Abstract
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.
Highlights
Plants employ both direct and indirect defenses against herbivores
To determine the silencing efficiency of Naggpps and Nafpps, total RNA was isolated from the leaves of the transformed plants and subjected to northern analysis (Fig. 2, insets); transcripts of both genes were substantially decreased in gene specific-silenced lines in comparison to EV controls
When inverted-repeat putrescine N-methyltransferase-silenced N. attenuata plants growing in a field plantation in the Great Basin Desert were elicited by methyl jasmonate (MeJA), the damage these plants received from native herbivores was as low as that of wild-type plants, suggesting the presence of other potent MeJA-inducible defenses (Steppuhn et al, 2004)
Summary
Plants employ both direct and indirect defenses against herbivores. Direct defenses may provide physical barriers, act to poison herbivores or impede their digestion, or make plant tissues otherwise unappetizing. In an example of a defensive synergism between metabolites, nicotine was found to be a effective defense in Nicotiana attenuata against the larvae of Spodoptera exigua because the alkaloid prevented the larvae from compensating for the ingestion of TPIs by increasing consumption of leaf material (Steppuhn and Baldwin, 2007). These complex interactions represent a principal reason why studies of metabolite defensive function in planta are thought to provide more reliable results. We examine the role of DTGs in the context of the other major secondary metabolites within the plant
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