Fig. 7
Identification of AvrL2. a RT-PCR analysis of AvrL2 family gene expression. Gene-specific primers were used to amplify products by PCR. Products were amplified from genomic DNA (gDNA) extracted from a CH5 F2 individual virulent on L2 flax (CH5F2-67, shown as ‘VIR’), a CH5 F2 individual avirulent on L2 flax (CH5F2-68, shown as ‘AVR’) 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. b Genetic mapping of individual AvrL2 family members using gene-specific SCAR markers. Multiplex PCR was performed using primers that amplify an internal control fragment of 861 bp. AvrL2 family members were amplified using gene-specific primers and product sizes are as follows: AvrL2-A, 733 bp; AvrL2-C, 722 bp; and AvrL2-D, 722 bp. The phenotype of each individual on L2 flax plants is indicated as ‘A’ for avirulent or ‘V’ for virulent. Approximate product sizes are shown in base-pairs. c Expression of avirulence gene candidates in flax leaves using Agrobacterium tumefaciens-mediated transient transformation. Expression constructs were generated using the full-length AvrL2-A cDNA sequence (full-length) or a sequence that lacked the region encoding the first 36 amino acids (dSP36). Constructs were expressed in near-isogenic flax lines that contained or lacked the L2 resistance gene (B12 × L2, shown as ‘L2’; or Bison, respectively). Plants were photographed 8 days after infiltration. d Expression of avirulence gene candidates in Nicotiana tabacum leaves using Agrobacterium tumefaciens-mediated transient transformation. Expression constructs were generated using the full-length AvrL2-A cDNA sequence (full-length) or sequences that lacked the region encoding the first 20, (dSP20), 26 (dSP26), 32 (dSP32) or 36 (dSP36) amino acids. For co-infiltrations, cultures containing AvrL2 and L2 constructs were mixed together in equal ratio. Leaves were photographed 5 days after infiltration