VIGS in monocots: barley, wheat, oat, Brachypodium distachyon. The picture shows silencing of the phytoene desaturase (PDS) gene (bottom) compared to a non-silenced control leaf (top):
From April 1 2007 to March 31 2010 PhD student Katrin Geisler will work on this project in Denmark and UK. Katrin received her Diploma March 2007 at the Max-Planck-Institute of Molecular Plant Physiology in Golm, Germany.
Avenacins are secondary metabolites found in oat roots that protect the plants against fungal diseases such as take-all.
The aim of the project is to develop a virus vector based on barley stripe mosaic virus (BSMV) that can be used to study the genes involved in avenacin biosynthesis in oat roots. It is well established that BSMV can silence genes in barley and wheat, but for this project we have obtained an oat infecting BSMV strain from Mike. C. Edwards USDA-ARS, Fargo ND.
Because take-all is a devastating disease of wheat and barley and no resistance is currently known in these two species, the hope is that knowledge on the avenacin biosynthesis in oat will allow transfer of the complete pathway to other plants. This might allow breeding cereals that are resistant to take-all and other devastating diseases.
The aim of the project is to generate molecular markers for the bc-resistance genes in P. vulgaris conferring specific resistance towards Bean common mosaic virus and Bean common mosaic necrosis virus.
A European Union Marie Curie Intra-European Fellowship to Dr. Andrzej Pacak is financing these studies of genes involved in phosphate uptake in barley and wheat.
Most cases of genetically engineered virus resistance involve an RNA-degradation mechanism (post-transcriptional gene silencing, PTGS) triggered by a virus-derived transgene. This mechanism can give very efficient virus resistance without any negative side effects on the plants. However recent findings have raised the possibility, that this kind of resistance may be compromised if plants are infected with viruses different from the one targeted by the resistance. We have studied these questions in transgenic potato (Solanum tuberosum) transformed with simple or with inverted-repeat constructs based on the coat protein gene from Potato virus Y.
RNA silencing techniques have been used for years to create transgenic plants with virus resistance . Recently, these techniques have also been shown to be able to generate resistance towards plant pests such as nematodes and, most recently, certain insect species. We are exploring the potential of RNA silencing for protecting crop plants against insect pests.
Enhanced nutrient transport for the benefit of crops and environment
Plants, which are effective in nutrient uptake, can contribute to improving the environment by reducing the loss of nutrients and the pollution of streams and lakes. Surplus of nutrients is one of the major culprits in the pollution of aquatic environments and phosphorus is one of the compounds causing problems. Phosphorus is on the other hand an essential nutrient for both plants and animals and addition of phosphorus to soil need to maintained in order to ensure proper crop growth and hence production of healthy feed for our domestic animal. In this project we aim for a solution, which improves the phosphate uptake by crops as well as the environmental quality. Phosphate uptake by plants can follow to pathways: Either directly through the roots or through fungi, which develop mycorrhizas in symbiosis with roots. In both cases it is molecules named phosphate transporters, which ensures that phosphate enters the root. The phosphate transporters must be very effective as the amount of plant available phosphate is usually small at the root surface. It is well known that plants differ in their ability to take up nutrients. Our hypothesis is that such variation may be caused by variation in the effectiveness of phosphate transporters. We will therefore investigate whether variation also exists among those genes encoding the phosphate transporters. We will utilize existing knowledge obtained by other researcher from their study of Arabidopsis and other model plants and we will use it to investigate phosphate transporters in pea and wheat. We have chosen pea and wheat because they are important crops and because we can use virus as nanoscopic tools in both of them to identify those phosphate transporters that are most important.
Approximately one fifth of all know plant viruses belong to the genus potyvirus and many of these viruses cause disease in agricultural and horticultural crops worldwide. Natural resistance providing protection against these viruses is present in many plants. A remarkably large fraction of these resistances are inherited as single recessive genes and some of these genes result in complete immunity to specific viruses. The recessive nature of these genes suggests the resistant plants fail to support virus replication of spread. Our research is aimed at clarifying the basis for this type of resistance to increase the potential for exploitation of this type of resistance in future plant breeding.
Resistance genes in pea
Construction of infectious clones
Pea seed-borne mosaic virus pathotypes
Bean yellow mosaic virus
Identification of susceptibility factors
In addition to using transgenic plants in research we also participate in developing new technology for producing transgenic plants. The current technology has not changed since insertion of new traits in plants begame possible nearly 20 years ago. Most procedures involve selection markers such as antibiotic resistance to select the few cells that have the new genes stably inserted. Although the probability is extremely low, the use of such antibiotic resistance genes could lead to microorganisms in the soil or in the gut acquiring this antibiotic resistance. We have constructed a vector for Agrobacterium-mediated transformation to eliminate this problem. In this vector an intron has been inserted into the coding region of the antibiotic resistance marker -Neomycin phosphotransferase II (NPTII). This new vector was shown to give the same transformation frequency in both potato and tobacco when compared to a similar vector without an intron in the antibiotic resistance gene (Libiakova et al 2001).
Clones of two isolates of East African cassava mosaic virus (EACMV) have been modified to serve as silencing vectors in cassava in collaboration with Dr. S. Winter at the DSMZ, Braunschweig. Very efficient silencing of a cassava magnesium chelatase gene has been demonstrated. Attempts to use this system to interfere with replication of other viral pathogens are in progress.