Pathogen-inducible expression of autoactive NLRs confers multi-pathogen resistance in tomato

Abstract

Many disease resistance genes utilized in crop breeding encode nucleotide-binding leucine-rich repeat receptors (NLRs), which are frequently race-specific and fail to provide durable protection in the field. Consequently, engineering broad-spectrum resistance has become a primary goal. While autoactive NLRs can provide broad-spectrum immunity by bypassing specific pathogen recognition, they often impose severe fitness costs. Here, we hypothesized that precise transcriptional control could mitigate these penalties and developed a synthetic strategy coupling pathogen-inducible (PI) promoters with autoactive NLRs. Through a transcriptomic meta-analysis, we identified and validated native tomato PI promoters that respond to diverse pathogens while remaining insensitive to abiotic stress. We established a tunable system by pairing these promoters varying in basal and inducible expression with NLRs exhibiting different degrees of autoactivity. Systematic characterization revealed that balancing these components is critical to prevent leaky immune activation in the absence of pathogens. Transgenic tomatoes expressing a weakly autoactive NLR under a PI phenylalanine ammonia-lyase promoter exhibited enhanced resistance to two taxonomically distinct pathogens without growth impairment. Disruption of the PI sequence largely abolished this resistance, confirming the necessity of pathogen-mediated transcriptional induction. Finally, we added an additional layer of tunability by engineering the copy number of inducible elements and the core promoter to fine-tune promoter strength while preserving inducibility. Together, our results showcase a versatile framework for engineering multi-pathogen resistance and provide new insights into using transcriptional control in overcoming the fitness costs associated with autoactive NLRs.