IDENTIFICATION OF THE SITE OF ACTION OF PTR TOX A IN WHEAT
L HARDISON and LM CIUFFETTI
Department of Botany and Plant Pathology, Oregon State University, Corvallis, USA
Background and objectives
The fungus Pyrenophora tritici-repentis is the causal agent of tan spot of wheat (Triticum aestivum) and produces the protein host-selective toxin, Ptr ToxA , which is essential for pathogenicity . The gene for Ptr ToxA has recently been cloned . We are currently investigating the site of action of ToxA in wheat tissue. Our current hypothesis is that the protein toxin is a ligand of a membrane-bound receptor in wheat, and thus we are using the yeast two-hybrid system to identify possible interactions between ToxA and a putative wheat receptor protein. Several factors led us to choose the diploid wild wheat Aegilops speltoides for this study: (i) we have identified accessions of Ae. speltoides that are either sensitive or insensitive to ToxA; (ii) Ae. speltoides is recognized as one of the three diploid progenitors of hexaploid wheat, and is the probable donor of the B genome of hexaploid wheat; (iii) the gene for resistance (and toxin insensitivity) to P. tritici-repentis was localized to chromosome 5BL in hexaploid wheat [3, 4]. Thus, the use of the diploid Ae. speltoides simplifies the genetic analysis of the toxin-wheat interaction, while providing a highly similar, if not identical, framework for the interaction in hexaploid wheat.
Materials and methods
We have screened 65 accessions of Ae. speltoides for sensitivity to Ptr ToxA, and have identified 16 accessions that are highly sensitive and 30 that are toxin-insensitive. Selfed F2 populations of sensitive Ae. speltoides var. speltoides exhibit strong necrosis when infiltrated with either purified toxin or crude culture filtrate. A cDNA library was prepared from young leaves of sensitive accessions and cloned into an expression vector as a fusion protein with the yeast GAL4 activation domain. The N+C domains  of the ToxA gene were cloned into an analogous vector containing the GAL4 DNA-binding domain. The yeast two-hybrid assay  was performed by co-transformation of a yeast strain with the cDNA library and the ToxA construct. Interactions between ToxA protein and a wheat protein were detected by expression of reporter genes.
Results and conclusions
We have used the yeast two-hybrid assay to identify interactions between the Ptr ToxA protein and proteins from toxin-sensitive diploid wheat. False-positive cDNAs were eliminated by repeated screening for reporter gene activity and by mating. Consequently, a number of wheat cDNA clones have been identified that appear to interact with ToxA. We are currently engaged in sequence analysis of these clones. These analyses should provide insights into relationships of these ToxA-interacting proteins (putative receptors) to one another, and possibly to the mode of action of Ptr ToxA.
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