Choice of host plants by phytophagous insects is essential for their survival and reproduction. This choice involves complex behavioral responses to a variety of physical and chemical characteristics of potential plants for feeding. For insects of the order Hemiptera, these behavioral responses involve a series of steps including labial dabbing and probing using their piercing mouthparts. These initial probing and feeding attempts also elicit a rapid accumulation of phytohormones, such as jasmonic acid (JA), and the induced defense metabolites they mediate. When Nicotiana attenuata plants are rendered JA deficient by silencing the initial committed step of the JA biosynthesis pathway, they are severely attacked in nature by hemipteran leafhoppers of the genus Empoasca. By producing N. attenuata plants silenced in multiple steps of JA biosynthesis and perception and in the biosynthesis of the plant's three major classes of JA-inducible insecticidal defenses, we demonstrate that the choice of plants for feeding by Empoasca leafhoppers in both nature and the glasshouse is independent of the accumulation of major insecticidal molecules. Moreover, this choice is independent of the presence of Candidatus Phytoplasma spp. and is not associated with detectable changes in plant volatiles but instead depends on the plant´s capacity to mediate JA signaling. We exploited this trait and used Empoasca leafhoppers to reveal genetic variation in JA accumulation and signaling hidden in N. attenuata natural populations. P lants provide a variety of resources, such as food, mating and oviposition sites, and shelter for a majority of phytophagous insect species. Host-plant selection by insects involves complex behavioral responses to a variety of physical and chemical characteristics of the host plant that operate at different spatial scales and include long-range olfactory (e.g., plant-derived volatiles perceived by odor receptors) and visual (e.g., plant shape, size, and color) cues and short-range chemotactic and gustatory (e.g., surface metabolites perceived by chemoreceptors) cues (1-3). The physical and chemical characteristics of plants that insects use for host selection depend on the feeding guild and the dietary behavior (e.g., polyphagy or oligophagy) of the insect species (4). For example, Drosophila melanogaster flies (order Diptera) use a wide range of olfactory cues such as methyl-, ethyl-, and propyl esters of short-chain fatty acids generated by microorganisms growing on decaying fruit (5), whereas Drosophila sechellia flies use a specific molecule (methyl hexanoate) emitted by its exclusive food plant, Morinda citrifolia (6).
Nicotiana attenuata has the capacity to respond specifically to herbivory by its natural herbivore, Manduca sexta, through the perception of elicitors in larval oral secretions. We demonstrate that Lectin receptor kinase 1 (LecRK1) functions during M. sexta herbivory to suppress the insect-mediated inhibition of jasmonic acid (JA)-induced defense responses. Gene function analysis performed by reducing LecRK1 expression in N. attenuata by both virus-induced gene silencing and inverted repeated RNA interference (ir-lecRK1 plants) revealed that LecRK1 was essential to mount a full defense response against M. sexta folivory; larvae growing on ir-lecRK1 plants were 40 to 100% larger than those growing on wild-type plants. The insect-induced accumulation of nicotine, diterpene-glucosides, and trypsin protease inhibitors, as well as the expression of Thr deaminase, was severalfold reduced in ir-lecRK1 plants compared with the wild type. The accumulation of JA and JA-Ile was unaffected during herbivory in ir-lecRK1 plants; however, salicylic acid (SA) accumulation was increased by twofold. The expression of nahG in ir-lecRK1 plants prevented the increased accumulation of SA and restored the defense response against M. sexta herbivory. The results suggest that LecRK1 inhibits the accumulation of SA during herbivory, although other mechanisms may also be affected.
The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPAR and RXR, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from 250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNnA binding model for PPAR based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPAR binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPAR copy number. Originally published at: Simicevic, Jovan; Schmid, Adrien W; Gilardoni, Paola A; Zoller, Benjamin; Raghav, Sunil K; Krier, Irina; Gubelmann, Carinne; Lisacek, Frédérique; Naef, Felix; Moniatte, Marc; Deplancke, Bart (2013). Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics. Nature Methods, 10(6):570-576.
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.
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