A Lycopersicon esculentum Mill. (tomato) cDNA clone with high similarity to a Nicotiana plumbaginifolia Viv. (tobacco) cytochrome P450 gene was isolated using 5' and 3' rapid amplification of cDNA ends (RACE). The isolated cDNA (GenBank Accession No. AF249329) has an open reading frame of 1494 base pairs (bp) and encodes a protein of 498 amino acids with 75% identity to the N. plumbaginifolia cytochrome P450 (CYP72A2) and 45% to a Catharanthus roseus G. Don (Madagaskar periwinkle) CYP72A1 protein sequence. By Southern-blot analysis, one or two highly homologous genes were detected in the L. esculentum genome. Expression of the cloned P450 gene was regulated by circadian rhythm and enhanced by wounding. Leaf transcripts were detected in the light but not dark. Highest transcript levels were observed 3 hours after mechanical wounding. No increase in expression was seen in response to applications of zeatin as with the N. plumbaginifolia gene. Of the tissues analyzed, shoot tips and young leaves and fruit had the highest detectable transcript levels. Attempts to transform more than 1400 cotyledon explants of L. esculentum with sense or antisense CYP72A2 gene constructs produced no transgenic plants.
This study was undertaken to remedy significant yield losses in commercial tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana tabacum L.) production caused by tomato spotted wilt virus (TSWV). One of the possible sources of resistance can be incorporation into the host plant of a viral nucleoprotein (N) gene by Agrobacterium-mediated transformation. Twelve primary transformants of tomato and 141 of tobacco were analyzed for the expression of the N gene and for resistance to the TSWV infection. The tests have demonstrated that transgenic plants were protected against virus infection irrespective of whether or not they contained detectable levels of the translational product.