CRISPR/Cas9-Mediated Mutagenesis of Two Alleles Encoding a Glutathione S-Transferase Confirms Their Identity as Fhb7 Conferring Fusarium Head Blight Resistance in Wheat

Fhb7 is a gene derived from the homoeologous group-7 chromosomes of tall wheatgrass ( Thinopyrum species), which confers resistance to Fusarium head blight (FHB) in wheat. Two resistance alleles at the Fhb7 locus ( Fhb7 Thp and Fhb7 The2) were identified from Th. ponticum and Th. elongatum , respectively, and introgressed into the wheat genome by homoeologous recombination. Fhb7 Thp was first cloned and reported to encode a glutathione S-transferase (GST), which detoxifies trichothecenes through de-epoxidation. However, a recent study has questioned the role of GST in Fhb7 -mediated FHB resistance. To decipher the function of two GST-encoding alleles, Fhb7 Thp and Fhb7 The2, in FHB resistance, we generated haploid plants from three spring wheat lines (CS-Fhb7, WGC002, and FW23-009-80) carrying Fhb7 Thp or Fhb7 The2 through wide hybridizations with transgenic maize expressing Cas9 and guide RNA ( gRNA ) targeting GST . Of 57, 35, and 74 haploid plants derived from CS-Fhb7, WGC002, and FW23-009-80, respectively, 16, 5, and 9 had an insertion/deletion at the target sites of GST . Greenhouse evaluation of doubled haploid (DH) lines derived from the haploid plants for FHB resistance indicated that the GST -edited DH lines were significantly more susceptible and accumulated more deoxynivalenol (DON) than their corresponding wildtypes and non-edited DH lines, confirming GST as the causal gene for Fhb7 -mediated FHB resistance. This study also demonstrated the power of wheat × maize hybridization coupled with genome editing in validating gene function in wheat.