A Kunitz trypsin inhibitor gene family from trembling aspen (Populus tremuloides Michx.): cloning, functional expression, and induction by wounding and herbivory

Three Kunitz trypsin inhibitor genes were isolated from trembling aspen (Populus tremuloides) by PCR and cDNA screening. Based on sequence similarity, they were grouped into two classes. Southern blots showed complex banding patterns and a high level of restriction fragment polymorphism between different aspen genotypes, suggesting that these trypsin inhibitors are members of a large, rapidly evolving gene family. One of the trypsin inhibitor genes, PtTI2. was over-expressed in Escherichia coli and its product shown to inhibit bovine trypsin in vitro. Both classes of PtTI genes are induced by wounding and herbivory, permitting rapid adaptive responses to herbivore pressure. The response appears to be mediated by an octadecanoid-based signaling pathway, as methyl jasmonate treatments induced the trypsin inhibitors. Wound-induced accumulation of trypsin inhibitor protein was also observed by western blot analysis. The pattern of expression, the apparent rapid evolution of TI genes, and the in vitro trypsin inhibitory activity are consistent with a role in herbivore defense. This work establishes the presence of a functional protein-based inducible defense system in trembling aspen.