Title page for ETD etd-41611182972760


Type of Document Dissertation
Author Lang, Saara Susanna
Author's Email Address salang@vt.edu
URN etd-41611182972760
Title Role of Subcellular Differentiation in Plant Disease Resistance
Degree PhD
Department Plant Physiology
Advisory Committee
Advisor Name Title
Alscher, Ruth G.
Grabau, Elizabeth A.
Lacy, George H.
Storrie, Brian
Cramer, Carole L. Committee Chair
Keywords
  • HMGR
  • 3-hydroxy-3-methylglutaryl CoA reductase
  • phytoalexins
  • vacuole
  • plant defense
  • immunolocalization
Date of Defense 1997-07-03
Availability unrestricted
Abstract
3-Hydroxy-3-methylglutaryl CoA reductase (HMGR,

EC 1.1.1.34) 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.

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