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ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Cereal Crops Research » Research » Publications at this Location » Publication #207303
Title: Functional Characterization of ToxA and Molecular Identification of its Intracellular Targeting Protein in Wheat

Author
item Tai, Yin Shan
item BRAGG, JENNIFER - UC BERKELEY
item MEINHARDT, STEVEN - NORTH DAKOTA STATE UNIV.

Submitted to: American Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2007
Publication Date: 3/31/2007
Citation: Tai, Y., Bragg, J., Meinhardt, S. 2007. Functional Characterization of ToxA and Molecular Identification of its Intracellular Targeting Protein in Wheat. American Journal of Plant Physiology 2:76-89.

Interpretive Summary: Wheat tan spot disease is caused by the fungal pathogen Pyrenophora tritici-repentis, which secretes a toxin called ToxA. This toxin plays a major role in production of the typical tan-colored necrotic spot symptom. ToxA is the first-identified fungal proteinaceous toxin and induces necrotic lesions (dead cells) surrounding the infection sites. The goal of this study was to better understand the molecular mechanism of ToxA-induced cell death in wheat. Since ToxA is a protein, the approach used in this study was to detect protein-protein interactions using a system termed yeast two-hybrid (Y2H) analysis. The objective of this approach is to identify the target protein of ToxA in wheat. By screening a cDNA library in the Y2H system, we identified plastocyanin (a component involved in electron transport in photosynthesis) as the ToxA target protein. This identification of plastocyanin as the target protein of ToxA explains why ToxA-induced cell death is light-dependent. In order to characterize and verify the ToxA-plastocyanin relationship, we also developed two new tools for wheat research: virus-induced gene silencing (VIGS) and virus-mediated transient expression assay. When VIGS was used to turn off production of plastocyanin, the plants showed symptoms similar to those of ToxA-induced cell death. When ToxA was transiently expressed in wheat leaves, results confirmed the existence of an intracellular target of ToxA in wheat and plants showed symptoms similar to those produced as a result of ToxA infiltration. We further characterized three amino acid residues that were critical for ToxA-induced cell death. Moreover, we analyzed two motifs in the ToxA protein molecule that were pivotal for ToxA-ToxA dimerization and in planta activity, respectively. This study provides a platform for the future development of resistant lines of wheat against this fungal pathogen. More importantly, we solved the puzzle of how a fungal pathogen evolved to attack the basic components of plant cells. Based on the knowledge gained in this study, someone someday might develop an herbicide that affects monocot plants but not dicots, or vice versa.

Technical Abstract: The fungus Pyrenophora tritici-repentis causes tan spot disease in wheat. Typical symptoms include tan-colored necrotic lesions surrounding small infection sites. The necrosis is induced by the host-selective proteinaceous toxin, ToxA. The ability of ToxA to be internalized into plant cells is required for the toxin to cause cell death. Using yeast two-hybrid (Y2H) analyses, we detected the oligomerization of ToxA, and we identified amino acid E145 and D149 residues that were critical for this ToxA-ToxA oligomerization. However, a G141A mutation in the RGD motif, which is important for necrosis, still can form the oligomer. We have developed a transient assay system to provide direct evidence that intracellular expression of ToxA causes cell death in wheat. By screening a cDNA library, we idnetified the wheat plastocyanin as the host target protein of ToxA. Plastocyanin serves as a component of the electron transport chain of photosynthesis. Virus-induced gene silencing of wheat plastocyanin showed a similar phenotype of ToxA-induced cell death. In Y2H analyses, the mutant G141A can not interact with plastocyanin, but E145R and D149K mutants, which can not form ToxA-ToxA oligomer, do interact with plastocyanin. However, all G141,E145, and D149 residues are required for ToxA-induced necrotic symptom in planta.