MOLECULAR-CLONING AND EXPRESSION OF A NEW CLASS OF ORTHO-DIPHENOL-O-METHYLTRANSFERASES INDUCED IN TOBACCO (NICOTIANA-TABACUM-L) LEAVES BY INFECTION OR ELICITOR TREATMENT

In tobacco (Nicotiana tabacum L. cv Samsun NN), three distinct enzymes account for ortho-diphenol-O-methyltransferase (OMT) activity. OMT I is the major enzyme of healthy leaves, whereas enzymes OMT II and III are preferentially induced during the hypersensitive reaction to tobac co mosaic virus (TMV). Using an anti-OMT III antiserum, we isolated a partial OMT III cDNA clone by immunoscreening an expression library ma de from mRNA of TMV-infected tobacco leaves. Using this OMT III clone as a probe, we isolated a full-length clone with a deduced amino acid sequence encompassing all of the sequences obtained by Edman degradati on of both purified proteins II and III. Thus, OMT II and III of tobac co are likely to be encoded by the same genes and to arise from differ ent posttranslational modifications. Sequence analysis showed that thi s OMT clone represents a new class of OMT enzymes (class II) with a lo w level of similarity (53-58%) to OMTs cloned previously from other di cotyledonous plants. Southern analysis indicated that a small family o f class II OMT genes inherited from ancestors related to Nicotiana syl vestris and Nicotiana tomentosiformis occurs in the tobacco genome. RN A blot analysis demonstrated that class II OMT genes, unlike class I O MT genes, are not expressed at a high constitutive level in lignified tissues of tobacco. Class II OMT transcripts were found to accumulate in tobacco leaves infected with TMV or treated with megaspermin, a pro teinaceous elicitor from Phytophthora megasperma, but not in leaves tr eated with salicylic acid, a molecule known to trigger many defense ge nes. In TMV-infected or elicitor-treated tissues, a marked increase in catechol-methylating activity accompanied the accumulation of class I I OMT gene products.