Heterologous expression and activity of Ptr ToxB from virulent and avirulent isolates of Pyrenophora tritici-repentis

Abstract

The fungal pathogen Pyrenophora tritici-repentis causes tan spot, an important foliar disease of wheat (Triticum aestivum). The fungus produces at least three host-specific toxins, including the chlorosis-inducing Ptr ToxB, a small protein encoded by the ToxB gene. A ToxB homolog is also found in race 4 isolates of P. tritici-repentis, which lack detectable toxin activity. It is not clear whether the absence of toxin activity in these isolates stems from differences in the ToxB gene sequence and (or) level of expression. To directly compare the chlorosis-inducing activity of different forms of Ptr ToxB, ToxB from a virulent race 5 isolate (Alg3-24) and a ToxB homolog from an avirulent race 4 isolate (90-2) were heterologously expressed as glutathione-S-transferase (GST)- and hexahistidine (His)-tagged fusion proteins in Escherichia coli. Infiltration of toxin solutions into the leaves of Ptr ToxB sensitive and insensitive wheat seedlings revealed that the overexpressed GST- and His-tagged toxin from Alg3-24 induced strong chlorosis symptoms in a genotype-specific manner, whereas the form of the toxin from isolate 90-2 induced only trace amounts of symptoms. This suggests that the absence of chlorosis-inducing activity in race 4 of P. tritici-repentis may be largely due to sequence differences in ToxB at the gene and protein level, regardless of the extent of gene expression.