1.1.6S
MOLECULAR ANALYSIS OF GENE-FOR-GENE SPECIFICITIES OF THE ALLELES OF THE FLAX L GENE FOR RUST RESISTANCE

J ELLIS1, G LAWRENCE1, J LUCK1, P DODDS1, M AYLIFFE1, K SHEPHERD2, D FROST1 and J FINNEGAN1

1CSIRO-Plant Industry, GPO Box 1600, Canberra, ACT, 2601 Australia; 2Department of Plant Science, Waite Campus, PMB1, Glen Osmond, 5064 Australia

Background and objectives
Thirteen different rust resistance specificities are genetically mapped to the L locus in flax. The locus contains a single resistance gene of the nucleotide-binding site-leucine-rich repeat class (NBS-LRR) and the gene has 13 allelic alternatives. The objectives of this study were to determine whether cloned alleles were sufficient for rust resistance and specificity determination, and to compare the sequences of the alleles and their protein products in an attempt to identify important regions for specificity determination and indications concerning the evolution of new alleles.

Results and conclusions
Twelve of the 13 L alleles for rust resistance were cloned and sequenced. An additional L allele (LH) from a variety of flax susceptible to all flax rust isolates was also sequenced, providing a total of 13 alleles for comparison. Ten allelic products were very similar to the product of the originally characterized L6 allele [1]. While variation occurred throughout the amino-acid sequences, it was more pronounced in the LRR region. Three alleles had undergone large deletions (L1 and L8) or sequence duplication (L2) in the LRR-encoding region of the gene. Transgenic plant experiments using the L2, L6 and L10 alleles indicated that the cloned genes were sufficient for specific rust resistance. Expression of two cDNA clones of L6 using the 35S promoter indicated that specificity was determined by the coding region and that alternative spliced mRNA products identified in L6 plants that involve the splicing or retention of intron 3 [1] may not be necessary for resistance.

The sequence comparisons and domain swap experiments indicated that the amino-terminal region and the LRR region both influence specificity. For example, L6 and L11 differ only in the LRR region or the protein product and determine distinct specificities. L6 and L7, however, differ only in the product of exon 1 and are identical in the rest of the protein, and also control distinct specificities. The sequence comparisons also indicate that allelic variation is generated by multiple crossing over or gene conversion events that shuffle pre-existing sequence variation into novel combinations.

References
1. Lawrence G et al., 1995. Plant Cell 7, 1195-1206.