Poplar protease inhibitor expression differs in an herbivore specific manner

Abstract

Background: Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp. Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar (P. nigra) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles.

Results: We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTIs via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTIs than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes.

Conclusions: We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.

Keywords: Coleoptera; Induced defenses; Kunitz-type trypsin inhibitors; herbivore specificity; woody plants; tree defenses; Lepidoptera; Proteinase inhibitors; Salicaceae.

Fig. 1

Insects used in this study and their damage pattern after 2 d feeding on black poplar leaves. Amata mogadorensis and Lymantria dispar (gypsy moth) remove large areas from the leaves, whereas Phratora vulgatissima (blue willow beetle) causes small, but numerous lesions

Fig. 2

Expression of Kunitz-type trypsin inhibitor (KTI) genes in black poplar leaves after feeding by gypsy moth caterpillars (L. dispar) or blue willow beetles (P. vulgatissima) compared to their respective controls (Control 1 and 2), and compared to actin (ACT) and elongation factor 1-α (EF1-α) as constitutively expressed ‘house-keeping genes’. Shown are the mean RPKM (reads per kilobase of transcript per million mapped reads; n = 4) as result of the transcriptome analysis

Fig. 3

Phylogenetic tree of poplar KTI proteins. PnKTIs identified in this study are shown in blue and asterisks mark full-length cDNA sequences. The tree was inferred using the Neighbor-Joining method and the JTT matrix-based method. Bootstrap values (n = 500) are shown next to each node. The tree is drawn to scale, with branch lengths in the units of the number of amino acid substitutions per site

Fig. 4

Transcript accumulation of Kunitz-type trypsin inhibitor genes (KTIs) of the a, b, and d subfamily in black poplar leaves after herbivory by two caterpillar species (L. dispar, A. mogadorensis) and one beetle species (P. vulgatissima). Shown are the gene expression normalized to Actin and relative to a control sample as boxplots (median with upper and lower quartile as bars; n = 6); results of the ANOVA are given in each graph. Different letters indicate significant differences among groups (P < 0.05; Tukey’s post-hoc test)