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 purified 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 of culture. Following in vitro refolding, NC-FP elicits cultivar-specific necrosis in wheat, with a specific activity similar to that of native Ptr ToxA. 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 does not function to eliminate activity of the protoxin, and (iii) post-translational modifications of Ptr ToxA are not essential for activity. A C domain construct with a cysteine residue mutated to glycine is inactive. This, plus the observation that toxin activity is sensitive to reducing agents, provides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity. The heterologous expression of Ptr ToxA provides a valuable tool for addressing a number of issues such as receptor binding studies, structure/function studies, and screening wheat cultivars for disease resistance.
Tuori
R P
RP
Department of Botany and Plant Pathology, Oregon State University, Corvallis 97331, USA.
Wolpert
T J
TJ
Ciuffetti
L M
LM
D002845:Chromatography; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins; D015151:Immunoblotting; D009183:Mycotoxins / Q000737:chemistry* / Q000378:metabolism; D009336:Necrosis; D018515:Plant Leaves / Q000633:toxicity*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D005656:Fungal Proteins; D009183:Mycotoxins; D011993:Recombinant Fusion Proteins; C105776:TOXA protein, Pyrenophora tritici-repentis