Fig. 2: Cloning and functional validation of wheat stem rust resistance gene Sr6.

a Wheat stem rust infection phenotype of wheat lines WL711, TA5602, TA5605, Fielder, CDC Landmark, Thatcher, Avocet S and Julius. Images of leaves were taken 12 days after inoculation. Infection types were analyzed using a 0–4 scale according to the system of Stakman et al. ³²: 0, no uredia; 1, small uredia; 2, small to medium-sized uredia; 3, medium-sized uredia; 4, large uredia; hypersensitive necrotic or chlorotic flecks of varying size present. Scale bar = 1 cm. The experiment was repeated independently twice with similar results. b Representative images showing the phenotype of ten loss-of-function TA5602 mutants. Images of stems were taken 12 days after inoculation. Scale bar = 1 cm. Spgt=Susceptible to Pgt. The experiment was repeated independently three times with similar results. c The gene structure of the BED-NLR. Exons are represented as rectangular bars, and the intron is shown as a black line between the exons. The predicted protein domains are indicated by different colors. Vertical bars represent 92 EMS-induced amino acid polymorphisms, upper bars = amino acid substitutions, lower bars = premature stop codon mutations. VIGS1 and VIGS2 indicate the positions of the two virus-induced gene silencing (VIGS) constructs. The sgRNA in red font indicates CRISPR/Cas9 targeting site. d Virus-induced gene silencing of BED-NLR resulted in susceptibility to Pgt isolate H3. Representative images showing the results of the VIGS experiment using barley stripe mosaic virus (BSMV). BSMV-γVIGS1 and BSMV-γVIGS2 = two silencing constructs targeting BED-NLR, BSMV-γGFP = control with a GFP silencing construct. Images were taken 12 days after inoculation. Scale bar = 1 cm. The experiment was repeated independently three times with similar results. e CRISPR/Cas9-mediated knock-out of BED-NLR in wheat cultivar Fielder resulted in susceptibility to Pgt isolate H3. The sgRNA was designed to target the coding sequence of the BED domain. Four mutant families (Mut1 – Mut4) carrying three types of deletions or insertions within the sgRNA region were susceptible to Pgt isolate H3. Images were taken 12 days after inoculation. Scale bar = 1 cm. The experiment was repeated independently twice with similar results.