Role of Subcellular Differentiation in Plant Disease Resistance

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Virginia Tech

3-Hydroxy-3-methylglutaryl CoA reductase (HMGR, EC catalyzes the reaction from hydroxymethylglutaryl CoA to mevalonate in the isoprenoid pathway. In solanaceous plants, one class of endproducts of the pathway are sesquiterpenoid phytoalexins, antibiotic compounds produced by plants in response to pathogens. We are interested in the role of the defense-inducible isoforms of HMGR in phytoalexin production and disease resistance. Transgenic tobacco, constitutively expressing the defense-inducible tomato hmgr isogene, hmg2, showed fewer and smaller lesions following tobacco mosaic virus (TMV) inoculation. There is little evidence of phytoalexins acting directly against viruses, but they may reduce the spread of viruses as part of the hypersensitive response resulting in death of the host cell. Transmission electron microscopy of leaf cells of the transgenic plants revealed a larger volume of cytosol and accumulation of electron-dense inclusion bodies within the vacuoles. No structures resembling crystalloid ER or karmellae, caused by overexpression of hmgr in mammalian or yeast cells, respectively, were observed. Similar inclusion bodies were found in the vacuoles of wild-type tobacco leaf cells adjacent to necrotic cells in a TMV lesion. Tobacco expressing a truncated (membrane domain) form of hmg2 did not show enhanced resistance to TMV or any ultrastructural changes, indicating the importance of catalytically active HMG2 in mediating these changes. Sesquiterpene cyclase (a key branch point enzyme controlling sesquiterpene phytoalexin biosynthesis) was not induced and the amount of capsidiol, the tobacco phytoalexin, was not elevated by expression of hmg2. After TMV-inoculation, HMGR activity and the amount of capsidiol were higher in the wild-type than in the transgenic plants. Consequently, the enhanced resistance to TMV was not due to constitutive capsidiol production. The transgenic plants may have been able to produce sesquiterpenoid phytoalexins faster due to constitutive hmg2- expression and restricted the spread of the virus earlier, so that only a few cells were sacrificed. The subcellular localization of the defense-specific HMG2 isoform was determined by tagging tomato hmg2 with a c-myc epitope, and constitutively expressing the construct in transgenic tobacco plants. In non-induced leaves, MYC-HMG2 was found localized in small clusters associated with the ER. In TMV-inoculated leaves MYC-HMG2 co-localized with sesquiterpene cyclase to the vacuolar inclusion bodies suggesting that they may contain a defense-induced, membrane-associated multienzyme complex dedicated to sesquiterpene production. Our results support the hypothesis of the multibranched plant isoprenoid pathway being partly regulated by pathway partitioning.

HMGR, 3-hydroxy-3-methylglutaryl CoA reductase, phytoalexins, vacuole, plant defense, immunolocalization