Fig. 5
Identification of AvrM14. a Physical and genetic maps of the region surrounding AvrM14. The position of AvrM14 is shown in red. b Alignment of the predicted AvrM14-A and AvrM14-B protein sequences. The predicted signal peptide is shaded green and polymorphic amino acids are shaded blue. The Nudix box (Gx5Ex5[U/A]xREx2EExGU; [83]) is shown in red, where X represents any amino acid and U represents an aliphatic, hydrophobic amino acid. c Genetic mapping of AvrM14 using a CAPS marker. PCR products were amplified from genomic DNAs and then digested with BspEI. The phenotype of each individual on M4 flax plants is indicated as ‘A’ for avirulent or ‘V’ for virulent. Approximate product sizes are shown in base-pairs. d RT-PCR analysis of AvrM14 gene expression. Products were amplified from genomic DNA (gDNA) extracted from strain C (virulent on M4 flax), strain H (avirulent on M4 flax) and CH5. Products were also amplified from cDNA made from RNA isolated from flax leaves 4 days post-infection with CH5. RT+ indicates a cDNA reaction that contained reverse transcriptase whereas RT- indicates an identical reaction that lacked reverse transcriptase. Approximate product sizes are shown in base-pairs. e Expression of avirulence gene candidates in flax leaves using Agrobacterium tumefaciens-mediated transient transformation. Expression constructs were generated using cDNA sequences that lacked the region encoding the predicted signal peptide (dSP). AvrM14-A is the allele found in strain H, which is avirulent on M1 and M4 flax. AvrM14-B is the allele found in strain C, which is virulent on M1 and M4 flax. Constructs were expressed in flax lines that lacked all known resistance genes (Hoshangabad, shown as ‘Hosh’), or contained only the M1 resistance gene (Williston Brown, shown as ‘M1’) or only the M4 resistance gene (Victory A, shown as ‘M4’). Plants were photographed 8 days after infiltration