Activation of multilayered plant immunity through spatiotemporal expression of Botrytis cinerea BcCrh1-derived dual epitopes

Botrytis cinerea is a major necrotrophic pathogen responsible for significant crop losses worldwide. Alternative strategies to control B. cinerea are urgently needed to reduce dependence on chemical fungicides, which are increasingly ineffective due to resistance and pose environmental risks. In this study, we identified two immunogenic epitopes derived from the B. cinerea cell death-inducing protein BcCrh1 and used them to engineer disease-resistant plants through a novel, spatially compartmentalized dual-epitope immune activation strategy. The first epitope is derived from a 35-amino acid intracellular peptide that exhibits both immunogenicity and cell death-inducing activity, which was mutated to separate these two properties. The second peptide represents an immunogenic portion of the protein that activates extracellular plant immunity. Transcriptomic and metabolomic analyses revealed that these epitopes trigger complementary defense pathways, and their co-expression integrates these responses into a robust, multilayered immunity, providing significantly enhanced protection compared with individual expression. Although constitutive expression of two epitopes conferred resistance, it also led to growth penalties. In contrast, pathogen-inducible expression of two epitopes preserved normal plant development while maintaining strong resistance to both B. cinerea and Pseudomonas syringae in Arabidopsis and tomato. This inducible strategy offers a major advantage by minimizing fitness costs while maximizing protection, highlighting the potential of spatially and temporally targeted epitope-based immune activation for durable and sustainable crop protection.