Targeting disease susceptibility genes in wheat through wide hybridization with maize expressing Cas9 and guide RNA

Two genes (TaHRC and Tsn1) conferring susceptibility to Fusarium head blight (FHB) and tan spot/Septoria nodorum blotch/spot blotch in wheat were targeted through wide hybridization with maize expressing Cas9 and guide RNA (gRNA). For each gene, two target sites were selected and corresponding gRNA expression cassettes were synthesized and cloned into a binary vector carrying the CRISPR/Cas9-mediated genome editing machinery. The constructed binary vectors were used to transform the hybrid maize Hi-II through an Agrobacterium-mediated approach to generate T0 and T1 plants, which were used to cross with wheat variety Dayn for targeting Tsn1 or the susceptible allele (TaHRC-S) of TaHRC, as well as with the near-isogenic line (Day-Fhb1) of Dayn for targeting the resistant allele (TaHRC-R) of TaHRC. Haploid embryos were rescued in vitro from the wide crosses to generate haploid plants. PCR amplification and sequencing indicated that 15-33% of the haploid plants contained the target gene with mutations at the target sites. This wheat × maize hybridization combined with genome editing approach provides a useful alternative tool not only for targeting susceptibility genes to improve disease resistance without regulatory issues but also for understanding gene function in wheat.