# Divergent functions of three Kunitz trypsin inhibitor (KTI) proteins in herbivore defense in poplar

## TL;DR

This study shows that two Kunitz trypsin inhibitor proteins in poplar trees reduce the growth of a harmful insect pest, while a third has no effect.

## Contribution

The study identifies distinct anti-herbivore functions of specific KTI proteins in poplar and their impact on plant growth.

## Key findings

- KTI_400 and KTI_600 reduce larval weight gain of Helicoverpa armigera when overexpressed in poplar.
- Double knockout of KTI_400 and KTI_600 increases larval weight gain and plant biomass.
- KTI_53200 has no significant effect on insect fitness or plant growth.

## Abstract

Climate warming promotes the expansion of insect pests. Among the inducible defense responses activated by attacked plants, Kunitz trypsin protease inhibitors (KTIs) play an outstanding role. KTIs affect food digestion and thereby control the fitness of herbivorous insects. Poplars contain an expanded family of KTIs, whose distinct intrinsic functions are under investigation. Here, we set out to identify KTIs with anti-herbivore activity and assessed the potential growth trade-off incurred by high KTI expression levels.

Using in-silico database searches, we identified 28 KTIs in the haploid genome of Populus x canescens; 21 of them were responsive to herbivory. The greatest induction by herbivory was observed for KTI_400, KTI_600 and KTI_0882 (P. trichocarpa orthologues Potri.019G124400, Potri.019G124600, Potri.019G088200), whereas a moderate response was found for KTI_53200 (Potri.017G153200 orthologue). Mechanical wounding and methyl-jasmonate treatments resulted in fast and strong induction of KTI_400 and KTI_600 and moderate or lacking responses in KTI_0882 and KTI_53200. Increased KTI expression levels were associated with upregulation of ALLENE OXIDE SYNTHASE, a key enzyme involved in jasmonate biosynthesis. On the contrary, exposure to compounds eliciting ethylene or salicylic acid signaling did not affect KTIs. We generated stable CRISPR-Cas12a-mediated knock-out and p35S-mediated overexpression lines of KTI_400, KTI_600 and KTI_53200 in Populus x canescens. Among the wildtype and transgenic lines, only kti_400 + kti_600 double knock-out lines produced greater biomass. Larvae of Helicoverpa armigera, a pest expanding in Europe due to a warmer climate, were allowed to feed on wildtype and transgenic poplar lines. Transgenic poplars overexpressing KTI_400 or KTI_600 resulted in reduced, and their double knockout lines in increased weight gain of the larvae. In contrast, overexpressing or knockout lines of KTI_53200 had no effect on larval weight gain compared with controls.

KTI_400 and KTI_600 are potent, natural in-planta anti-herbivorous agents. Their expression is associated with larval growth reductions. Modulation of KTI_53200 levels had no direct effects on the fitness of leaf-feeding H. armigera or on plant growth. This study sheds light on the potential application of KTI in plant defenses and biocontrol against H. armigera in trees and presents new options to investigate growth-defense theories.

The online version contains supplementary material available at 10.1186/s12870-025-07955-z.