Accelerating Resistance Breeding: Emerging Methods to Identify and Validate Plant Immunity Genes

Plant pathogens are a major cause of crop yield loss, making disease resistance breeding crucial for crop improvement. Plants have evolved innate immune systems, mediated by immune-related genes such as nucleotide-binding site leucine-rich repeat (NLR), pattern-recognition receptors (PRR) and susceptibility genes, which are essential resources for breeding disease-resistant plants. To identify immunity genes, extensive genetic approaches that examine the association between resistance phenotypes and genomic regions have been applied with great success. While genetic methods remain important for identifying immunity genes, novel strategies that rely on functional rather than genetic association with disease resistance offer unique advantages. For example, mutagenesis with <i>R</i> gene enrichment sequencing (MutRenSeq) enabled the identification of wheat resistance genes <i>Sr22</i> and <i>Sr45</i> by comparing the NLRomes of resistant and susceptible lines while single-cell RNA sequencing resolved cell-type-specific responses to pathogen infection and revealed <i>ZmChit7</i>, especially in maize epidermal and guard cells. These approaches reach beyond existing natural variation, can accelerate experimental timelines, reduce the experimental scale, and provide mechanistic insights into pathogen resistance. This review discusses emerging techniques that generate focused candidate immunity gene lists or accelerate their validation, as both are required to identify causal variants for resistance breeding. We consider advances in RenSeq-derived methods, spatial omics, proximity labelling, computational prediction, Clustered regularly interspaced short palindromic repeats (CRISPR) screens, and cell death assays. These approaches are reshaping resistance breeding pipelines beyond association-based discovery. By discussing the strengths and limitations of these emerging methods and their combinations, we outline current opportunities and future directions to help plant pathologists to more effectively identify and validate plant immunity genes.