Can plants serve as a vector for prions causing chronic wasting disease?

Abstract

Prions, the causative agent of chronic wasting disease (CWD) enter the environment through shedding of bodily fluids and carcass decay, posing a disease risk as a result of their environmental persistence. Plants have the ability to take up large organic particles, including whole proteins, and microbes. This study used wheat (Triticum aestivum L.) to investigate the uptake of infectious CWD prions into roots and their transport into aerial tissues. The roots of intact wheat plants were exposed to infectious prions (PrP^TSE) for 24 h in three replicate studies with PrP^TSE in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrP^C) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrP^TSE) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrP^TSE from the roots to the stem to be detectable by the methods employed. Undigested PrP^TSE did not associate with roots. The present study suggests that if prions are transported from the roots to the stems it is at levels that are below those that are detectable by western blot, IDEXX or Bio-Rad diagnostic kits.

Keywords: :

• prion
• wheat
• transmission
• chronic wasting disease
• risk assessment
• cervid
• food safety

Introduction

Chronic Wasting Disease (CWD) is a fatal neurodegenerative prion disease affecting wild and farmed cervids. Specifically, CWD has been found in deer (mule and white-tail), elk and moose.¹ Experimental transmission has also been demonstrated in reindeer.² Prion diseases, also termed Transmissible Spongiform Encephalopathies (TSE), are caused by misfolding of the cellular prion protein, PrP^C, into the disease-causing conformation designated PrP^TSE. Misfolded PrP^TSE is broadly accepted as the disease causing agent responsible for misfolding of PrP^C into PrP^TSE.^3,4

Prevalence of CWD is high in endemic areas like Wyoming (USA) where, in a number of herds, 50% of wild mule deer are CWD positive.¹ Indirect transmission of CWD has been confirmed in studies where deer became infected following exposure to areas which previously held CWD-positive deer.^5,6 CWD PrP^TSE remains infectious for more than two years ex vivo in the environment representing a long-term hazard for transmission.⁵ Infected cervids have been reported to shed PrP^TSE in bodily fluids, disseminating infectious prions in urine, feces, blood and saliva.^7-10

PrP^TSE binds to whole soil and interaction with some soil minerals has been shown to increase the infectivity of PrP^TSE.^11,12 Although PrP^TSE binds to soil, it requires 60 d for CWD PrP^TSE to become fully bound in sandy soil as compared with 30 d for prions associated with transmissible mink encephalopathy (TME).¹³ In seawater, scrapie PrP^TSE remains stable with a one log reduction only observed after 30 d, suggesting unbound PrP^TSE may persist and flow with surface or ground water.¹⁴ Taken together, these factors suggest PrP^TSE may remain unbound within the soil environment for substantial periods.

Plants acquire nutrients from the surrounding soil environment. Nitrogen (N) is one of the main macronutrients vital for plant growth and reproduction. There are multiple forms of N in the soil environment and the importance of organic N, including amino acids and peptides, to plant health is well documented.^15,16 The nutritional value of protein N to plants is contentious but, the potential for uptake has been demonstrated.^17-19 Uptake of green fluorescent protein into roots has been shown for Arabidopsis thaliana and Hakea actites.¹⁹ Earlier work suggested ovalbumin is not only taken up by roots, but is subsequently transported to upper regions of tomato plants when roots are damaged.¹⁸

The ability of plants to take up larger organic particles raises the possibility that PrP^TSE may enter plants through roots. This study used wheat as a model to investigate the potential uptake of CWD PrP^TSE into roots and its subsequent transport to the stem. If such a phenomenon was confirmed it would suggest the potential for plants to act as a vector for the transmission of CWD in cervids.

Results

PrP^C exposures. Wheat plants with roots exposed to recombinant mouse PrP^C contained a prion band in the root protein extract as identified by western blotting (Fig. 1, lane 3). The prion band in the root extract had a slightly smaller molecular weight than PrP^C in the solution roots were exposed to (Fig. 1, lane 5). When the outer surface of roots was exposed to 10 µg/mL proteinase K (PK) for 5 min at room temperature, no prion signal in the root protein extract was observed (Fig. 1, lane 4). No prion signal was seen in stem or leaf protein extracts (data not shown).

Figure 1. Recombinant PrP^C binds to the outside of wheat roots. Wheat plant roots were exposed to 50 µg/mL PrP^C for 24 h then roots were either digested with (10 µg/mL) proteinase K (PK) for 5 min or left undigested. Control plants were exposed to the same solution lacking PrP^C. Western Blotting on root protein extracts used the 8H4 mAb (1:10000). The last lane confirming antibody specificity is from the same blot with irrelevant lanes omitted. Results are representative of three independent replicates (n = 3). Lanes 1 and 2: control plants; Lanes 3 and 4: PrP^C exposed plants; Lane 5: positive control (50 ng PrP^C).

PrP^TSE exposures. In wheat plants exposed to PrP^TSE, the root extract had three prion reactive bands <30 kDa that were not present in control extracts (Fig. 2, lanes 1 and 2). Plant proteins cross-reacted with P4 mAb in both the root and stem extracts with prominent bands at ~40 kDa and ~29 kDa (Fig. 2). When root extracts were digested with PK (10 µg/mL, 37 °C, 30 min) there were no PK-resistant proteins present in either the control or CWD exposed samples (Fig. 2, lanes 3 and 4). Only PK-sensitive cross reaction bands were present in the lower stem extract (Fig. 2, lanes 5–8).

Figure 2. Prion signal found in wheat roots exposed to CWD PrP^TSE was protease sensitive but no prion signal in lower stem (Stem) extract was visible. CWD PrP^TSE was purified from brain homogenate (BH) with Bio Rad-TeSeE® purification and re-suspended in phosphate buffered saline to a 1% solution (w/v) based on the initial BH solution. Wheat plant roots were exposed to the purified solution for 24 h. Normal BH processed with TeSeE® served as a negative control. Plant protein extracts were digested with proteinase K (PK) (10 µg/mL, 30 min, 37 °C) to determine PK-resistance of any proteins. Western blotting of plant protein extracts (plant total protein extraction kit) was done using P4 mAb (1:5000) and Prionics®-Check Western kit. Results are representative of three independent replicates (n = 3). Lanes 1, 3, 5, 7: plants exposed to normal BH processed with Bio Rad kit; Lanes 2, 4, 6, 8: plants exposed to CWD infected BH processed with Bio Rad kit; Lane 9: CWD infected BH (0.1%) processed with Bio Rad kit.

Wheat plants were also exposed to either infectious or non-infectious elk brain homogenates (BH), which were either digested with PK to leave only PK-resistant PrP^TSE protein, or not digested, to represent all brain proteins (Fig. 3). The root extract from plants exposed to PK-digested CWD BH had prion reactivity with bands <30 kDa not present in the control extract (Fig. 3A, lanes 1 and 5). Similarly, the lower stem extract from the same PK-digested CWD BH treatment had three prion reactive bands which were not present in the control lower stem extract when the outside of the stem was not rinsed with dH₂O (Fig. 3A, lanes 6 and 10). When the outside of the stem was rinsed with dH₂O following exposure, the suspected PrP^TSE bands were no longer visible in the lower stem extract, leaving only cross-reaction bands (Fig. 3B, lanes 3 and 4). There were no definitive bands in the root or stem extracts of the other treatments (normal BH-digested and undigested; and CWD BH-undigested) which differed from the control protein extracts (Fig. 3A, lanes 2–4 and 7–9).

Figure 3. Proteinase K (PK) digested CWD PrP^TSE in a brain homogenate (BH) solution interacts with wheat roots but signal from lower stem (Stem) disappears when the stem is rinsed. (A) Wheat plant roots were exposed for 24 h to BH solutions either CWD positive (CWD BH) or CWD negative (Normal BH) and both digested with PK (50 µg/mL, 30 min, 37 °C) or not. Control plants were exposed to dH₂O for 24 h. Only the roots were rinsed for 1 min in dH₂O, with protein extracted from both the root and lower stem. Western blotting of plant extracts was done using P4 mAb (1:5000) and Prionics®-Check Western kit. Lanes 1 and 6: plants exposed to H₂O; Lanes 2 and 7: plants exposed to Normal BH; Lanes 3 and 8: plants exposed to Normal BH digested with PK; Lanes 4 and 9: plants exposed to CWD BH; Lanes 5 and 10: plants exposed to CWD BH digested with PK; Lane 11: 0.1% Normal BH; Lane 12: 0.1% Normal BH digested with PK; Lane 13: 0.1% CWD BH; Lane 14: 0.1% CWD BH digested with PK. (B) Wheat plant roots were exposed to PK-digested (50 µg/mL, 30 min, 37 °C) CWD BH for 24 h then both the roots and stem were rinsed in dH₂O for 1 min. Western blotting of extracted protein used P4 mAb (1:5000) and Prionics®-Check Western kit. Results are representative of three independent replicates (n = 3). Lanes 1 and 3: plants exposed to H₂O; Lanes 2 and 4: plants exposed to CWD BH digested with PK; Lane 5: 0.1% CWD BH digested with PK.

The plant total protein extraction kit (Sigma Aldrich) resulted in PrP^TSE becoming sensitive to a mild PK digestion (10 µg/mL, 30min, 37 °C) (data not shown). An alternate extraction with 1% sodium dodecyl sulfate (SDS) was used in order to preserve PK-resistance of any PrP^TSE present in plant tissue associated with the PK-digested CWD BH treatment (Fig. 4). With the 1% SDS extraction, no cross-reaction bands were observed in the control root extract (Fig. 4, lane 1). Prion protein in the root extract was resistant to mild PK treatment (10 µg/mL, 30min, 37 °C) (Fig. 4, lanes 2–3). There were no PK-resistant proteins in the lower stem and only cross-reaction bands were visible when extracts were not digested (Fig. 4, lanes 4–7).

Figure 4. Proteinase K (PK) digested CWD PrP^TSE interacts with wheat roots and remains slightly PK resistant after extraction while no CWD PrP^TSE was detected in the lower stem (Stem). CWD positive brain homogenates (BH) were digested with PK (50 µg/mL, 30 min, 37 °C) prior to being exposed to wheat roots for 24 h. Wheat roots and stems were rinsed with dH₂O for 1 min then protein was extracted with 1% SDS. Extracts were digested with PK (10µg/mL, 30 min, 37 °C) to determine any PK-resistant bands prior to blotting. Western blotting on protein extracts was done using P4 mAb (1:5000) and Prionics®-Check Western kit (Prionics). Images are from the same blot with irrelevant lanes omitted. Results are representative of three independent replicates (n = 3). Lanes 1, 4, 6: plants exposed to H₂O; Lanes 2, 3, 5, 7: plants exposed to CWD BH digested with PK; Lanes 8 and 9: 0.1% CWD BH in presence of 1% SDS digested with PK.

Protein extracted with 1% SDS from plants exposed to PK-digested CWD BH was analyzed using Bio-Rad TeSeE® and IDEXX HerdChek® diagnostic tests (Table 1). TeSeE® analysis revealed all root and stem extracts were negative (Table 1). When PK digestion was omitted from TeSeE® protocol, the root extract from the PK-digested CWD BH treatment was positive and control root extract was negative (Table 1). Stem extracts were negative when PK treatment was omitted from the TeSeE® protocol (Table 1). All samples were negative when tested with IDEXX HerdChek® (Table 1).

Table 1. Analysis of plant protein extracts (1% SDS) from proteinase K-digested (PK) CWD BH treatment with Bio-Rad and IDEXX diagnostic tests

	Root		Stem
	Control	PK-digested CWD BH	Control	PK-digested CWD BH
Bio-Rad TeSeE® (no PK)	-	+	-	-
Bio-Rad TeSeE®	-	-	-	-
IDEXX HerdChek®	-	-	-	-

Results are representative of three independent replicates (n = 3).

Discussion

This study shows that protease-digested CWD PrP^TSE interacts with wheat roots, both when exposed in a brain homogenate (BH) and purified solution. Similarly, when in a purified solution, recombinant PrP^C interacted with the outside of wheat roots. Transport of PrP^TSE or PrP^C from roots to stems was not detectable using the methods employed. This suggests that plants with damaged root hairs do not uptake PrP^TSE or transport it to stems at high concentrations, an outcome that may change if more extensive root damage occurs.

One interesting observation was that only PK-digested PrP^TSE associated with roots, while undigested PrP^TSE did not (Fig. 3). Proteinase K digestion is known to alter the structure of PrP^TSE by removing the N-terminus.²⁰ With this altered structure, PrP^TSE may have exhibited an enhanced affinity for wheat roots. When considering the strong interaction between recombinant PrP^C and the roots, it is worth noting that this prion protein is also N-terminally truncated as compared with mammalian PrP^C (Fig. 1). The fact that PrP^TSE and PrP^C lacking the N-terminus interact with roots is supported by studies showing the N-terminus plays a role in the binding of PrP^TSE to soil components.^11,13 Alternatively, in an undigested BH it is possible that other brain proteins may occupy binding sites, thereby blocking any interaction of PrP^TSE with the root surface. Degradation of PrP^TSE by microbes has been suggested to occur during composting, suggesting that partial degradation of PrP^TSE in the soil environment is possible,²¹ an outcome that may increase its interaction with plant roots.

Initially, total plant protein extractions were undertaken using a kit to maximize the amount of protein obtained (Figs. 2 and 3). The high concentration of detergents in the extraction buffer affected the PK-resistance of prions, as other studies have confirmed.²² A 1% SDS solution was used as an alternate extraction procedure to preserve PK-resistance of PrP^TSE based on other studies that extracted PrP^TSE from compost (Fig. 4).²¹ It should be noted that 1% SDS extracted protein less efficiently than the extraction kit and likely lowered our detection limit in plant tissue as demonstrated by the decreased intensity of cross-reaction bands in the root and stem (Fig. 4). Efforts to specifically identify the proteins responsible for the cross-reaction bands in the root and stem were not undertaken owing to the lack of identifiable targets for the P4 antibody epitope as determined by BLAST analysis of the wheat proteome. The closest match for the cross reaction bands at ~40 kDa and ~29 kDa were the Cysteine Proteinase protein (71% coverage, 80% similarity) and R2R3-MYB protein (57% coverage, 100% similarity), respectively. Western blotting without the P4 antibody failed to produce the > 20 kDa cross reaction bands in the plant stem, confirming that the cross-reaction was primarily attributable to P4 (data not shown).

When extracted with 1% SDS, the PrP^TSE signal from the roots was reduced with a mild PK digestion (10 µg/mL, 30 min, 37 °C) (Fig. 4). When digested under the same conditions, the PrP^TSE signal from the solution roots were exposed to also decreased in the presence of SDS (Fig. 4). There was a larger decrease in PrP^TSE signal as a result of digestion of the root extract as compared with PrP^TSE in solution (Fig. 4). Together, these results suggest PrP^TSE was more PK-sensitive when associated with wheat roots than when in solution. The interaction with roots could have altered PrP^TSE conformation making it more PK-sensitive. This hypothesis is supported by the negative reading obtained from the IDEXX HerdChek® test, which identifies PrP^TSE based on misfolded conformation by Seprion-capture (Table 1). The lack of any positive samples from IDEXX HerdChek® suggests even though there are PK-resistant prions in the roots, the conformation of the prion species is no longer identifiable as CWD PrP^TSE. This finding is also supported by Bio-Rad TeSeE® which also did not identify any of the plant tissues as positive for PrP^TSE. Conversely, when Bio-Rad TeSeE® protocol was altered by removing PK-treatment of samples, roots from the PK-digested CWD BH treatment were positive for prions, while control roots were negative (Table 1). The Bio-Rad TeSeE® manufacturer’s protocol was altered by removing PK-digestion in order to determine if more PK-sensitive prion species could be identified. The observation that western blotting of the PK-digested CWD BH treatment solution exhibited the only reactivity to P4 mAb with PK-resistant PrP^TSE present, along with TeSeE® results confirms these roots contained PrP^TSE. These results suggest the PrP^TSE in the root extract underwent significant changes with the impact of these conformational changes on infectivity being unknown.

The loss of the possible PrP^TSE signal in the unwashed stem of plants exposed to PK-digested CWD BH after a simple dH₂O wash was surprising (Fig. 3). It is unlikely that this observation is due to external contamination from the media solution as care was taken to have only the roots submerged in media. The fact that other treatments with prion reactive protein in the media (undigested CWD and normal BH) did not have a prion signal in unwashed stems that were handled in the same manner, supports that these findings are not due to contamination from the treatment solution (Fig. 3A).

The interaction of protease-digested PrP^TSE with wheat roots may have implications for the behavior of prions in the environment. Previous studies have shown CWD PrP^TSE remains infectious for over two years outside of the host.⁵ Other work has also outlined that CWD PrP^TSE are relatively resistant to degradation in a brain homogenate, but that the N-terminus is rapidly cleaved.²⁰ In subsequent work, the role of the N-terminus in soil interactions was investigated with truncated hamster adapted transmissible mink encephalopathy (TME) HY PrP^TSE exhibiting a greater affinity for sand and sandy loam soil as compared with undigested TME HY PrP^TSE.¹³ This suggests that removal of the N-terminus of PrP^TSE as a result of microbial activity could affect the mobility of prions within soil. Our work shows PK-digested CWD PrP^TSE binds to wheat roots when soil is not present, but the effect of soil particles on this interaction are unknown. Based on the documented affinity of PrP^TSE to different soils and minerals, like montmorillonite, it is likely there would be competition between wheat roots and soil for binding of PrP^TSE.^11,12 We predict N-terminally truncated CWD PrP^TSE would have the best possibility of binding to wheat roots in a silty clay loam soil, as PK-digested PrP^TSE has a lower affinity for this soil type.¹³

The lack of detectable PrP^TSE inside the stem of wheat plants suggests that if it is transported to the stem, it is at levels that were below the level of detection for the methods employed in the current study. It is possible that trace amounts of PrP^TSE could be transported to the stem. We also observed cross-reactivity of plant proteins with the P4 mAb antibody during western blotting, suggesting that plants do contain proteins with linear epitopes similar to prions. The Prionics^®-Check WESTERN kit had a detection limit on the elk PrP^TSE source used in this study of approximately 10² mg-/mg+ brain tissue (based on mono-glycosylated band) similar to other work on BSE with the same kit (Fig. S1).²³ Given the bioassay titration was 10^7.2 i.c. ID₅₀/g in transgenic mice, infectious levels of prions could be contained in plant tissue that would not have been identified by western blotting. Cervids grazing on plants with trace PrP^TSE at this level could potentially develop CWD.

Early studies have suggested transport of lysozyme and ovalbumin proteins to aerial portions of tomato plants occurs and is aided by root damage.¹⁸ Recently, uptake of whole bacteria into plants and transport distal from the roots was shown as pathogenic Salmonella enterica entered the roots of sweet basil plants and moved internally to the stem and leaves.²⁴ Similar to protein uptake, root damage has also been identified as a factor enhancing the uptake of viruses and bacteria into plants.^25,26 In soil, the activity of nematodes or other parasites could form lesions in roots that could serve as a port of entry for prions into plants.²⁷ In our study, plants were grown in solid agarose media and subsequently exposed to PrP^TSE solution, an approach that deliberately inflicted root damage to fine root hairs but avoided major damage to the main branches. Previous work in our lab, has shown that ovalbumin can enter wheat roots and be transported to the stem using the same growth and exposure system employed in this study.²⁸ This difference may be explained by the aggregating properties of prions. It has been shown in previous studies that the size of an infectious aggregate for PrP^TSE is ~26 protein monomers.²⁹ This behavior could be responsible for differences in the uptake of ovalbumin monomers (~45kDa) as compared with prion aggregates.

In conclusion, our data shows that PrP^TSE is not transported to aerial tissues at concentrations detectable by western blot, however, it still remains possible for infectious levels to be achieved, especially if significant root damage occurs. The findings suggest that PK digestion may facilitate the interaction of PrP^TSE with plant roots which is interesting based on the affects this also has on affinity of these molecules for soil. This study used wheat as a model but it would be valuable to test native grasses such as crested wheat grass and fescue which make up a significant portion of the diet of elk and deer. It is probable that both biotic and abiotic factors affect uptake of PrP^TSE into plants and the introduction of other factors such as soil organisms and particles into the system is something to consider in future investigations. Regardless of system, if uptake occurs, animal infectivity assays are needed to confirm if the prion protein remains in an infective state after plant uptake. These experiments provide the first known investigation of the involvement of plants in prion transmission and provide a solid base for future studies.

Materials and Methods

Plant growth

Triticum aestivum L. (cv AC Andrew) seeds were germinated for five d after surface sterilization with 70% ethanol and 3% hypochlorite. Germinated seeds were transferred to Magenta^® boxes containing sterile Murashige and Skoog (MS) agarose media adjusted to pH 5.8.³⁰ Filter-sterilized media (0.2 µm) was combined with autoclaved phytagel (P8169, Sigma Aldrich) to a final concentration of 0.3%. All plants were grown axenically for 30–45 d prior to uptake experiments. Growth boxes were placed in a growth room with light conditions of 16 h d/8 h night (150 µmol m⁻² s⁻¹) at 20 °C ± 2 °C.

PrP^C exposures

Recombinant mouse PrP^C (amino acids 23–230) was expressed in BL21 Escherichia coli (Stratagene) and purified as previously described.³¹ The PrP^C gene contained a His₆ affinity tag at the C-terminus that was removed by a thrombin cleavage site after purification on a Ni-NTA agarose column (Qiagen). After re-folding, PrP^C was dialyzed against dH₂O and concentrated using Amicon Ultra centrifugal filters (3kDa, Millipore). Purity was confirmed by SDS-PAGE and concentration was determined by Bradford assay.³² Wheat plants were removed from growth boxes, roots were rinsed with dH₂O and patted dry with paper towel. Roots were exposed to 4 mL of 50 µg/mL PrP^C for 24 h in nitrogen-free MS media: 29.21 mM sucrose, 2.99 mM CaCl₂·2H₂O, 1.50 mM MgSO₄·7H₂O, 1.25 mM KH₂PO₄, 0.11 mM EDTA ferric sodium salt, 0.10 mM H₃BO₃, 0.10 mM MnSO₄·H₂O, 29.91 nM ZnSO₄·7H₂O, 5.00 nM KI, 1.03 nM Na₂MoO₄·2H₂O, 0.11 nM CoCl₂·6H₂O, and 0.10 nM CuSO₄·5H₂O.³⁰ Plants were removed from the PrP^C solution and roots were rinsed for 1 min in dH₂O. Roots from one half of the plants were exposed to 10 µg/mL proteinase K (0.7 U/s L, PK, Life Technologies) for 5 min followed by another rinse with dH₂O to remove loosely adherent protein on the outer surface. Plants were separated into roots, stems and leaves and each fraction was flash frozen in liquid nitrogen. Separated fractions were pulverized with a mortar and pestle and protein was extracted using a plant total protein extraction kit (Sigma Aldrich). Protein samples were heated at 96 °C for 5 min in sample buffer (2.2% SDS, 48 µM Tris, 10% glycerol, 1% 2-mercaptoethanol, pH 6.8) prior to loading on a polyacrylamide gel. Samples were electrophoresed using a 4–12% NuPAGE® Bis-Tris polyacrylamide gels at 200 V for 50 min in 1X MOPS buffer (Life Technologies). Gels were transferred (wet) to nitrocellulose membranes (0.2 µm pore, Genscript, Piscataway, USA) at 100V for 60 min in 1X NuPAGE® transfer buffer (Life Technologies) with 10% methanol. Western blotting was done with 8H4 mAb (1:10000, Sigma Aldrich) and an Advanced One-Hour Western Kit (Genscript).

Prion source

All experiments using infectious material were completed in enhanced BSL 2 containment laboratories at the National Centre for Animal Disease Lethbridge Laboratory, Canadian Food Inspection Agency and material was also obtained through this collaboration. Elk (Cervus canadensis) brain stems positive for CWD were homogenized in PBS (CWD positive) or dH₂O (CWD negative) with a Dounce tissue grinder to make 20% solutions. The titer of CWD positive elk homogenate used in this study was 10^7.2 i.c. ID₅₀/g as determined by bioassay in Tg(CerPrP)1536^+/− transgenic mice expressing elk PrP. For experiments with purified PrP^TSE, both CWD positive and negative brain homogenates (BH) were processed using a TeSeE® purification kit (Bio-Rad, Mississauga, Canada) following manufactures specifications based on its use in other publications for quick removal of other brain homogenate components.^33,34 Pelleted PrP^TSE was re-suspended in Dulbecco’s phosphate-buffered saline (PBS, Sigma Aldrich), vortexed, and subject to 8, 10 s rounds of sonication at maximum intensity using a Misonix Sonicator 3000 Ultrasonic Cell Disruptor (Misonix). Solutions containing purified PrP^TSE were diluted to be equivalent to 1% (w/v) of the BH input material. For BH experiments, digestion of homogenates before plant exposure were done using the homogenized 20% brain solutions at 37 °C for 30 min with 50 µg/mL PK (Life Technologies). The BH were then diluted to 1% (w/v) with dH₂O for plant exposure.

PrP^TSE exposure and extraction

Wheat plants were removed from growth boxes, roots were rinsed in dH₂O and patted dry with a paper towel. Plants with roots were placed in conical tubes containing 4 mL of either CWD positive or negative 1% BH or 1% purified solution so as to ensure the roots were immersed in the solution for 24 h. Exposures were independently replicated three times. After exposure, roots and stems were immediately separated with a razor blade and handled separately to avoid cross-contamination. Roots and the lower half of stems were then rinsed in dH₂O for 1 min and flash frozen in liquid nitrogen. Frozen tissue was pulverized using a tissue TUBE™ Impactor (Covaris Inc.). Protein was extracted either using a plant total protein extraction kit (Sigma Aldrich) following manufactures specifications or by incubation of tissue with 4 µL 1% sodium dodecyl sulfate (SDS)/mg tissue for 15 min at room temperature. For both the kit and 1% SDS extraction, the protein concentration was kept consistent for roots and stems by using the same volume of extraction buffer per mg of tissue.

Detection

Protease digestion of protein extracts was done with 10 µg/mL PK at 37 °C for 30 min. Samples were heated at 96 °C for 5 min in sample buffer (2.2% SDS, 48 µM Tris, 10% glycerol, 1% 2-mercaptoethanol, pH 6.8) prior to loading onto a polyacrylamide gel. Samples were electrophoresed using a 12% NuPAGE® Bis-Tris polyacrylamide gels at 150 V for 60 min in 1X MOPS buffer with NuPAGE® antioxidant (Life Technologies). Gels were transferred (wet) to polyvinyl difluoride membranes (PVDF, 0.45 µm pore, Millipore) at 150V for 60 min in 1X NuPAGE® Transfer buffer (Life Technologies) with 10% methanol. Western blotting was done using P4 mAb (1:5000, R-biopharm), Prionics®-Check WESTERN kit (Prionics) and were developed with CDP-Star (Roche).

Samples extracted with 1% SDS were subjected to diagnostic testing with Bio-Rad TeSeE® and IDEXX HerdChek® (Westbrook). Sample analyses using Bio-Rad TeSeE® were done in duplicate, with one replicate following manufacture’s specifications and the other without the addition of PK (undigested). Samples were also analyzed using Seprion-capture technology with the IDEXX HerdChek® Ag Test and were performed according to manufacturer’s specifications.

Abbreviations:
CWD	=	chronic wasting disease
PrPTSE	=	infectious prion protein
PrPC	=	cellular prion protein
TSE	=	transmissible spongiform encephalopathy
TME	=	transmissible mink encephalopathy
N	=	nitrogen
MS	=	murashige and skoog media
PK	=	proteinase K
BH	=	brain homogenate
PBS	=	phosphate-buffered saline
SDS	=	sodium dodecyl sulphate
TME HY	=	hyper strain TME

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by funding from the Alberta Prion Research Institute and the Peer-Review Project plan of Agriculture and Agri-Food Canada. NSERC and Alberta Innovates GSS scholarships awarded to J. R. are also gratefully acknowledged. Technical assistance by Renee Clark is greatly appreciated.

Supplemental Materials

Supplemental materials may be found here: http://www.landesbioscience.com/journals/prion/article/27963/

10.4161/pri.27963