Abstract
BackgroundPlants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown.ResultsWe used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the M. sexta-N. attenuata interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated ≥ 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction.ConclusionsThe analysis of the early changes in the transcriptome of N. attenuata after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.
Highlights
Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae
Wounding is a prerequisite for Fatty acid-amino acid conjugates (FACs)-elicitation; analysis of wounded leaves was used to differentiate between genes regulated by mechanical damage from those regulated or deferentially by FACs
The total number of SuperSAGE tags obtained after sequencing the libraries in a single 454 plate and eliminating i) incomplete reads, ii) twin-ditags, and iii) ditags without complete library-identification DNA linkers was 354,930; comprising 227,536 tags from the wounding (W) library and 127,394 from the FAC-elicited (F) library (Table 1)
Summary
Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. Nicotiana attenuata is an annual native to Southwestern USA that germinates from seed banks in response to factors in wood smoke after fires [1] Because of this germination behavior and a strong intra-specific competition, N. attenuata allocates resources primarily to sustain rapid growth and seed setting and as a consequence, it has developed a large number of induced defense responses to ward off the unpredictable attacks from herbivores [2]. Fatty acidamino acid conjugates (FAC) are major components in the OS of M. sexta larvae and they are necessary and sufficient to induce most of the defense responses triggered by feeding M. sexta caterpillar in N. attenuata [14]. Disentangling the effect of mechanical tissue damage and FAC elicitation will provide critical information on how plants control changes in its metabolism to more efficiently reduce the negative fitness consequences of herbivore attack
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