Functional Analysis of the Kunitz Trypsin Inhibitor Family in Poplar Reveals Biochemical Diversity and Multiplicity in Defense against Herbivores

Ian T Major,C Peter Constabel

doi:10.1104/pp.107.106229

Abstract

We investigated the functional and biochemical variability of Kunitz trypsin inhibitor (KTI) genes of Populus trichocarpa x Populus deltoides. Phylogenetic analysis, expressed sequence tag databases, and western-blot analysis confirmed that these genes belong to a large and diverse gene family with complex expression patterns. Five wound- and herbivore-induced genes representing the diversity of the KTI gene family were selected for functional analysis and shown to produce active KTI proteins in Escherichia coli. These recombinant KTI proteins were all biochemically distinct and showed clear differences in efficacy against trypsin-, chymotrypsin-, and elastase-type proteases, suggesting functional specialization of different members of this gene family. The in vitro stability of the KTIs in the presence of reducing agents and elevated temperature also varied widely, emphasizing the biochemical differences of these proteins. Significantly, the properties of the recombinant KTI proteins were not predictable from primary amino acid sequence data. Proteases in midgut extracts of Malacosoma disstria, a lepidopteran pest of Populus, were strongly inhibited by at least two of the KTI gene products. This study suggests that the large diversity in the poplar (Populus spp.) KTI family is important for biochemical and functional specialization, which may be important in the maintenance of pest resistance in long-lived plants such as poplar.

Highlights

We investigated the functional and biochemical variability of Kunitz trypsin inhibitor (KTI) genes of Populus trichocarpa 3 Populus deltoides
We determined that poplar has at least 22 KTIs (Fig. 1), but with sequence ambiguities the number of KTIs could be as high as 30
Kunitz TIs have been studied in many different plants and contexts, often with a focus on their potential for biotechnology-based pest control for agriculture; the diversity of KTIs within a plant species has not been extensively examined

Summary

Introduction

We investigated the functional and biochemical variability of Kunitz trypsin inhibitor (KTI) genes of Populus trichocarpa 3 Populus deltoides. Five wound- and herbivore-induced genes representing the diversity of the KTI gene family were selected for functional analysis and shown to produce active KTI proteins in Escherichia coli. These recombinant KTI proteins were all biochemically distinct and showed clear differences in efficacy against trypsin-, chymotrypsin-, and elastase-type proteases, suggesting functional specialization of different members of this gene family. This study suggests that the large diversity in the poplar (Populus spp.) KTI family is important for biochemical and functional specialization, which may be important in the maintenance of pest resistance in long-lived plants such as poplar. The KTI family showed surprising sequence variation, including many nonsynonymous substitutions and indels, which appear to translate into functional diversity

Methods

Results

Conclusion