Peptidomics approaches for the identification of bioactive molecules from Diaphorina citri

Authors: Fleites, Laura; JOHNSON, RICHARD - University Of Washington; KRUSE, ANGELA - Cornell University; Nachman, Ronald; Hall, David; MACCOSS, MICHAEL - University Of Washington; Heck, Michelle

Submitted to: Journal of Proteome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/10/2020
Publication Date: 4/3/2020
Citation: Fleites, L.A., Johnson, R., Kruse, A.R., Nachman, R.J., Hall, D.G., Maccoss, M., Heck, M.L. 2020. Peptidomics approaches for the identification of bioactive molecules from Diaphorina citri . Journal of Proteome Research. 19/4; 1392-1408. https://doi.org/10.1021/acs.jproteome.9b00509.
DOI: https://doi.org/10.1021/acs.jproteome.9b00509

Interpretive Summary: Huanglongbing, also known as citrus greening, is the most serious disease of citrus and is devastating citrus production worldwide. To date, there is no effective control strategy, and citrus growers are struggling to manage the disease. In the United States, the disease is caused by a bacterium which is spread from tree to tree by the small sap-sucking insect, the Asian citrus psyllid. Our group is focused on understanding the interactions between the bacterium and the insect, with the goal of stopping the ability of the insect to spread the disease. This study's aim was to describe the collection of small proteins in the psyllid. Typical methods for studying proteins largely miss the smaller end of the size range. A new method was developed and optimized for the measurement and detection of small proteins. The method enabled the detection of over 120 insect neuropeptides. Several of the neuropeptides found have already been engineered for increased stability by other ARS scientists, and have been shown to be lethal to insects that are closely related to the psyllid. The psyllid neuropeptides detected by the new method provide a starting point for similar engineering of stable analogs that may be lethal to the psyllid. The bacteria have no other natural route for spread within and among groves, and if a tool can be developed to control the insect, the disease could be eliminated.

Technical Abstract: The citrus industry worldwide has been devastated by the spread of Huanglongbing (HLB), a disease primarily associated with ‘Candidatus Liberibacter asiaticus’ and spread by the hemipteran insect Diaphorina citri. The importance of D. citri in the disease cycle has driven multiple ‘omics-based studies; however, the peptidome, a dynamic set of small polypeptides continuously produced by proteolysis and other cellular processes, has not yet been described. In this work, we developed and compared series of workflows for the extraction of endogenous peptides from D. citri. High-resolution mass spectrometry analysis revealed bias among methods reflecting the physiochemical properties of the peptides: while TCA/acetone-based methods resulted in enrichment of C-terminally amidated peptides, a modification characteristic of biologically active peptides, larger peptides were overrepresented in the aqueous phase of chloroform/methanol extracts, possibly indicative of reduced coanalytical degradation during sample preparation. Mining of the datasets also revealed 122 candidate neuropeptides, including PK/PBAN family neuropeptides and kinins, biostable analogs of which have been previously shown to have insecticidal properties in another hemipteran, the pea aphid. Taken together, the information obtained in this work yields new insights into peptidomics methods development and psyllid physiology and may lead to the development of novel management tools for HLB disease.