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The Arg-Gly-Asp--Containing, Solvent-Exposed Loop of Ptr ToxA Is Required for Internalization

March 2008 , Volume 21 , Number  3
Pages  315 - 325

Viola A. Manning,1 Sara M. Hamilton,1 P. Andrew Karplus,2 and Lynda M. Ciuffetti1

1Department of Botany and Plant Pathology and 2Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331, U.S.A.

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Accepted 30 October 2007.

Internalization of the proteinaceous host-selective toxin, Ptr ToxA (ToxA), into sensitive wheat mesophyll cells is correlated with toxin activity. The solvent-exposed, Arg-Gly-Asp (RGD)-containing loop of ToxA is a candidate for interaction with the plasma membrane, which is a likely prerequisite to toxin internalization. Based on the percentage of cells affected by a given number of ToxA molecules in a treatment zone, the number of ToxA molecules bound to high-affinity sites was estimated at 3 × 106 per cell and the Kd for binding was estimated to be near 1 nM. An improved heterologous expression method of proteins that contain mutations in ToxA, coupled with a newly developed semiquantitative bioassay, revealed that some amino acids in the RGD-containing loop contribute more to toxin activity than others. Protease protection assays that detect internalized protein and inhibition of toxin uptake indicated that, for each ToxA variant tested, the extent of toxin activity correlates with the amount of internalized protein. RGD-containing peptide inhibition of both activity and internalization supported these findings. These data support the hypothesis that ToxA interacts with a high-affinity binding site on wheat mesophyll cells through the RGD-containing, solvent-exposed loop, resulting in toxin internalization and eventual cell death. The inability to detect phosphorylation of ToxA in vitro and in vivo suggests that a putative CKII phosphorylation site in the RGD-containing loop is required for internalization, not phosphorylation.

Additional keywords:inverse gene-for-gene, necrotroph, Pyrenophora tritici-repentis, receptor-mediated endocytosis

© 2008 The American Phytopathological Society