Performing Department
(N/A)
Non Technical Summary
Aphids are like the vampires of the plant, using a specialized piercing-sucking mouthpart to feed on the phloem for long periods of time (8+ hours). During this process, the plant and aphid are each mounting defenses against the other, with the plant trying to stop the aphid from feeding and the aphid trying to stop the plant from defending itself. From the plant side, it will produce reactive oxygen species (ROS) and calcium (Ca2+) that send signals to the rest of the plant to defend itself, as well as stop the flow of phloem to the aphid. The aphid in turn is spitting into the plant a complex mixture of saliva that can stop these defenses, either by reducing the ROS or binding up the Ca2+, amongst other strategies. Currently, what is known about aphid saliva is that is contains a diverse mixture of proteins, each with a unique function to aid the aphid in feeding. However, recently I have discovered another component of the saliva that may have an important role in aphid manipulation of the plant called small molecules. Small molecules, including compounds known as metabolites, can interact with plant proteins or defense compounds to manipulate plant physiology. One of the most abundant metabolites I found in aphid saliva was citric acid (CA) and malic acid (MA). These two metabolites are essential components of metabolism in many organisms, including the plant. They serve to produce energy for the plant, as well as precursors of other important compounds in the plant like amino acids and antioxidants, molecules that scavenge ROS. I also found a diversity of dipeptides (DPs) in aphid saliva, an emerging class of compounds that can serve as antioxidants and/or regulate cellular metabolism. Intriguingly, none of the DPs I have discovered in aphid saliva have a known function. Overall, my preliminary findings suggest that aphid salivary metabolites are a promising new avenue of research in plant-aphid interactions for their potential to mitigate the plant defense response. This research area is important as it has relevance to basic research, investigating how plant physiology is regulated by aphid saliva, as well as applied research in discovering new novel compounds, such as DPs, that are invovled plant adaptation or susceptibilityto aphids.In order to investigate the role of aphid salivary metabolites in the plant-aphid interaction, I will employ a variety of techniques to characterize first (1) how aphid fitness is impacted on plants with increased CA, MA, and DPs in the plant and (2) how the plant fitness changes in response to these compounds. For objective 1, I will use aphid bioassays that measure the growth and survival of aphids on plants with increased CA, MA, and/or DPs. I will also use a powerful tool called Electrical Penetration Graph (EGP) to measure aphid feeding, which indicates aphid success on the host plant. For objective 2, I will investigate how the CA, MA, and DPs are impacting plant physiology and defense responses to aphids. CA and MA are both central carbon metabolites, which can be incorporated into plant metabolism. Using stable-isotope carbon, I can trace aphid-salivary metabolites in the metabolic flux of the plant and identify which compounds, specifically antioxidants, are made in the plant from aphid saliva. Additionally, CA, MA, and DPs are found to dampen the ROS and Ca2+ response of the plants, and thereby plant defenses. To investigate this potential role of salivary metabolites, I will use fluorescent reporter lines of Arabidopsis to visualize ROS and Ca2+ signaling intensity and duration in plants with and without increased CA, MA, and DPs after aphid infestation. If these metabolites are found to decrease the ROS and Ca2+ response, I will further validate their function in a reciprocal approach using ROS and Ca2+ signaling mutants. Together, these results will begin to uncover the role of salivary metabolites in the plant-aphid interaction and provide methodology for studying the salivary metabolites of other aphid species in agronomically important crops.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
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Goals / Objectives
The goal of this project istocharacterize the influence of the most abundant aphid salivary metabolites on plant-aphid interactions. To do this,I will evaluate their impact on host-plant suitability for Myzus persicae, the green peach aphid (GPA), in two plant species(Objective 1) as well as the influence on plant physiology and metabolism related to aphid defense (Objective 2). GPA feed readily on the laboratory model plants Arabidopsis and Nicotianabenthamiana. The benefit of Arabidopsis is the large toolbox of genetic and transgenic material that will allow me to study plant responses. For N. benthamiana there are readily available methods for transient gene expression and CRISPR/Cas9 mutagenesis by Agrobacterium infiltration into leaves. Additionally, Arabidopsis and N. benthamiana belong to the two different plant families that exploit different suit of specialized metabolites to defend against aphids, glucosinolates and nicotine, respectively.Objective 1: Assess effects of TCA cycle intermediates and dipeptides on aphid fitnessHypothesis: TCA cycle intermediates and dipeptides will increase aphid fitness on host plants through improved feeding and juvenile developmentObjective 2: Evaluate TCA cycle intermediates and dipeptides on plant response to aphidsHypothesis: TCA cycle intermediates and dipeptides will alter host-plant metabolome and dampen Ca2+ signaling and ROS accumulation
Project Methods
Objective 1: To begin unraveling the role of aphid-derived TCA cycle intermediates and dipeptides in mitigating plant defenses, I will assess the impact of citric acid and malic acid, as well as the most abundant dipeptide in GPA saliva, Glu-Tyr, on aphid fitness. First, I will perform GPA no-choice bioassays using N. benthamiana leaves, with and without increasing of the three target metabolites. The no-choice bioassay requires aphids to feed on the plant for a number of days and, because metabolites are readily metabolized in the plant, I will use detached N. benthamiana leaves with their petioles set in solutions of each metabolite. The detached leaf assay has been previously performed by the Jander lab with reliable results. For the no-choice bioassay,in brief, 25 newly larviposited GPA will be caged on detached leaves of N. benthamiana set in solutions of each metabolite ranging from 100 µM to 10 mM and left to reproduce. I will daily monitor survival and growth of the nymphs. After 5-6 days, when nymphs reach adulthood, five adults will be left to reproduce for five more days. At the end of the bioassay, I will confirm the increase of the targeted metabolite in the leaf tissue. Tissue will be harvested, flash-frozen in liquid nitrogen, and processed with the methyl tert-butyl ether (MTBE)-based extraction protocolfor quantification of targeted metabolites using Skirycz lab methods with UPLC-MS on a Thermo Q-Exactive quadrupole-orbitrap mass spectrometer. Uninfested leaves in solution will be used as controls. This bioassay will determine metabolites and concentrations that significantly influence the fitness of GPA on N. benthamiana. Using these findings, I will perform the bioassay with Arabidopsis to compare between two plant families.I will also evaluate the feeding behavior of aphids on N. benthamiana with the targeted metabolites using the Electrical Penetration Graph (EGP) technique. EPG is a well-established tool to facilitate the visualization of plant feeding by piercing-sucking insects, such as aphids, in which I am well practiced. Simply put, a partial circuit is generated in the plant and when an aphid with a gold wire (20 µm thick) glued to its abdomen feeds on the plant tissue, the circuit is completed and results in output waveforms that correspond to different stages of aphid feeding. These include stylet probing, salivation into the phloem, and ingestion of the phloem. Because EPG it is typically performed on the first 8 hrs of aphid feeding and is best done on whole plants, I will syringe-infiltrate metabolites into the N. benthamiana leaf. Two hours before EPG recording, metabolites will be infiltrated into the leaf at concentrations that significantly influenced aphid fitness in the bioassays. Each EPG run will last 8 hrs and be replicated 20 times to account for plant variation. Individual EPG runs produce a file that will be annotated with the "EPG Stylet a+" software for relevant feeding parameters.Objective 2: My preliminary study using [13C12]sucrose fed to aphids demonstrates that TCA cycle intermediates and dipeptides found in aphid saliva accumulate in plant tissue by 24 hrs post-infestation. Additionally, some 13C-labeled metabolites that accumulated in the plant tissue after aphid feeding were not previously detected in aphid saliva, including the putatively-identified ROS-scavenging glutathione. This strongly suggests that aphid-derived metabolites are being incorporated into plant metabolism. However, as aphids are salivating sucrose into the plant along with TCA cycle intermediates, it is not clear whether aphid-derived citric acid and/or malic acid are primary contributors to the accumulation of secondary metabolites. To further investigate the metabolic consequences of aphid-derived salivary metabolites being injected into the plant, I will perform time-course assays similar to the one described in Figure 2B. Adult GPA will be fed for 5 days with [13C12]sucrose in an artificial diet to provide sufficient time for citric acid, malic acid, and other metabolites to be labeled. Paired leaf discs of both Arabidopsis and N. benthamiana will be infested with either [13C12]sucrose-fed aphids or control aphids from unlabeled sucrose diet. After 0, 2, 6, 12, and 24 hrs, both aphids and leaf discs will be collected and weighed in 2 mL tubes, flash-frozen in liquid nitrogen, and processed with the non-targeted MTBE extraction. Metabolites will be measured with LC-MS/MS. I will match unique mass features extracted from chromatograms with our in-house reference library of 2000 small molecules. Data analysis will focus on detecting and quantifying isotopomers of metabolites central to the TCA cycle, dipeptides, sugars, and metabolites relevant to plant stress response such as antioxidants. Results will be visualized as the relative abundance of isotopomers and the total 13C enrichment of each metabolite in the studied network. This will provide me with a flux map of how aphid-derived metabolites are being incorporated into and likely influencing plant metabolism. TCA cycle intermediates are found to bind with Ca2+, and dipeptides have previously been reported to dampen Ca2+ signaling. To determine whether citric acid, malic acid, and Glu-Tyr reduce Ca2+ signaling, I will utilize a fluorescent reporter line of Arabidopsis to visualize Ca2+ signaling. The ratiometric GCaMP3 fluorescent protein based [Ca2+]cyt sensorexpressed under the pSUC2 promoter can detect [Ca2+]cyt changes in the phloem. Using this reporter, I willvisualize and quantify changes in [Ca2+]cyt in an Arabidopsis leaf, with and without increased citric acid, malic acid, or Glu-Tyr in response to aphid infestation. For this, 12 h prior to imagining, the petiole from a pSUC2:: GCaMP3 Arabidopsis leaf will be set in metabolite solutions with concentrations ranging from 100 µM to 10 mM (may change depending on work in Objective 1). If there is a significant reduction in [Ca2+]cyt it may be because the metabolites are acting as chelators to bind Ca2+ or due to acidification that can dampen Ca2+ signaling. Therefore, using another ratiometric sensor in Arabidopsis, pHluorin, I will measure the changes in pH in a similar experiment as for Ca2+. ROS is an early defense response to aphid infestationand successful aphid infestations often are dependent on reducing ROS accumulation. TCA cycle intermediates and dipeptides have both been shown to reduce the oxidative response of the plant in to stress.I will measure the ROS response in Arabidopsis using multiple methods. First, I will measure extracellular ROS accumulation using the luminol-based protocolwhich uses an elicitation solution that reacts with ROS to produce quantifiable luminescence. Additionally, because the subcellular location of ROS accumulation influences the defense response, I will use a ratiometric green fluorescent protein sensitive to the reductive/oxidative (redox) status of a cellular compartment (roGFP2). Arabidopsis reporter lines with roGFP2 localized to the chloroplast, cytosol, peroxisome, and mitochondria will allow me to evaluate the impact of citric acid, malic acid, and Glu-Tyr on mitigating the redox response of the plant to aphids. The luminol-based assay and roGFP2 assays will all be performed using high-throughput paired leaf assays in multi-well plateswhich are suitable for work with aphids. Leaf discs will be punched from mature Arabidopsis leaves and floated in metabolite solutions of concentrations ranging from 100 µM to 10 mM for a 12 hrs prior to aphid inoculation.Based on my initial experiments to investigate the effects of citric acid, malic acid, and dipeptides on ROS formation, I will conduct follow-up research to investigate the specific aspects of this pathway that are affected. For this, I will make use of readily available Arabidopsis mutants with deficiencies in ROS signaling.