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Figure 5 Uptake of Prions by Plants Grown in PrPSc-Contaminated Soil Show full caption 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 four 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 (PrPC), were digested with PK, as indicated in The soil of barley grass plants, grown from seeds, was carefully contaminated on day 5 with 20 ml of 5% 263K brain homogenate and as control with the same amount of normal brain homogenate (NBH). One or 3 weeks after infection, plant samples were taken, dried, and minced. The grinded tissue corresponding to either the stem (A) or leaves (B) was analyzed for the presence of PrPby 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 four 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), were digested with PK, as indicated in Experimental Procedures

The experiments described above were done by exposure of the surface of leaves and roots with different solutions containing prions. To evaluate whether living plants can uptake PrPfrom contaminated soil, we grew barley grass plants on soil that was contaminated by addition of 263K brain homogenate. Plants were grown for 1 or 3 weeks under conditions that carefully prevented any direct contact of the aerial part of the plant with the soil. After this time, pieces of stem and leaves were collected and analyzed for the presence of PrPby PMCA. As shown in Figure 5 A, all plants grown for 3 weeks in contaminated soil contained PrPin their stem, albeit in small quantities that required four serial rounds of PMCA for detection. One of the four plants analyzed contained a detectable amount of PrPin the leaves ( Figure 5 B), indicating that prions were uptaken from the soil and transported into the aerial parts of the plants, far from the soil. These results differ from a recent article reporting that infectious prions were not detectable in above the ground tissues of wheat plants exposed to CWD prions (). The lack of detection in this article is most likely due to the low sensitive techniques (western blots or ELISA) employed to analyze the presence of PrP. Indeed, as we reported previously, PMCA has a power of detection, which is several millions times higher than western blots or ELISA (). In order to estimate the amount of PrPpresent in stem and leaves coming from contaminated soil, we performed a quantitative PMCA study, as previously described (). Unfortunately, by comparing the PMCA amplification in the absence or the presence of plant tissue, it is possible to conclude that stems and leaves substantially interfered with the PMCA procedure, and thus the calculation cannot be very precise ( Figure S4 ). Indeed, after two rounds of PMCA we cannot detect any protease-resistant PrP, but on the third round we observed the maximum amplification (10), presumably because at this round the concentration of PMCA inhibitors has been reduced enough to permit good amplification. At this point, we can estimate that the amount of PrPthat reaches the stem and leaves from contaminated soil is equivalent to the PrPconcentration present in a 10to 10dilution of sick brain homogenate. Nevertheless, this result is interesting, because it indicates that the amount of prions uptaken from soil and transported to aerial parts of the plant is within the infectious range. Indeed, titration studies showed that the last infectious dilution of a 263K brain homogenate is ∼10).