Functional analyses of tomato 3-hydroxy-3-methylglutaryl coenzyme a reductase (HMGR) genes in transgenic plants engineered for altered HMGR expression

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Virginia Tech


3-Hydroxy-3-methylglutaryl CoA reductase (HMGR, EC mediates the first regulatory step (HMG-CoA reduction to mevalonate) in isoprenoid biosynthesis. The tomato genome contains at least four differentially regulated hmg isogenes encoding HMGR. Functions of tomato hmg2 in defense responses were studied by promoter analyses of hmg2:GUS gene fusions, overexpression of hmg2 cDNA, and antisense inhibition of hmg1 and hmg2 in transgenic plants. Activity of the hmg2 promoter is developmentally regulated showing expression in seedling cotyledons and hypocotyls, in trichomes, and in reproductive tissues including pollen, stigmas, ovules, petals and mature seeds. hmg2:GUS activity is rapidly induced by wounding or in response to pathogenic viruses or bacteria. hmg2:GUS expression is localized to tissue surrounding lesions generated through interactions with either TMV or the bacterial pathogen, Erwinia carotovora subsp. carotovora (Ecc). Tomato hmg2 cDNA was cloned by PCR, expressed in E. coli to confirm its HMGR activity, inserted behind the double enhanced CaMV 35S promoter, and engineered into tobacco. Southern and northern analyses confirmed transformation and message expression. Enzyme activity was enhanced compared to nontransformed plants. Selected transgenic plants were significantly reduced for Ecc tissue maceration. The size of necrotic lesions induced by TMV was also significantly reduced compared to the nontransformed or vector controls. Thus, genetic manipulation of the rate-limiting step in a major defense pathway provides a novel strategy for enhancing disease resistance. We also generated transgenic tobacco and tomato containing antisense constructs for tomato hmg1 and hmg2 to study their effect on disease resistance. Full-length hmg2 and 5' regions of hmg1 or hmg2 were inserted in the antisense orientation behind a 35S promoter. Tomato expressing the full-length hmg2 antisense showed lower HMGR enzyme activity and were more susceptible to soft rot by Ecc than control plants. In contrast, expression of either antisense hmg/ or antisense hmg2 in the heterologous tobacco system resulted in plants with enhanced resistance to Ecc and reduced TMV lesion sizes. These results may indicate that antisense inhibition is non-specifically exerted on isogenes other than the defense-specific HMGR gene.



tomato HMGR, disease resistance, genetic engineering, over-expression, antisense inhibition