Mutation and sequencing-based cloning and functional studies of a rust resistance gene in sunflower (Helianthus annuus)

Rust, caused by the fungus Puccinia helianthi Schwein., is one of the most devastating diseases of sunflower (Helianthus annuus L.), affecting global production. The rust R gene R-11 in sunflower line HA-R9 shows broad-spectrum resistance to P. helianthi virulent races and was previously mapped to an interval on sunflower chromosome 13 encompassing three candidate genes annotated in the XRQr1.0 reference genome assembly. In the current study, we combined ethyl methane sulfonate (EMS) mutagenesis with targeted region capture and PacBio long-read sequencing to clone the R-11 gene. Sequencing of a 60-kb region spanning the R-11 locus from the R-11-HA-R9 rust-resistant line and three EMS-induced susceptible mutants facilitated the identification of R-11 and definition of induced mutations. The R-11 gene is predicted to have a single 3996-bp open reading frame and encodes a protein of 1331 amino acids with CC-NBS-LRR domains typical of genes conferring plant resistance to biotrophic pathogens. Point mutations identified in the R-11 rust-susceptible mutants resulted in premature stop codons, consistent with loss of function leading to rust susceptibility. Additional functional studies using comparative RNA sequencing of the resistant line R-11-HA-R9 and R-11-susceptible mutants revealed substantial differences in gene expression patterns associated with R-11-mediated resistance at 7 days post-inoculation with rust, and uncovered the potential roles of terpenoid biosynthesis and metabolism in sunflower rust resistance.