Source: MICHIGAN STATE UNIV submitted to NRP
SMALL MOLECULES IN APHID SALIVA: COMPOSITION AND ROLE IN PLANT-APHID INTERACTIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032906
Grant No.
2024-67012-43106
Cumulative Award Amt.
$225,000.00
Proposal No.
2023-09758
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2026
Grant Year
2024
Program Code
[A1100]- Plant Health and Production and Plant Products: Post doctoral Fellowships
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
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
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21119991040100%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1999 - Tobacco, general/other;

Field Of Science
1040 - Molecular biology;
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.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:Undergraduate/graduate researchers - individuals who need exposure to plant-insect interactions and methodologies for studying this relationship. Children - Kindergarten through high school students who are curious about science and the impact research has on our communities and agriculture. Michigan State University - Researchers whom I have worked with and presented research to on a regular basis Entomological Society of America - my scientific community that I converse with about my work and present my findings biannually to at branch and national meetings Changes/Problems:The only major change in approach for this research has beend the use of EPG for analyzing the aphid fitness after changes to citrate. Due to moving universities, my current lab did not have a set up for EPG and therefore my project moved to evaluating aphid fitness only through choice and no-choice assays. However, because we no longer believe citrate to be a calcium chelator, it is likely that EPG would not show us more significant results than choice and no-choice assays. What opportunities for training and professional development has the project provided?This project has allowed me two major opportunities for career development. First, my work was selected for the Clarence Suelter Postdoctoral Fellowship through the Biochemistry and Molecular Biology department, which gave me $5000 for professional development. Second, my work was selected to be shared at the Journal of Experimental Botany 75th Anniversary symposium. Additionally, this work at Michigan State University has given me several opportunities to collaborate and grow in my metabolomics capacity. Our Mass Spec facility manager, Tony Schillmiller, has trained me on several LC-MS and GC-MS machines and software for data analysis. I could not do any of this without Tony. How have the results been disseminated to communities of interest?These results have been shared at several conferences, as follows. Fischer, H., Bonnema, S., Waffle, S., Jander, G., and Skirycz, A. 2025. Evaluating the role of the glyoxylate pathway in the plant-aphid interaction. Journal of Experimental Botany's 75th Anniversary Conference. Edinburgh, UK. (Poster presentation) Fischer, H., Schillmiller, A., Jander, G., and Skirycz, A. 2025. Are you my partner? Searching for the function of a novel aphid-salivary metabolite. North Central Branch Entomological Society of America, Lincoln, NE. (Invited Oral Presentation) Fischer, H., Jander, G., and Skirycz, A. 2024. Aphid salivary metabolites in the plant. Entomology Society of America National Meeting, Phoenix, AZ. (Oral presentation) Fischer, H., Jander, G., and Skirycz, A. 2024. The Aphid Salivary Metabolome. North Central Branch Entomological Society of America, Fort Collins, CO. (Oral presentation) Also, my student presented her part of the project at the MSU UURAF conference and won first place. Bonnema, S., Fischer, H., and Skirycz, A. 2025.Exploring the Impact of Plant Central Metabolism on Aphid Fitness. MSU UURAF Annual Conference. East Lansing, MI. (Poster presentation) What do you plan to do during the next reporting period to accomplish the goals?The next stages of my work will include the following: Feed labeled citrate to plants during aphid infestation to confirm that citrate is pulled into the glyoxylate pathway and made into sucrose Quantify the accumulation of sucrose in icl and pck1 mutants to identify the role of sucrose accumulation in aphid fitness Perform a transcriptomics analysis of Arabidopsis with the same treatment groups as the previous metabolomics study to identify potential regulation by γEY Write these results into two papers (1) outlining the fate of aphid salivary metabolites in the plant and (2) the role of the novel aphid-salivary metabolite γEY in the plant-aphid interaction

Impacts
What was accomplished under these goals? Where are salivary metabolites going in the plant? 13C labeling to identify the enrichment of pathways in the plant utilizing aphid saliva from Myzus persicae, the green peach aphid (GPA). LC-MS and GC-MS were used to track 13C from aphids in the plant and identified the glyoxylate pathway and gluconeogenesis as highly enriched, leading to over 40% of carbon in sucrose coming from aphids. Interestingly, the sucrose was not fully labeled, confirming the sucrose was being produced in the plant and not spit in by the aphid, which would then be fully labeled sucrose. Other metabolites that were highly enriched include nucleosides and nucleotides, such as inosine, that had 30% enrichment after aphid feeding. Inosine has been implicated in plant stress response. Additionally, several metabolites were identified that came directly from aphids, such as raffinose, a trisaccharide previously implicated in plant resistance to aphids, and gluconate, an oxidize sugar that could also be involved in stress adaptation of the plant. Furthermore, these labeled metabolites are found in tissue that is restricted from aphids, showing that these metabolites are mobile through the phloem and could reach the rest of the plant. Lastly, several metabolites were strongly increased by aphid infestation, such as carnitine and citrulline, but did not incorporate aphid-derived salivary carbon, showing that the pull of aphid saliva is specific to certain pathways. Overall, these findings are exciting because the glyoxylate pathway has previously been identified as an upregulated pathway from aphid infestation in several crops, including switchgrass, soybean, and tomato. To see aphid saliva be incorporated specifically into this pathway, resulting in sugar, further validates this pathway as important in the plant-aphid interaction. How is the glyoxylate pathway involved in plant-aphid interactions. To determine how the glyoxylate pathway impacts aphid and plant fitness, I utilized Arabidopsis mutants, including: aconitase1 and 3 (aco1 and aco3), citrate synthase2 and 3 (csy2 and csy3), isocitrate lyase (icl), cytosolic malate dehydrogenase1/2 (cmdh1/2), and phosphoenolpyruvate kinase1 (pck1). Using a simple no-choice aphid bioassay with 10 synchronized adult GPA infested on Arabidopsis for 5 days. After that, the number of juveniles produced on the plant were counted. After several rounds of bioassays, I found that csy2, csy3, icl, mdh1/2, and pck1 all had significant resistance to GPA compared with Col-0 wild-type plants. I found that pck1 had a strong effect of 20% reduction in juvenile production. To determine if the aphids performed poorly do to unhealthy plants, plants were photographed 5 days after infestation (dai), 8 dai, and tracked until all the plants collapsed. Interestingly, none of the plants had a sickly phenotype compared with Col-0 and they all collapsed on the same day as Col-0 (12 dai). This is exciting because aphid-resistance mechanisms are desperately needed that do not cause reduction to plant fitness, while also not exerting a strong selection pressure on aphid populations. More work is needed to see if reduced sucrose after aphid feeding is the cause of aphid fitness reduction or if plants in this pathway are increased in other defensive metabolites. What dipeptides are aphids spitting into the plant? Initial investigations into dipeptides in GPA saliva identified glutamyltyrosine (EY) as a possible target because it was found in aphid saliva consistently and was found to increase significantly in the plant only after aphid infestation. However, further work to validate this compound in the plant found that while it was EY, it was a γ-EY, not an α-EY. This seemingly small change in structural isomer is exciting because it tells me how γEY is being produced. Dipeptide formation can occur in a few ways in the plant, typically categorized as proteinogenic breakdown products or as biosynthesis products While α-EY would most likely be the result of proteinogenic breakdown, γEY is a recycling product of the glutathione pathway. This also means that the production of γEY can be regulated more steadily in the aphid and is likely why it has consistent accumulation in the plant. Additionally, a screen of several other aphids found that γEY is present in several species of aphids, including Acyrthosiphon pisum and Brevicoryne brassicae, with γEY being confirmed in the plant after B. brassicae feeding on Arabidopsis. This is exciting because it demonstrates a conserved metabolite in aphids that they spit into the plant and gives me pathway of biosynthesis that could potentially be disrupted. What is γEY doing in the plant? After discovering γEY as a potential target for studying dipeptides in the plant-aphid interaction, I then went on to try and identify what it could be doing to the plant. In human systems, γEY is found to regulate several proteins such as being an agonist of calcium receptors on the human tongue and an inhibitor of dipeptidyl peptidase IV (DPP IV). I went on to try and identify potential protein targets in the plant that aphids might want to regulate. To do this, I performed an isothermal shift assay (iTSA) that uses protein thermostability properties to determine if a ligand is binding with a protein target. This study discovered about 160 proteins that are potentially binding with γEY. To determine if these proteins are actually binding with γEY, I heterologously expressed the proteins, purified them, and used nano-Differential Scanning Fluorimetry (nanoDSF) to evaluate the impact of γEY on protein conformational stability. This technique takes advantage of protein autofluorescence from intrinsic tryptophan and tyrosine. This process is ongoing, but already an important enzyme in photorespiration, serine:glyoxylate aminotransferase (AGT1) has been identified as promising target. For the rest of this project, I have collaborated with Dr. Aaron Leipmann from Eastern Michigan University who is an expert in AGT1 and is performing the enzyme assays to determine if γEY enhances or inhibits AGT1 activity. Additionally, ACO1 and ACO3, which were already mentioned in the above study with the glyoxylate pathway are potential targets for γEY. Expression of these proteins has proved challenging due to the requirement of FeS clusters as a cofactor. The next stages of this project are aimed at addressing this issue. Is γEY causing a metabolic shift in the plant? To further characterize the impact of γEY on the plant, I performed a metabolomics assay of plants after aphid infestation with and without γEY, giving four treatment groups: uninfested control, aphid-infested control, uninfested + γEY, and aphid-infested + γEY. A PCA plot of these groups showed that while aphid infested control tissue and aphid infested + γEY were not significantly different, uninfested + γEY tissue was more similar to aphid infested tissue. Additionally, the metabolites significantly increased in uninfested + γEY include aspartic acid and several metabolites involved in cell well strengthening. These results are important because they show γEY plays a role in plant-stress response to aphids by causing a significant metabolic shift in the plant, possibly for plant stress adaptation.

Publications