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Comparing the response of Citrus ×limon and Citrus ×sinensis to Trioza erytreae infestation using a proteomic approach

Publication . Magalhães, Tomás; Dandlen, Susana; L, Anjos; Power, Deborah; Pereira, José Alberto; Duarte, Amilcar; Marques, Natália

Citrus production is on high alert because of the devastating disease Huanglongbing (HLB), caused by the bacteria Candidatus Liberibacter spp.. With no viable treatment, current management practices rely on the control of its vectors, such as the African citrus psyllid,Trioza erytreae (Del Guercio 1918), which is already in the Mediterranean region (Portugal and north of Spain). This vector develops better in some citrus hosts, with Citrus ×limon described as the preferred host. To better understand the molecular response of citrus hosts to the psyllid, the phloem proteome of lemon (Citrus ×limon) and orange (Citrus ×sinensis) plants infested with T. erytreae was compared with equivalent non infested plants. Infestation was established with isolated plants by exposing them to 10 T. erytreae adults. Nymphs of T. erytreae at the 4-5th instar stage were removed from plants and infested leaf phloem was extracted. In control plants phloem was extracted from leaves of a similar size and developmental stage. The experiment was done under controlled conditions of temperature, light and humidity. Phloem was analyzed by nanoLC-MS/MS. A total of 48 and 1265 differentially abundant proteins (DAP) were identified in lemon and orange plants, respectively, with 18 proteins common to both species. The topmost enriched GO terms retrieved for upregulated proteins in lemon plants were assigned to organic acid and cellular amino acid metabolic processes. The topmost enriched GO terms in orange plants included organonitrogen compound metabolic process, cellular component assembly, establishment of protein localization, while downregulated terms were associated with carbohydrate metabolic process. This study revealed that T. erytreae infestation promoted distinct modifications in the phloem proteome of lemon and orange plants. This work is part of a group of studies that focus on this insect-plant interaction that aims for more informed and improved T. erytreae control.

2022-11-08Conference object

Restricted access

Proteomics of fish white muscle and Western blotting to detect putative allergens

Publication . Anjos, Liliana; Loukissas, A. Ζ.; Power, Deborah

A detailed workflow is provided for preparation from teleost fish white muscle of extracts for proteomics analysis. The protocol generates samples that can be analyzed by SWATH (Sequential Window data independent Acquisition of the Total High-resolution-Mass Spectra), a modern MS-based quantitative label free technology. The main steps for the extraction of three independent protein fractions, (1) soluble sarcoplasmic, (2) soluble myofibrillar, and (3) insoluble material, from fish white muscle are detailed. Coupled to the protein extraction protocol a Western blotting approach is outlined for detection of common fish allergens, in this case β-parvalbumin, in the white muscle sarcoplasmic protein fraction.

2022Journal article

Open access

Extracting protein from microalgae (Tetraselmis chuii) for proteome analysis

Publication . L, Anjos; Estêvão, J.; Infante, Carlos; Mantecón, Lalia; Power, Deborah Mary

Microalgae have high potential as a resource for sustainable and green protein for food or bioactive molecules. Nonetheless, despite the high protein content of microalgae (40 - 70% dry weight) progress in the characterization of their protein composition remains challenging. This is due to the highly variable chemical composition of microalgae strains and factors such as their rigid thick cell wall, polysaccharide content, protein stability, pH. The method described herein was developed to optimize protein extraction for proteome analysis of microalgae (Tetraselmis chuii) biomass. The effects on protein solubility of solvent type (organic, denaturing, and non-denaturing) combined with three customized microalgae disruption methods were investigated. The proteome targeted high quality protein extracts were for hydro-soluble proteins recovered by cell disruption using bead milling coupled to centrifugation (protein yield approximate to 13%). The developed method is inexpensive, efficient (yielding high-quality protein extracts with a low content of interfering compounds) and from an industrial perspective easily scalable and compatible with other applications. To add value to the end product we additionally propose the use of stabilizing agents to maintain protein solubility during refrigerated storage and a method targeting the fractionation of low molecular weight proteins. An inexpensive easy-to-do 5 step protocol for microalgae protein extracts. A protein extraction method free from dangerous or highly polluting chemicals. Production of high yield aqueous protein extracts suitable for proteomics. (C) 2022 The Authors. Published by Elsevier B.V.

2022Journal article

Open access