Project description:Nicotiana attenuata and Solanum nigrum were challenged with different insects: Manduca sexta and Tupiocoris notatus in different combinations Goals of the study are first to haracterize the transcriptional response of two native Solanaceous plants (Nicotiana attenuata and Solanum nigrum) to attack from two herbivorous pests common on Solanaceous crops (Manduca sexta and Tupiocoris notatus). And second to identify genes involved in herbivore vaccination phenomenon. After germination, inbred and genetically characterized lines of N. attenuata DI92 and S. nigrum Sn30 originally collected from southwestern Utah in 1988, and Jena Germany in 2000, respectively, were grown in a greenhouse. Eggs of Manduca sexta (abbreviated M. s.) came from the North Carolina State University - Entomology Insectary and were hatched under 37°C. Nymphs and adults of Tupiocoris notatus (abbreviated T. n.) came from laboratory colonies started in 2000 with individuals collected from our Utah field sites. Plant were challenged with these different insect and combinations of them. All plants for a given treatment were enclosed in glass-and-mesh insect cages to avoid cross-infection. After 24 h or 5 days the herbivores and their frass were removed, and the above ground biomass of each plant was flash-frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Three independent replicate cages (biological replicates) were used for each of the 7 treatments resulting in a total of 21 cages per species. RNA extraction was performed according to the protocol using Trizol. The RNA samples represent a pooled sample from 4 plants grown in a cage. Keywords: Direct comparison
Project description:DICER-like proteins produce small RNAs that silence genes involved in development and defenses against viruses and pathogens. Which DCLs participate in plant-herbivore interactions remains unstudied. We identified four distinct DCL genes and stably silenced their expression by RNAi in Nicotiana attenuata, a model system for the study of plant-herbivore interactions. Silencing DCL1 expression was lethal to the plants. Manduca sexta larvae performed significantly better on ir-dcl3and ir-dcl4 plants, but not on ir-dcl2 plants compared to wild type plants. Phytohormones, defense metabolites and microarray analyses revealed that when DCL3 and DCL4 were silenced separately, herbivore resistance traits were regulated in distinctly different ways. Crossing of the lines revealed complex interactions in the patterns of regulation. Single ir-dcl4 and double ir-dcl2/ ir-dcl3 plants were impaired in JA accumulation, while JA-Ile was increased in ir-dcl3 plants. Ir-dcl3 and ir-dcl4 plants were impaired in nicotine accumulation; silencing DCL2 in combination with either DCL3 or DCL4 restored nicotine levels to those of WT. Trypsin proteinase inhibitor activity and transcripts were only silenced in ir-dcl3 plants. We conclude that DCL2/3/4 interact in a complex manner to regulate anti-herbivore defenses and that these interactions significantly complicate the already challenging task of understanding smRNA function in the regulation of biotic interactions. Gene expression in leaves of Nicotiana attenuata wild type, irDCL3 and irDCL4 plants was measured at 1 hour after elicitation with oral secretions of Manduca sexta larvae. Three independent experiments were performed with wild type plants and three independent experiments were performed with irDCL3 and irDCL4 plants. A total of 811 genes were identified as differentially regulated in irDCL3 and irDCL4 compared to wild type plants.
Project description:DICER-like proteins produce small RNAs that silence genes involved in development and defenses against viruses and pathogens. Which DCLs participate in plant-herbivore interactions remains unstudied. We identified four distinct DCL genes and stably silenced their expression by RNAi in Nicotiana attenuata, a model system for the study of plant-herbivore interactions. Silencing DCL1 expression was lethal to the plants. Manduca sexta larvae performed significantly better on ir-dcl3and ir-dcl4 plants, but not on ir-dcl2 plants compared to wild type plants. Phytohormones, defense metabolites and microarray analyses revealed that when DCL3 and DCL4 were silenced separately, herbivore resistance traits were regulated in distinctly different ways. Crossing of the lines revealed complex interactions in the patterns of regulation. Single ir-dcl4 and double ir-dcl2/ ir-dcl3 plants were impaired in JA accumulation, while JA-Ile was increased in ir-dcl3 plants. Ir-dcl3 and ir-dcl4 plants were impaired in nicotine accumulation; silencing DCL2 in combination with either DCL3 or DCL4 restored nicotine levels to those of WT. Trypsin proteinase inhibitor activity and transcripts were only silenced in ir-dcl3 plants. We conclude that DCL2/3/4 interact in a complex manner to regulate anti-herbivore defenses and that these interactions significantly complicate the already challenging task of understanding smRNA function in the regulation of biotic interactions.
Project description:The purpose of this experiment was to assess the effect of a synergistic combination of natural pyrethrin and an ethylacetate extract of Piper nigrum seeds (a botanical insecticide). This effect was compared to the effects of P. nigrum or pyrethrin used alone. Due to the synergistic nature of the mixture, it was predicted that gene expression profiles in this treatment would reflect this effect. Keywords: insecticide response, stress-response
Project description:Idenfication and characterization of oscillating transcripts after elicitation with oral secretions from the sepcialist herbivore, Manduca sexta larvae
Project description:Idenfication and characterization of oscillating transcripts after elicitation with oral secretions from the sepcialist herbivore, Manduca sexta larvae Source leaves, sink leaves and roots were collected every 4 h for one days.
Project description:Identifying whether FAC-induced early defence signalling genes were regulated by JA Gene expression in leaves of Nicotiana attenuata wild type and ir-AOC plants was measured at 30 min after elicitation with FAC (C18:3-Glu) and water (control). Three biological replicates were performed with both wild type and ir-AOC plants. In total 12 microarraies were hybridized.