Work package 3

Search for innovative selection techniques (in vitro selection, candidate resistance genes and pathogenesis related protein) for resistance against the fungus and toxin accumulation

Objective:

We intend to develop an in vitro selection technique for FHBR and to investigate molecular mechanisms responsible for Fusarium resistance. In particular, candidate trichothecene toxin resistance genes will be studied: ABC transporter proteins (acting as drug efflux pump), glutathione-S-transferases and MRP-homologues (with a suspected role in detoxification and vacuolar sequestration of glutathione conjugates), and RPL3 (ribosomal protein L3, the ribosomal target of trichothecenes). In addition the role of defense related genes, pathogenesis related (PR) proteins and especially the role of antifungal proteins will be studied, which are potentially not only inhibitory for fungal growth but also inhibit DON-production by Fusarium. Also the role of low molecular defense compounds will be tested by development of markers for wheat homologs of benzoxazinoid phytoalexin biosynthesis genes of maize. Further candidate genes will be identified using RNA fingerprinting techniques.

Description of work:

Subtask 1. A novel in vitro strategy for early screening of FHB resistance in wheat

Preliminary results demonstrated a close correlation between resistance towards M. nivale on the leaves and FHBR on the ear. Leaf segments of wheat lines with known FHBR will be inoculated with a M. nivale spore suspension and incubated under defined conditions. Components of partial disease resistance will be measured.  

Subtask 2. Candidate genes for resistance, toxification, detoxification and toxin efflux

Resistance to DON and other trichothecene mycotoxins produced by Fusarium seems to be a an important component of FHB resistance. Resistance conferring mutations in the ribosomal protein L3, the target of toxin, have been found in yeast. We will characterize the small gene family (6 genes) encoding wheat RPL3, in order to search for special alleles in resistant germplasm. A yeast heterologous complementation assay should allow testing of the relevance of identified sequence changes. Markers for individual RPL3 genes will allow to determine whether QTLs for toxin or field resistance are detected. Glutathione S transferases (GST) are suspected to have a role in detoxification of trichothecenes, therefore markers for wheat ESTs with homology to Arabidopsis GSTs will be developed. Similarly, the large gene families encoding two types of ABC transporter proteins will be studied: 1) MRP homologues, suspected to function as transporters of glutathione conjugates into the vacuole, and 2) PDR5-like proteins, functioning as drug efflux pumps located in plasma membrane.  

Subtask 3. Candidate resistance genes for inhibition of toxin accumulation and fungal growth

We furthermore will use GUS-tagged isolates of F. graminearum and F. culmorum with DON toxin antibody assay to screen germplasm and segregating populations for the presence of antifungal proteins. We will identify lines containing substances which inhibit fungal growth and also those which inhibit toxin synthesis. The antifungal proteins will be further characterized. Similarly, low molecular defense compounds of wheat (benzoxazinoid phytoalexins) are suspected to have a role as inhibitors of fungal growth and/or toxin production. Wheat ESTs homologous to genes responsible for phytoalexin synthesis genes of maize will be used for marker development and testing on segregating populations. The pattern of expression of the candidate genes and genes identified in an unbiased RNA fingerprinting approach will be used to study the dynamics of the plant pathogen interaction and to produce “expression markers” for FHB resistance.