ISOLATION OF THE CDNAS ENCODING (-HYDROXYMAACKIAIN 3-O-METHYLTRANSFERASE, THE TERMINAL STEP FOR THE SYNTHESIS OF THE PHYTOALEXIN PISATIN INPISUM-SATVIUM()6A)

Pisatin is the major phytoalexin produced by pea upon microbial infect ion. The enzyme that catalyzes the terminal step in the pisatin biosyn thetic pathway is (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM). We report here the isolation and characterization of two HMM cDNA clon es pHMM1 and pHMM2) made from RNA obtained from Nectria haematococca-i nfected pea tissue. The two clones were confirmed to encode HMM activi ty by heterologous expression in Escherichia coli. The substrate speci ficity of the methyltransferases in E. coli was similar to the activit y detected in CuCl2-treated pea tissue. Nucleotide sequence analysis o f Hmm1 and Hmm2 revealed an open reading frame of 1080 bp and 360 amin o acid residues which would encode 40.36 kda and 40.41 kDa polypeptide s, respectively. The deduced amino acid sequence of HMM1 has 95.8% ide ntity to HMM2, 40.6% identity to Zrp4, a putative O-methyltransferase (OMT) in maize root, and 39.1% to pBH72-F1, a putative OMT induced in barley by fungal pathogens or UV light. Comparison of the deduced amin o acid sequences of the cDNA clones to OMTs from other higher plants i dentified the binding sites of S-adenosylmethionine (AdoMet). Southern blot analysis showed two closely linked genes with strong homology to Hmm in the pea genome.