|Tai, Yin Shan|
Submitted to: Plant and Animal Genome VX Conference Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 10/28/2006
Publication Date: 1/6/2007
Citation: Tai, Y., Bragg, J., Lu, H., Edwards, M.C., Faris, J.D., Friesen, T.L., Meinhardt, S.W. 2007. Functional Characterization of Ptr ToxA and Molecular Identification of its Intracellular Targeting Protein in Wheat. Plant and Animal Genome VX Conference Abstracts. p.
Technical Abstract: Wheat transformation is still a highly challenging barrier for analyses of gene function in wheat. Unlike the situation with dicot plants and other model organisms, RNA silencing and transient assay of gene expression are not well-established. We develop a virus-induced gene silencing (VIGS) system and a virus-mediated expression system for transient assays in wheat. In this presentation, we will highlight the applications of these two systems for our ongoing projects. Particularly, we will focus on the study of the fungal toxin, ToxA. The necrotrophic fungus Pyrenophora tritici-repentis causes tan spot in bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L. var. durum). Typical disease symptoms include tan-colored, diamond-shaped necrotic lesions surrounding small, dark-brown infection sites. The necrotic symptom is due to the production of a host-selective toxin (HST), ToxA, which is the first proteinaceous HST cloned from the fungi. The ability of ToxA to be internalized within plant cells is required for the toxin to cause cell death. Using yeast two-hybrid analyses, we detected the formation of a ToxA dimer. We determined two amino acid residues (E145 and D149) were critical for dimer formation. However, a G141A mutation in the RGD motif (residues 140 to 142), which is important for cell death, still allows dimer formation. We also developed a system for transient assays to provide direct evidence of cell death initiated by the intracellular expression of ToxA in wheat. By screening a cDNA library, we identified a wheat protein, designated TAI (ToxA interactor), as the host target protein of ToxA. TAI is nucleus-encoded, but located in the chloroplast. The mutant G141A cannot interact with TAI, but mutants of E145R and D149K do interact with TAI in the yeast two-hybrid system. However, in planta both the RGD motif and the region of residues that includes E145 and D149 are required for cell death. A hypothetical mechanism underlying wheat sensitivity to ToxA and ToxA-induced cell death will be discussed.