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Silencing Geranylgeranyl Diphosphate Synthase in Nicotiana attenuata Dramatically Impairs Resistance to Tobacco Hornworm^1,[W],[OA]

Department of Molecular Ecology (A.R.J., K.G., C.H., I.T.B.), and Department of Biochemistry (A.S.), Max Planck Institute for Chemical Ecology, D–07745 Jena, Germany

In bioassays with artificial diets, the 17-hydroxygeranyllinalool diterpenoid glycosides (HGL-DTGs) of Nicotiana attenuata function as antifeedants for the plant's adapted herbivore, tobacco hornworm (Manduca sexta). To determine whether HGL-DTGs have a defensive function in planta, we suppressed HGL-DTG production by silencing the source of the geranylgeranyl diphosphates (GGPPs) required for geranyllinalool biosynthesis, a key intermediate. We used virus-induced gene silencing to suppress transcript levels of GGPP synthase gene (Naggpps) and farnesyl diphosphate (FPP) synthase gene (Nafpps), northern blotting and real-time polymerase chain reaction to quantify transcript accumulations, and radio gas chromatography to analyze prenyltransferase specificity. Silencing Nafpps had no effect on the accumulation of HGL-DTGs but decreased leaf steroid content, demonstrating that DTG-synthesizing enzymes do not use GGPP derived from FPP and confirming FPP's role as a steroid precursor. Unlike plants silenced in the phytoene desaturase gene (Napds), which rapidly bleached, Naggpps-silenced plants had reduced HGL-DTG but not carotenoids or chlorophyll contents, demonstrating that Naggpps supplies substrates for GGPP biosynthesis for HGL-DTGs, but not for phytoene or phytol. Expression of Naggpps in Escherichia coli revealed that the recombinant protein catalyzes the GGPP synthesis from isopentenyl diphosphate and dimethylallyl diphosphate. When fed on silenced plants, hornworm larvae gained up to 3 times more mass than those that fed on empty vector control plants or plants silenced in Nafpps, the trypsin protease inhibitor gene, or the putrescine N-methyltransferase gene. We conclude that HGL-DTGs or other minor undetected diterpenoids derived from GGPP function as direct defenses for N. attenuata and are more potent than nicotine or trypsin protease inhibitors against attack by hornworm larvae.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors is: Ian T. Baldwin (baldwin{at}ice.mpg.de).

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www.plantphysiol.org/cgi/doi/10.1104/pp.107.108811

^* Corresponding author; e-mail baldwin{at}ice.mpg.de.

Received September 8, 2007; accepted October 21, 2007; published October 26, 2007.

This article has been cited by other articles:

				S. Mitra and I. T. Baldwin Independently Silencing Two Photosynthetic Proteins in Nicotiana attenuata Has Different Effects on Herbivore Resistance Plant Physiology, October 1, 2008; 148(2): 1128 - 1138. [Abstract] [Full Text] [PDF]

				M. Skibbe, N. Qu, I. Galis, and I. T. Baldwin Induced Plant Defenses in the Natural Environment: Nicotiana attenuata WRKY3 and WRKY6 Coordinate Responses to Herbivory PLANT CELL, July 1, 2008; 20(7): 1984 - 2000. [Abstract] [Full Text] [PDF]