RNA Interference of the Unfolded Protein Response in Acyrthosiphon pisum

Aphid species are distributed worldwide and are persistent agricultural pests. Previous studies have demonstrated the efficacy of dsRNA as a tool to cause selective death in target organisms through RNA interference ( Balthazor , J. R., et al 2018). Whole RNA was isolated from adult A. pisum and used to synthesize cDNA. Synthesized cDNA was then used as a template to produce dsRNA for use in feeding studies with pea aphids . dsRNAs complementary to target mRNAs were fed to adult aphids and survivorship was assessed. Concentrations of target mRNAs were assessed by quantitative real time PCR (qPCR

RNA Interference of X Box Binding Protein 1 in Acyrthosiphon pisum

Pea aphids, Acyrthosiphon pisum , are a significant pest to legumes Febaceae throughout the world, primarily due to the species serving as a vector to many Febaceae viruses. Current management of A. pisum includes use of insecticides and the introduction of natural predators, neither of which is ideal because it can have detrimental effects on organisms other than A. pisum . The utilization of RNA Interference (RNAi) presents an alternative, pest specific targeting of A. pisum . X Box Binding Protein 1 (XBP1) is involved in the regulation of the unfolded protein response to promote proper folding in the endoplasmic reticulum (ER). RNAi targeting the XBP1 gene could result in the accumulation of misfolded proteins in the ER lumen, which downstream results in death of the cell and organism. RNA was isolated from A. pisum and reverse transcribed to synthesize cDNA. The cDNA was used as a template for XBP1 primers to synthesize XBP1 dsRNA for use in RNAi feeding studies. RNA interference of XBP1 resulted in a fifty percent decrease in fecundity and a thirty percent decrease in survival of Acyrthosiphon pisum . RNA interference of XBP1 has shown to be a promising alternative to the management of A. pisu

Studies of human Armet and of pea aphid transcripts of saliva proteins and the Unfolded Protein Response

Doctor of PhilosophyBiochemistry and Molecular Biophysics Interdepartmental ProgramGerald R. ReeckArmet is a bifunctional protein that is apparently universally distributed among multicellular animal species, vertebrate and invertebrate alike. A member of the Unfolded Protein Response, (UPR) Armet promotes survival in cells that are under endoplasmic-reticulum (ER) stress. I have carried out biophysical studies on human Armet looking for compounds that bind to Armet and hence could reduce its anti-apoptotic function, thus potentially joining the growing class of pro-apoptotic drugs. Performed primarily with 1H-15N HSQC NMR, ligand studies showed that approximately 60 of the 158 residues are potentially involved with binding. The 60 residues are distributed throughout both domains and the linker suggesting multi-domain interaction with the ligand. Circular dichroism studies showed heat denaturation in a two-step unfolding process with independent unfolding of both domains of Armet with Tm values near 68°C and 83 C with the C-terminal domain unfolding first, as verified by 1H-15N HSQC NMR measurements. I also provide the first identification of UPR transcripts in pea aphids, Acyrthosiphon pisum, the genetic model among aphids. I measured transcript abundance with hope of finding future transcriptional targets for pest mitigation. I identified 74 putative pea aphid UPR components, and all but three of the components have higher transcript levels in aphids feeding on plants than those that fed on diets. This activated UPR state is attributed to the need for saliva proteins for plant feeding. Because aphids are agriculturally significant pests, and saliva is pivotal to their feeding on host plants, genes that encode saliva proteins may be targets for pest mitigation. Here I have sought the aphid’s saliva proteome by combining results obtained in several laboratories by proteomic and transcriptomic approaches on several aphid species. With these data I constructed a tentative saliva proteome for the pea aphid by compiling, collating, and annotating the data from several laboratories. I used RNA-seq to verify the transcripts in pea aphid salivary glands, thus expanding the proposed saliva proteome from approximately 50 components to around 130 components, I found that transcripts of saliva proteins are upregulated during plant feeding compared to diet feeding