Heterologous expression of functional Ptr ToxA

Ptr ToxA, a proteinaceous host-selective toxin (HST) produced by the fungus Pyrenophora tritici-repentis, was expressed in Escherichia coli and purifi ed as a polyhistidine-tagged, fusion protein (NC-FP). NC-FP, consisting of both the N and C domains of the ToxA open reading frame (ORF), is produced as an insoluble protein in E, coli at approximately 10 to 16 mg per liter o f culture. Following in vitro refolding, NC-FP elicits cultivar-specific ne crosis in wheat, with a specific activity similar to that of native Ptr Tox A, A fusion protein consisting of only the C domain has approximately 10 to 20% of the activity of native Ptr ToxA, These data suggest that (i) the N domain is important for maximal activity of Ptr ToxA, (ii) the N domain doe s not function to eliminate activity of the protoxin, and (iii) post-transl ational modifications of Ptr ToxA are not essential for activity. A C domai n construct with a cysteine residue mutated to glycine is inactive. This, p lus the observation that toxin activity is sensitive to reducing agents, pr ovides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity. The heterologous expressio n of Ptr ToxA provides a valuable tool for addressing a number of issues su ch as receptor binding studies, structure/function studies, and screening w heat cultivars for disease resistance.