Large-scale identification and functional analysis of NLR genes in blast resistance in the Tetep rice genome sequence

Abstract

Significance Rice resistance against blast, a devastating fungal disease, is typically mediated by nucleotide-binding site leucine-rich repeat (NLR) proteins. Most previous studies focused on individual NLR genes, but single R genes typically confer no durable resistance owing to their narrow resistance spectrum. In this study, we sequenced the genome of Tetep, a widely used resistance donor, to decipher the molecular basis of its broad-spectrum and durable blast resistance. Large-scale cloning and functional analysis of annotated NLRs uncovered a large number of functional NLR genes and interactive NLR networks in the genome. Moreover, pedigree tracing of elite cultivars indicated the more NLRs inherited from Tetep the better resistance of the cultivar. Various datasets were provided for facilitating breeding for new resistant cultivars. Tetep is a rice cultivar known for broad-spectrum resistance to blast, a devastating fungal disease. The molecular basis for its broad-spectrum resistance is still poorly understood. Is it because Tetep has many more NLR genes than other cultivars? Or does Tetep possess multiple major NLR genes that can individually confer broad-spectrum resistance to blast? Moreover, are there many interacting NLR pairs in the Tetep genome? We sequenced its genome, obtained a high-quality assembly, and annotated 455 nucleotide-binding site leucine-rich repeat (NLR) genes. We cloned and tested 219 NLR genes as transgenes in 2 susceptible cultivars using 5 to 12 diversified pathogen strains; in many cases, fewer than 12 strains were successfully cultured for testing. Ninety cloned NLRs showed resistance to 1 or more pathogen strains and each strain was recognized by multiple NLRs. However, few NLRs showed resistance to >6 strains, so multiple NLRs are apparently required for Tetep’s broad-spectrum resistance to blast. This was further supported by the pedigree analyses, which suggested a correlation between resistance and the number of Tetep-derived NLRs. In developing a method to identify NLR pairs each of which functions as a unit, we found that >20% of the NLRs in the Tetep and 3 other rice genomes are paired. Finally, we designed an extensive set of molecular markers for rapidly introducing clustered and paired NLRs in the Tetep genome for breeding new resistant cultivars. This study increased our understanding of the genetic basis of broad-spectrum blast resistance in rice.