Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

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.

Turnip yellows virus (TuYV) is one of the most important pathogens of oilseed rape worldwide. The virus has a large host range including many crop species (e.g., oilseed rape, pea, chickpea) and weeds from more than twenty plant families. Other than oilseed rape, we detected TuYV in many commonly grown weed species that share the fields and vegetation period together with canola crops in Czech and Slovak Republics. TuYV was detected by reverse-transcription polymerase chain reaction (RT-PCR) in at least 26 species including main crop hosts (oilseed rape), intercrops and weeds such as Amaranthus retroflexus, Atriplex patula (Amaranthaceae), Arctium lappa, Lactuca serriola, Taraxacum officinale, Tripleurospermum inodorum (Asteraceae), Phacelia tanacetifolia (Boraginaceae), Brassica napus, Capsella bursa–pastoris, Descurainia Sophia, Raphanus raphanistrum, Sinapis alba, Sisymbrium officinale, Thlaspi arvense (Brassicaceae), Silene alba, Stellaria media (Caryophyllaceae), Euphorbia helioscopia (Euphorbiaceae), Geranium rotundifolium (Geraniaceae), Lamium purpureum (Lamiaceae), Fumaria officinalis, Papaver rhoeas (Papaveraceae), Veronica persica (Plantaginaceae syn. Scrophulariaceae), Fallopia convolvulus (Polygonaceae), Solanum nigrum (Solanaceae), Urtica dioica (Urticaceae) and Viola arvensis (Violaceae). The detection of TuYV was further confirmed by RT-qPCR as well as Sanger sequencing of the PCR fragments. We discovered four new weed species as hosts of TuYV such as T. inodorum, S. alba, G. rotundifolium and E. helioscopia, representing their three respective plant families. The readthrough domain (RTD) gene sequence analysis of the Czech and Slovak TuYV isolates from oilseed rape and weed species showed similar within-group nucleotide divergence (7.1% and 5.6%, respectively) and the absence of geographical- or host-based phylogenetic clustering. The high-throughput sequencing of the P. rhoeas sample enabled the obtention of a nearly complete genome of TuYV and revealed the mixed infection of TuYV with turnip mosaic virus and cucumber mosaic virus. Our results thus show that weed species are an important TuYV reservoir and play a significant role in the spread and incidence of the disease in field crops such as oilseed rape.