5.4.6
MODIFICATION OF THE RIBOSOMAL TARGET AND DRUG EFFLUX PUMPS: MECHANISMS OF RESISTANCE TO TRICHOTHECENE MYCOTOXINS

R MITTERBAUER1, T KARL1, M LEMMENS2, H BUERSTMAYER2 and G ADAM1

1Center of Applied Genetics, University of Agricultural Sciences (BOKU), Vienna, Austria; 2Institute for Agrobiotechnology, Tulln, Austria

Background and objectives
Trichothecenes are a highly diverse class of toxic, sesquiterpenoid secondary metabolites that are produced mainly by plant pathogenic fungi. The contamination of important agricultural products such as wheat, barley or maize with the trichothecene mycotoxin deoxynivalenol (DON) due to infection with Fusarium graminearum and F. culmorum is a worldwide problem. Trichothecene mycotoxins act as inhibitors of eukaryotic protein biosynthesis and their formation is considered to be a virulence factor in a number of plant diseases [1]. We wanted to determine whether trichothecene resistance contributes to resistance towards Fusarium head blight (scab) in wheat and, using yeast as a model system, to identify potential molecular mechanisms of trichothecene resistance.

Results and conclusions
We determined the toxin resistance of a large number of wheat cultivars representing the full spectrum of Fusarium field resistance with an assay based on seed germination on DON-containing medium. Our results confirm that DON resistance is a relevant component of scab resistance, and may explain up to 40% of the variation observed between wheat varieties. Investigating trichothecene resistance in the yeast Saccharomyces cerevisiae we found that semi-dominant toxin-resistant yeast mutants either contain alterations in the target of trichothecenes, a ribosomal protein, or show increased drug efflux due to overexpression of an ABC transporter protein.

We have identified the mutation in the yeast ribosomal protein conferring trichothecene resistance and have introduced a plant homologue with the corresponding mutation into tobacco using Agrobacterium-mediated transformation. At present it is unclear if differences in the ribosomal resistance exist in the wheat breeding material. A restriction fragment length polymorphism between a highly sensitive and a highly resistant wheat cultivar used for generating a doubled haploid mapping population has been detected using the ribosomal protein gene as a probe.

The second mechanism, pleiotropic drug resistance mediated by broad specificity drug efflux pumps, seems to be the predominant mechanism operating in wheat. We observed a strong correlation between trichothecene resistance and resistance to other inhibitors (e.g. cycloheximide) that are substrates of the yeast ABC transporter protein encoded by the PDR5 gene. Evidence for the existence of homologous ABC transporter genes in plant genomes that most likely play a prominent role in plant defence against many toxic metabolites will be presented. Furthermore, we will report on our attempts to express the yeast PDR5 gene in tobacco.

Our identification of candidate trichothecene resistance genes could be extremely important for both classical resistance breeding strategies employing marker-assisted selection, and biotechnological approaches for improving Fusarium resistance of cereals.

References
1. Proctor RH, Hohn TM, McCormick SP, 1995. Molecular Plant-Microbe Interactions 8, 593-601.