Amino acid residues required for the activity of avrD alleles

Certain Pseudomonas syringae pathovars harbor avrD alleles belonging to two different homology classes. The nonfunctional avrD allele of P. s. pv. glycinea is highly homologous to active class II avrD alleles but has five unique amino acid substitutions. Three of these five amino acid changes were shown to be absolutely required for restoration of avrD activity to the P. s. pv. glycinea allele by oligonucleotide site-directed mutagenesis. They were cysteine 19 to arginine, alanine 280 to valine, and leucine 304 to serine. In addition, changing leucine 301 to phenylalanine was required for high activity. However, alteration of the leucine at position 245 of the P. s. pv. glycinea allele to serine, present in the active alleles, did not affect avrD activity. Results from recombinant gene constructs between the nonfunctional P. s. pv. glycinea avrD gene and the functional allele from P. s. pv. phaseolicola identified six other amino acid residues that may form contextual motifs important for AvrD function. These were a four amino acid stretch comprised of glutamate 41, alanine 42, asparagine 43 and arginine 44 in addition to aspartate 243 and phenylalanine 301. Some divergence is tolerated within the four amino acid motif, but phenylalanine 301 appears to be necessary for highly active class II avrD proteins.