Efficient Confirmation of Plant Viral Proteins and Identification of Specific Viral Strains by nanoLC-ESI-Q-TOF Using Single-Leaf-Tissue Samples

Abstract

Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456.

A Novel Isolate of Bean Common Mosaic Virus Isolated from Crownvetch (Securigera varia L. Lassen)

“…This procedure was repeated twice for each plant, producing two protein samples per plant. In all samples, BCMV viral proteins were detected using a medium-resolution nano-Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight (nanoLC-ESI-Q-TOF), as described before [18], confirming the pathogenicity of the isolate. Proteins were extracted from young leaves using the double crushing step extraction protocol [18] including the RuBisCO depletion step using phytate and Ca 2+ ions.…”

“…In all samples, BCMV viral proteins were detected using a medium-resolution nano-Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight (nanoLC-ESI-Q-TOF), as described before [18], confirming the pathogenicity of the isolate. Proteins were extracted from young leaves using the double crushing step extraction protocol [18] including the RuBisCO depletion step using phytate and Ca 2+ ions. Mass spectrometry measurements were carried out using the UHPLC Dionex Ultimate 3000 RSLCnano (Dionex, Sunnyvale, CA, USA) connected to the mass spectrometer ESI-Q-TOF Maxis Impact (Bruker, Billerica, MA, USA), and the spectra were acquired as before [18].…”

“…Proteins were extracted from young leaves using the double crushing step extraction protocol [18] including the RuBisCO depletion step using phytate and Ca 2+ ions. Mass spectrometry measurements were carried out using the UHPLC Dionex Ultimate 3000 RSLCnano (Dionex, Sunnyvale, CA, USA) connected to the mass spectrometer ESI-Q-TOF Maxis Impact (Bruker, Billerica, MA, USA), and the spectra were acquired as before [18]. The peptides in raw spectra were identified and quantified using MaxQuant 1.6.17.0 [19] for Windows using the same parameters as before [18].…”

“…Mass spectrometry measurements were carried out using the UHPLC Dionex Ultimate 3000 RSLCnano (Dionex, Sunnyvale, CA, USA) connected to the mass spectrometer ESI-Q-TOF Maxis Impact (Bruker, Billerica, MA, USA), and the spectra were acquired as before [18]. The peptides in raw spectra were identified and quantified using MaxQuant 1.6.17.0 [19] for Windows using the same parameters as before [18]. The spectra were searched against all known Nicotiana proteins downloaded from the UniProt protein knowledgebase [20] together with all known BCMV protein sequences, all known BCMV protein sequences downloaded from the NCBI Protein database [21], and BCMV SVK isolate protein sequence.…”

Characterization of an isolate of Lettuce big-vein associated virus (LBVaV) detected in naturally infected tomato (Solanum lycopersicum L.) in Slovakia

“…To confirm the presence of LBVaV in the original sample by another independent method, the virus was successfully detected in leaf sample of tomato plant by a medium resolution nano-Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight (nanoLC-ESI-Q-TOF) as described by Cejnar et al (2020). A proteomic search for viruses potentially present in the sample resulted in the identification of a specific peptide fragments with the unique sequence of 16 aminoacids characteristic for the portion of LBVaV L protein (polymerase).…”