Honors College Thesis

 

Mutagenesis and Crystallization of Ptr ToxB, a host-selective toxin of Pyrenophora tritici-repentis Public

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  • Pyrenophora tritici-repentis (Ptr) is the fungal causal agent of the disease tan spot in wheat and causes necrosis and chlorosis in infected leaves. These symptoms are caused by host-selective toxins (HSTs), molecules that serve as the sole determinants of pathogenicity. Ptr has three known HSTs, the proteinaceous Ptr ToxA and Ptr ToxB, and the non-proteinaceous Ptr ToxC. Ptr ToxB causes chlorosis on sensitive cultivars and has a protein homolog, toxb, which is present in a non-pathogenic race of Ptr. Using mutagenesis, six chimeric proteins were created from the ToxB and toxb genes. The study of the six chimeric proteins allowed us to investigate which specific regions of ToxB were responsible for the protein’s induction of chlorosis in sensitive wheat cultivars. The presence of a higher molecular weight band on SDS-PAGE of toxb and chimeras containing the N-terminus of toxb suggest that this region in toxb is responsible for improper folding. The assessment of activities of the chimeras and site-directed mutants by bioassay further suggest that this region is also important for the lack of protein activity seen in toxb. Crystallization with the goal of determining the tertiary structure solution of ToxB has despite much effort proven unsuccessful.
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