2.5. Virus Detection in Pea Plants

Total RNA was extracted from the inoculated and youngest systemically infected leaves using TRIzol^® reagent (Ambion, Life Technologies, USA) followed by a normalized reverse transcription-quantitative PCR (RT-qPCR) method. Individual leaves, previously collected at 15 and 21 days post inoculation (dpi) and stored at −80 °C, were frozen in liquid nitrogen and then ground with zirconium oxide beads. Then, the leaf powder was treated with 8–10% (v/w) TRIzol^® reagent and chloroform. The nucleic acids were precipitated by adding 1 vol of isopropanol and 4 µL of linear polyacrylamide (5 mg/mL, Ambion) and resuspended in 30 µL of nuclease-free water. All RNA samples were then treated with 2U of TURBO DNase enzyme (TURBO DNA-free Kit; Ambion) to remove DNA contamination. DNase-treated total RNA was quantified with the QuBit^® fluorometer 2.0 (Invitrogen, Carlsbad, CA, USA) and the Quant-iT RNA HS Assay Kit according to the supplier’s procedure.

RNA quantification was performed in duplicate for each gene and sample by one-step real-time quantitative RT-PCR (qRT-PCR) in 384-well optical plates (CFX384 Touch™ Real-Time PCR Detection System, Bio-Rad, Hercules, CA, USA) using SYBR^® Green technology (iTaq One-Step RT-PCR Kit, BioRad). Each reaction was performed in 10 μL of 1× SYBR^® Green RT-PCR reaction mix, 0.125 µL of iScript Reverse Transcriptase, 300 nM each qPCR primer (Table S3), and 2 μL of either standards or diluted samples (30 ng/uL of total RNA). Cycling parameters were 50 °C for 10 min followed by reverse transcriptase inactivation for 5 min at 95 °C and then PCR cycling by 45 cycles of 10 s at 95 °C, 30 s at 62 °C. Fluorescence data were obtained via a melt-curve step from 65 °C to 95 °C with a gradual increase in temperature 0.5 °C/5 s and data acquired every 5 s. Normalized relative quantification (NRQ) for each sample was estimated using the standard curve and after transformation of the raw data using the Lin-RegPCR program [30,31] according to the Pfaffl formula [30]. A logarithmic transformation was applied to NRQ dataset.

Viral RNA was checked by Sanger sequencing as follows. One microgram of total RNA extract positive for the virus by RT-qPCR, was reverse transcribed using Rev-CP₄₇₅₅ primer (5′-GATTCGGGTACGCCTTCATAG-3′) and SuperScript II RNase H—Reverse Transcriptase (Invitrogen, Life Technologies, Waltham, CA, USA), according to the manufacturer’s instructions. The CP ORF (ORF-3) was amplified with primers Rev-CP₄₇₅₅ and For-PEMV1₃₉₂₂ using Platinum^® Taq DNA Polymerase (Invitrogen) and 2 μL of 1:10 dilution cDNA. Cycling parameters were 94 °C for 2 min followed by 35 cycles of 30 s at 94 °C, 30 s at 60 °C, and 1 min at 72 °C, then 7 min at 72 °C. The PCR product was assessed by electrophoresis and visualization in a non-denaturing agarose gel (1%). The PCR products were sequenced using both forward and reverse PCR primers at the Iowa State University DNA Facility. CLC Main Workbench software package version 6.0 (CLC bio, Aarhus, Denmark) was used to analyze chromatograms for each sample.

Thirty to 150 milligrams of infected leaf tissue collected from pea plants at 15 or 21 dpi was ground with liquid nitrogen, suspended in two volumes of 0.2 M sodium acetate buffer pH 6.0, then centrifuged at 12,000× g for 1 min. The total protein concentration of each sample (1:3 dilution) was estimated using Bradford reagent (Bio-Rad, USA) with bovine serum albumin (BSA) standards from 12.5 to 50 ng/μL. The samples were calibrated to the same amount of total proteins using 0.2 M sodium acetate buffer. The calibrated samples were mixed with 10× sample reducing agent and 4× lithium dodecyl sulfate buffer (LDS; Novex, Life Technology, Carlsbad, CA, USA) and boiled at 70 °C for 10 min. Fifteen microliters of the protein suspensions were loaded and separated in a 4–12% Bis-Tris Gel with 1× compound 2-(N-morpholino)ethanesulfonic acid (MES) buffer supplemented with an antioxidant (Novex). The same wild type (WT) positive control was loaded as reference in all 10 gels used for two independent experiments. Next, the proteins were transferred into an Amersham Protran Premium 0.45 NC membrane (GE Healthcare Life Sciences, Freiburg, Germany). The primary PEMV polyclonal rabbit antibody (AC Diagnostics, Inc., Fayetteville, AR, USA) as well as the secondary antibody goat HRP-conjugated anti-rabbit (Invitrogen, USA) used for western blots were diluted 1:2000. The chemiluminescence was detected with the Pierce ECL western blotting substrate (ThermoFisher Scientific, Rockford, lL, USA). GelQuant software was used to determine the density of bands at approximatively 22 kDa corresponding to CP. Five technical quantification replicates were done for each sample.

PEMV virions were partially purified from plants according Liu et al. [32]. Briefly, individual infected pea plants were harvested at 15 and 21 dpi. The plant material was ground in liquid nitrogen and resuspended in 1× w/v 0.2 M sodium acetate buffer (pH 6.0) and 1 volume of chloroform. The homogenized tissue suspension was clarified at 1200× g for 45 min (Sorval ST40R, ThermoFisher Scientific, Osterode am Harz, Germany), and the supernatant was mixed with 33.3% v/v of Triton X-100 then centrifuged again at 2000× g for 15 min. The virions were pelleted by ultracentrifugation at 78,000× g for 2 h at 4 °C and resuspended in 30 μL of 0.2 M sodium acetate buffer (pH 6.0). The suspensions were negatively stained with 2% uranyl acetate and examined using a JEOL 1200 transmission electron microscope (Iowa State University Microscopy and NanoImaging Facility). Negative control plants were mock-inoculated donor plants. Positive control plants were inoculated with wild-type viruses.

The capacity for transmission by aphids was measured at 36–37 dpi, by allowing aphids to feed first on infected “donor” plants, and then on “recipient” plants. This time point was selected on the basis of previous experiments following within-host viral accumulation of PEMV1 WT in the non-inoculated upper leaves, which showed that viral density reached the maximum after approximately 21 days (unpublished data). We estimated the transmission efficiency of the wild-type and 16 CP mutant viruses using the pea aphid Acyrthosiphon pisum Harris [11,12]. This aphid colony A. pisum was reared on broad bean, Vicia faba in a growth chamber at 24 °C with a 12 h light/12 h dark cycle.

A mixture of non-viruliferous nymphs and adults was sampled and starved for 2 h in glass tubes in groups of 40. These aphids were then placed on the top three leaves of each individual infected pea plant and kept in individual 4.5 × 14 cm insect rearing nylon bags during the time of acquisition. After a five-day acquisition feeding period, four or five aphids were placed on a 10–11-day-old healthy P. sativum recipient plant. We used one to three donor plants for each clone, and transferred aphids from each donor plant onto four to six recipient plants. After 72 h, approximatively 0.6 grams of systemic insecticide (Mantra^® 1G, Nufarm Americas Inc., Alsip, IL, USA) was added into each pot. Recipient plants were scored for PEMV1 infection 21 days after aphid infestation by evaluation of symptoms and viral detection by RT-PCR. Negative control plants were mock-inoculated donor plants.