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

Abstract

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 R₁₁ 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 R₁₁ gene. Sequencing of a 60-kb region spanning the R₁₁ locus from the R₁₁ -HA-R9 rust-resistant line and three EMS-induced susceptible mutants facilitated the identification of R₁₁ and definition of induced mutations. The R₁₁ 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 R₁₁ 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 R₁₁ -HA-R9 and R₁₁ -susceptible mutants revealed substantial differences in gene expression patterns associated with R₁₁ -mediated resistance at 7 days post-inoculation with rust, and uncovered the potential roles of terpenoid biosynthesis and metabolism in sunflower rust resistance.

Keywords: ethyl methane sulfonate mutation; gene cloning; resistance gene; rust; sunflower; target region sequencing.