Grass plants bind, retain, uptake, and transport infectious prions

Abstract

Prions are the protein-based infectious agents responsible for prion diseases. Environmental prion contamination has been implicated in disease transmission. Here, we analyzed the binding and retention of infectious prion protein (PrP(Sc)) to plants. Small quantities of PrP(Sc) contained in diluted brain homogenate or in excretory materials (urine and feces) can bind to wheat grass roots and leaves. Wild-type hamsters were efficiently infected by ingestion of prion-contaminated plants. The prion-plant interaction occurs with prions from diverse origins, including chronic wasting disease. Furthermore, leaves contaminated by spraying with a prion-containing preparation retained PrP(Sc) for several weeks in the living plant. Finally, plants can uptake prions from contaminated soil and transport them to aerial parts of the plant (stem and leaves). These findings demonstrate that plants can efficiently bind infectious prions and act as carriers of infectivity, suggesting a possible role of environmental prion contamination in the horizontal transmission of the disease.

Fig. 1. Detection of PrP^Sc bound to leaves or roots by PMCA

(A) Serial dilutions of 263K brain homogenate (BH, 10⁻¹ to 10⁻⁸) done in PBS were incubated with either wheat grass roots (15mg weight) or leaves (2cm²) during 16h at room temperature. Thereafter, unbound material was discarded, leaves and roots were thoroughly washed 5 times with water and deposited into tubes containing 120μL of 10% normal hamster brain homogenate. The presence of plant-attached PrP^Sc was detected by serial rounds of PMCA, as described in Methods. Positive PrP^Sc signal was detected by Western blot after proteinase K (PK) digestion. (B) Serial dilutions of 263K brain homogenate (10⁻¹ to 10⁻⁸) were directly loaded into tubes containing NBH PMCA substrate and wheat grass roots and leaves not previously exposed to PrP^Sc. The purpose of this experiment was to study the level of amplification expected for the total amount of PrP^Sc contained in each dilution of sick brain homogenate. (C) To investigate the possible induction of PrP^Sc formation by plant material and to rule out cross-contamination, we exposed leaves and roots to 10% normal brain homogenates and subjected the material to several rounds of PMCA as described in panel A. The figure shows two replicates of the same experiment (Rep 1 and 2). No PMCA amplification was detected for any of the samples. F: Non-amplified control. 1, 2 and 3: number of PMCA rounds performed. Each round consisted of 96 PMCA cycles (2 days). All samples were digested with PK, except the normal brain homogenate (NBH, PrP^C) used as a migration control.

Fig. 2. PrP^Sc contaminated plants induce prion disease by oral ingestion

Survival curve (A) of hamsters orally inoculated with leaves or roots exposed to 263K BH. Plant tissue was exposed to prions as described in Fig. 1 and in Methods. Three units of leaves and roots were used to orally inoculate healthy hamsters. The positive control group consisted on hamsters orally inoculated with 750μL of 5% 263K BH. Negative control groups were inoculated with leaves and roots incubated with normal brain homogenate. All sick animals exhibited the typical 263K clinical signs, including ataxia, hyperactivity, aggressiveness and sensitivity to noise, and were sacrificed at the terminal stage of the disease. Hamsters injected with leaves and roots treated with healthy brain homogenates did not show any clinical signs up to 550 days post-inoculation. The differences in the survival curves of animals infected with 263K brain homogenate versus those infected with prion-contaminated leaves or roots were statistically significant (P = 0.0136 and 0.047, respectively) as analyzed by the Log-rank (Mantel-Cox) test. (B) Brains from hamsters orally infected with roots and leaves exposed to prions displayed neuropathological alterations typical of prion disease, including characteristic synaptic and diffuse patterns of PK-resistant PrP^Sc deposition (antibody 6H4, left panels), astrogliosis (middle panels) and spongiosis (right panels). These alterations were not observed in animals fed with plant tissue exposed to normal brain homogenate. Magnification 20x in all panels. (C) Biochemical analysis confirmed the presence of PrP^Sc accumulation in the brain of all animals showing signs of prion disease. The figure shows a Western blot of different brain dilutions from a representative animal per group. All samples were digested with PK, except the normal brain homogenate (PrP^C) used as a migration control.

Fig. 3. PrP^Sc contained in urine and feces of prion-infected animals bind to leaves and roots

(A) Wheat grass roots (R) and leaves (L) were incubated for 1h with 1mL of urine or 1mL of 20% feces homogenate from sick hamsters experimentally infected with 263K prions. Controls included similar experiments using urine and feces from healthy animals. After exposure, roots and leaves were thoroughly washed 5 times with water, dried and the presence of plant-attached PrP^Sc was detected by serial rounds of PMCA. The figure shows the results of 2 replicated experiments (1 and 2). In the right blot of this panel we show the results of the positive control experiment aiming to directly detect PrP^Sc in urine (U) and feces (F) from 263K infected animals. We also include several negative controls for the PMCA reaction, containing only the normal brain homogenate (NBH) used as substrate, to rule out cross-contamination or de novo formation of PrP^Sc. (B) A similar experiment as described in panel A was done using urine and feces from white-tailed deer clinically affected by CWD. In this case, leaves (L) and roots (R) were incubated in 1:2.5 diluted urine or with 5% feces homogenates. The middle blot shows the positive control experiment in which PrP^Sc was detected directly in urine and feces from CWD affected deer. No PrP^Sc signal was detected for various negative controls in which the PMCA reaction was carried out in the absence of infectious samples (right panel). Both panel A and B show the results obtained in the 1^st, 2^nd, 3^rd and 4^th round of PMCA. Each round consisted of 96 PMCA cycles (2 days). All samples were digested with PK, except the normal brain homogenate (PrP^C) used as a migration control.

Fig. 4. PrP^Sc bind to living plants

The leaves of living wheat grass plants were sprayed three times with 10⁻² diluted 263K brain homogenate. Plants were left to grow for a period of 0, 3, 7, 14, 21, 28, 35 and 49 days. Thereafter, leaves were collected washed 5 times with water, dried and used to detect PrP^Sc signal by serial rounds of PMCA. The experiment was done in two independent replicates (Rep 1 and 2) for each time point. F: Non-amplified control. 1, 2 and 3: number of PMCA rounds performed. Each round consisted of 96 PMCA cycles (2 days). All samples were digested with PK, except the normal brain homogenate (PrP^C) used as a migration control.

Fig 5. Uptake of prions by plants grown in PrP^Sc-contaminated soil

The soil of barley grass plants, grown from seeds, was carefully contaminated on day 5 with 20mL of 5% 263K brain homogenate and as control with the same amount of normal brain homogenate (NBH). One or three weeks after infection, plant samples were taken, dried and minced. The grinded tissue corresponding to either the stem (panel A) or leaves (panel B) was analyzed for the presence of PrP^Sc by PMCA. Western blots of four different samples (1, 2, 3 or 4) of stems or leaves taken from plants grown for 1 or 3 weeks in 263K BH (or NBH as control) are shown. The results of 4 consecutive serial rounds of PMCA are depicted. Each round consisted of 96 PMCA cycles (2 days). All samples, except the normal brain homogenate used as a migration control (PrP^C), were digested with PK, as indicated in Experimental Procedures.