Fig. 3. Validation of AvrSr13 and AvrSr22 candidates.

a, Wheat protoplasts (cv. Fielder) were co-transformed with YFP, an Avr gene (AvrSr13, AvrSr22, AvrSr27-2 or AvrSr50) and an R gene (Sr13c, Sr22 or Sr27) or empty vector. b, YFP was co-transformed with AvrSr13 or AvrSr50 into protoplasts derived from wheat lines Kronos (KR, containing native Sr13a), Fielder (FL) or transgenic FL containing the Sr13c transgene (FL-Sr13c). c, The YFP reporter was co-transformed with AvrSr22 or AvrSr27-2 into protoplasts derived from wheat lines Schomburgk (SB, containing native Sr22), FL, transgenic FL containing the Sr22 transgene (FL-Sr22), or transgenic Robin containing a five-R-gene cassette including Sr22 (RB-Big5)³. Plots in a, b and c show the percentage of YFP-positive living cells determined by flow cytometry. Mean ± s.e.m. of 3 replicates, with significant differences indicated for relevant pairwise comparisons (two-tailed unpaired t-test assuming equal variances). All gene constructs were delivered at an MOT of 36 million plasmid molecules per cell. d, Agrobacterium-mediated transient co-expression of Sr27, Sr13c or Sr22 proteins (C-terminally fused to YFP) with AvrSr27-2, AvrSr13 or AvrSr22 (N-terminally fused to YFP) or YFP alone in N. tabacum leaves. Agrobacterial cultures were delivered at OD₆₀₀ of 0.4 (R gene constructs) or 0.7 (Avr gene constructs).