Mutation and sequencing-based cloning and functional studies of the rust resistance gene R11 in sunflower (Helianthus annuus)

Authors: Shamimuzzaman, Md - Shamim; MA, GUOJIA - North Dakota State University; Underwood, William; Qi, Lili

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/24/2023
Publication Date: 4/20/2023
Citation: Shamimuzzaman, M., Ma, G., Underwood, W., Qi, L. 2023. Mutation and sequencing-based cloning and functional studies of the rust resistance gene R11 in sunflower (Helianthus annuus). Plant Journal. 115(2):480-493. https://doi.org/10.1111/tpj.16238.
DOI: https://doi.org/10.1111/tpj.16238

Interpretive Summary: Foliar rust disease is one of the most important diseases limiting sunflower yields worldwide. This disease is mainly managed through host-plant resistance conferred by single dominant resistance genes (R genes). Understanding the genetic mechanism of resistance genes is critical to successful resistance breeding and the control of rust disease. The rust R gene R11 in sunflower line HA-R9 shows broad-spectrum resistance to rust virulent races and was previously mapped to a small region in sunflower chromosome 13. In the current study, we combined EMS mutagenesis with targeted region sequencing of the genomic region spanning the R11 gene to identify the R11 gene and define induced mutations. The R11 gene is predicted to have a single sequence of 3,996 base pairs that encodes a typical disease resistance protein. Point mutations identified in the R11 rust-susceptible mutants resulted in loss of function leading to rust susceptibility. Additional functional studies revealed interesting insights into sunflower defense responses associated with resistance conferred by R11.

Technical 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 EMS (ethyl methane sulfonate) 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 3,996-bp open reading frame and encodes a protein of 1,331 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 (RNA-Seq) 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.