Engineered rice RGA5-HMA domain for expanded pathogen effector recognition and pesticide-free disease resistance

Overview

This research proposes a structure-based protein engineering strategy to broaden the disease resistance profile of rice by modifying the HMA (heavy metal-associated) domain of the rice immune receptor RGA5. RGA5 naturally recognizes specific effector proteins secreted by the rice blast fungus Magnaporthe oryzae, but its recognition spectrum is limited. By redesigning the RGA5-HMA domain, the project aims to expand its binding specificity to cover multiple MAX-family effectors (AVR-Pia, AVR-Pik, AVR-Pib, and AvrPiz-t). The engineered HMA domain will then be introduced into rice seeds via gene editing, producing rice varieties with stacked, broad-spectrum resistance to rice blast disease. The ultimate goal is to reduce pesticide use in rice cultivation, supporting more sustainable agriculture.

Technical specifications

• Target receptor: RGA5, a rice nucleotide-binding leucine-rich repeat (NLR) immune receptor that uses its HMA domain to detect pathogen effectors
• Effector targets: MAX-family effectors from Magnaporthe oryzae, including AVR-Pia, AVR-Pik, AVR-Pib, and AvrPiz-t, which share a conserved structural fold
• Engineering approach: Structure-guided design of RGA5-HMA domain mutants, informed by homology with Pik-HMA and existing Pik-HMA specificity-switching studies
• Screening method: Yeast two-hybrid assays to identify HMA mutants with altered effector binding profiles
• Validation pipeline: In vitro and in vivo binding assays combined with functional analysis to confirm expanded recognition
• Delivery to crops: Gene editing of rice seeds to incorporate the engineered RGA5-HMA domain, enabling co-cultivation of multiple resistant varieties
• Key demonstrated result: One RGA5-HMA mutant has already been identified that recognizes AVR-Pib instead of its natural target AVR-Pia, confirming proof of concept

Technology readiness level

The project is at an early-to-mid stage of technology development. Proof of concept has been achieved, with a single RGA5-HMA mutant successfully shown to switch effector recognition from AVR-Pia to AVR-Pib. The next phase involves screening the designed mutant library to identify additional variants that recognize AVR-Pik and AvrPiz-t, followed by comprehensive binding and functional validation. The full study is expected to take approximately two years to complete. The team is actively seeking a partnership to provide expertise in rice seed development through gene editing, which would accelerate translation from laboratory validation to field-ready rice varieties.