Molecular analyses of toxin(s) produced by Pyrenophora tritici-repentis
1998
Ciuffetti, L.M. | Tuori, R.P. | Gaventa, J.M. (Oregon State Univ. Corvallis, Oregon (USA))
We are interested in the key events that regulate specificity in the Pyrenophora-wheat interaction. The host-specific toxin(s) (HST) produced by yrenophora tritici-repentis is a protein. In the continuing effort to assess the role of toxin production in the pathogenesis of P. tritici-repentis, we have purified a major necrosis toxin produced by our fungal isolates, designated ToxA. Analysis of purified toxin by mass spectroscopy indicated a molecular weight of 13.2 kD. Western analysis indicated that antibodies raised against purified toxin reacted with and are specific to a 13.2 kD band associated with toxic activity. cDNA libraries were constructed from toxin- producing isolates of the fungus, and ToxA antibodies were used to identify and subclone cDNAs for the ToxA protein. A subgenomic library was prepared from a toxin-producing isolate of the fungus, and a cDNA insert was used to select a genomic copy of the ToxA gene that included its endogenous promoter region. A tox- isolate of P. tritici- repentis was transformed with the ToxA gene. All tox+ transformants tested produced wildtype levels of the active ToxA protein in culture. Additionally, tox+ transformants were shown to be fully pathogenic when inoculated onto susceptible wheat cultivars. Present efforts are directed toward the elucidation of possible regulation of the ToxA gene in planta, and to determine whether multiple forms or toxins produced by P. tritici-repentis are due to post- translational modification of the ToxA gene product.
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