# Pathogen-inspired engineering of plant protease enhances late blight resistance

## TL;DR

Scientists engineered a plant protease to resist pathogen inhibitors, boosting tomato resistance to late blight.

## Contribution

A novel pathogen-inspired engineering approach enhanced plant protease resistance to pathogen inhibitors.

## Key findings

- Engineered C14 protease (eC14) is less inhibited by EpiCs and increases tomato resistance to Phytophthora infestans.
- Pain1 and Pain2 proteases from the pathogen avoid inhibition by EpiCs, inspiring the design of eC14.
- eC14's resistance to EpiCs inhibition is achieved by incorporating residues from Pain1.

## Abstract

Genetic engineering to produce crops that are pathogen resistant is an important strategy for world food security. Here, we engineered tomato-secreted immune protease C14 to become less sensitive to inhibition by cystatin-like inhibitor extracellular protease inhibitors of cysteine proteases (EpiCs) secreted by the oomycete late blight pathogen Phytophthora infestans, and demonstrated that this engineered C14 (eC14) provides increased immunity. Importantly, this engineering was inspired by our identification of two proteases (Pain1 and Pain2) secreted by P. infestans that contribute to virulence and have reduced sensitivity to EpiCs inhibition. Thus, a pathogen avoiding self-inhibition can inspire crop engineering.

The apoplast is an important battlefield in plant–pathogen interactions. The late blight oomycete pathogen Phytophthora infestans, for instance, secretes cystatin-like protease inhibitors EpiC1 and EpiC2B to suppress C14, a papain-like immune protease secreted by tomato. Here, we found that P. infestans also secretes two distinct papain-like proteases termed Pain1 and Pain2, which are transcriptionally induced during infection. Both Pains promote P. infestans infection, but not when their catalytic residues are mutated. Strikingly, EpiC1 and EpiC2B preferentially inhibit tomato C14 rather than self-produced Pains, suggesting that they coevolved with Pains to avoid self-inhibition. To mimic the avoidance of inhibition by EpiCs, we engineered C14 (eC14) with seven Pain1 residues that potentially disturb the EpiCs–C14 interface. This eC14 is less sensitive to inhibition by EpiCs and enhances resistance to P. infestans infection. This strategy demonstrates that a pathogen-inspired protein engineering approach can increase crop resistance to plant pathogens.