Incubation of 32P-dsRNA in Soil Solutions

32P-dsRNA while at the same time minimizing 32P-dsRNA adsorption to soil particles, we first incubated 32P-dsRNA in soil solutions collected as the supernatant of centrifuged soil suspensions (32P-dsRNA concentrations during incubation would result from two co-occurring processes: (i) dsRNA hydrolysis by extracellular hydrolases and (ii) microbial uptake and utilization of dsRNA and its hydrolysis products. We note that while hydrolases either specific to or competent toward dsRNA have been identified, To assess degradation and microbial utilization of dissolvedP-dsRNA while at the same time minimizingP-dsRNA adsorption to soil particles, we first incubatedP-dsRNA in soil solutions collected as the supernatant of centrifuged soil suspensions ( Scheme 1 A). Centrifugation removed most, but not all, particulates: the solution contained a small number of soil particles and active microorganisms ( Figures S3–5 ). Because abiotic dsRNA hydrolysis is slow under the experimental pH and temperature conditions (i.e., on a time scale of years), (41) we anticipated decreasing dissolvedP-dsRNA concentrations during incubation would result from two co-occurring processes: (i) dsRNA hydrolysis by extracellular hydrolases and (ii) microbial uptake and utilization of dsRNA and its hydrolysis products. We note that while hydrolases either specific to or competent toward dsRNA have been identified, (43−45) their abundance and activity in soils are not documented.

32P-ssRNA with the ssRNA-specific32P counts in the gel (i.e., intensity values integrated over the gel length) in the ssRNA sample degraded by RNase T1 were the same as in the untreated ssRNA sample. We therefore concluded that most 32P-containing hydrolysis products were retained in the gel under the selected run conditions. We first verified that PAGE coupled to phosphorimaging allows investigating hydrolysis reactions by detecting both loss of the intact molecule and formation of lower molecular weight (LMW) fragments. To this end, in a control experiment, we treatedP-ssRNA with the ssRNA-specific (46) RNase T1 hydrolase to generate smaller ssRNA fragments. We note that we monitored ssRNA hydrolysis by RNase T1 because a dsRNA-specific hydrolase was not available to us. RNase T1 treatment decreased the amounts of intact ssRNA and produced LMW fragments ( Figures 1 A, B). The totalP counts in the gel (i.e., intensity values integrated over the gel length) in the ssRNA sample degraded by RNase T1 were the same as in the untreated ssRNA sample. We therefore concluded that mostP-containing hydrolysis products were retained in the gel under the selected run conditions.

32P-dsRNA was still present after incubation for 0.5 h, it was no longer detectable after incubation for 24 h. Total 32P activity retained in the gel decreased by 45 (±8)% from 0.5 to 24 h. In comparison to ssRNA hydrolysis by RNase T1 (32P-activity was detected at a short migration distance (<0.1 cm into the gel). The intensity at this location approximately doubled during incubation from 0.5 to 24 h. Based on the short migration distance, we hypothesized that these higher molecular weight (HMW) 32P-containing molecules were biomolecules that microorganisms synthesized from 32P-dsRNA and/or its hydrolysis products. To provide support that these products resulted from microbial uptake and utilization, we conducted a series of experiments in which microbial activity in the solutions was reduced either by filter-sterilization or by X-ray preirradiation of the soils, as described in the following paragraphs. As expected, the ssRNA-specific RNase T1 did not degrade dsRNA ( Figure 1 A, B). However, incubation of dsRNA in the soil solution resulted in decreasing dissolved concentrations of intact dsRNA ( Figures 1 C, D), indicating that dsRNA degradation may occur in the soil solution. While intactP-dsRNA was still present after incubation for 0.5 h, it was no longer detectable after incubation for 24 h. TotalP activity retained in the gel decreased by 45 (±8)% from 0.5 to 24 h. In comparison to ssRNA hydrolysis by RNase T1 ( Figure 1 B), incubation of dsRNA in the soil solution resulted in few detectable LMW hydrolysis products, demonstrated by low intensities at longer migration distances ( Figure 1 D). Instead, a sharp peak inP-activity was detected at a short migration distance (<0.1 cm into the gel). The intensity at this location approximately doubled during incubation from 0.5 to 24 h. Based on the short migration distance, we hypothesized that these higher molecular weight (HMW)P-containing molecules were biomolecules that microorganisms synthesized fromP-dsRNA and/or its hydrolysis products. To provide support that these products resulted from microbial uptake and utilization, we conducted a series of experiments in which microbial activity in the solutions was reduced either by filter-sterilization or by X-ray preirradiation of the soils, as described in the following paragraphs.

32P-dsRNA in soil solutions for 0.5 or 24 h, we passed aliquots of the soil solutions through 0.22 μm syringe filters and quantified dissolved 32P-activity in the filtrate (32P-activity in the filtrate corresponded to 79 ± 3% of the initial total 32P-activity added to the solution as 32P-dsRNA; therefore, most of the added 32P remained dissolved over the 0.5 h incubation. The minor loss of dissolved 32P-activity observed at this early time point may have resulted from either adsorption of 32P-containing molecules to the filter apparatus or transfer of 32P-containing molecules to cells or particles. In contrast, after incubation for 24 h, no 32P-activity was quantifiable in the filtrate, demonstrating that the solution was completely depleted of dissolved 32P-dsRNA and 32P-containing degradation products. Consistently, no 32P-containing HMW products (nor other products) were detected by PAGE analysis of the filtrate (32P-dsRNA from 0.5 to 24 h resulted in substantial transfer of the 32P-activity into cells or onto suspended particles that were removed by filtration. After incubatingP-dsRNA in soil solutions for 0.5 or 24 h, we passed aliquots of the soil solutions through 0.22 μm syringe filters and quantified dissolvedP-activity in the filtrate ( Figure 1 E). Filtration removed microbial cells ( Figure S3 ) and larger particles from the solutions. After incubation for 0.5 h, theP-activity in the filtrate corresponded to 79 ± 3% of the initial totalP-activity added to the solution asP-dsRNA; therefore, most of the addedP remained dissolved over the 0.5 h incubation. The minor loss of dissolvedP-activity observed at this early time point may have resulted from either adsorption ofP-containing molecules to the filter apparatus or transfer ofP-containing molecules to cells or particles. In contrast, after incubation for 24 h, noP-activity was quantifiable in the filtrate, demonstrating that the solution was completely depleted of dissolvedP-dsRNA andP-containing degradation products. Consistently, noP-containing HMW products (nor other products) were detected by PAGE analysis of the filtrate ( Figure 1 F). These findings imply that incubation ofP-dsRNA from 0.5 to 24 h resulted in substantial transfer of theP-activity into cells or onto suspended particles that were removed by filtration.

32P-dsRNA. When these solutions were filtered again after incubation, 79 ± 8% of the 32P-activity remained in the final filtrate (32P-activity that remained dissolved. In a solution that was only filtered prior to incubation, the formation of the HMW products was reduced by ∼75% relative to the unfiltered solution (32P-activity remained in the filtrate and few HMW products formed in preincubation filtered solutions, we did not detect intact dissolved 32P-dsRNA in the preincubation filtered solution or its filtrate (32P-dsRNA suggested that it had been degraded by extracellular hydrolases that were not removed in the preincubation filtration step. However, unlike small hydrolysis products produced from 32P-ssRNA hydrolysis by RNase T1 (32P-containing hydrolysis products were retained on the PAGE gel in the preincubation filtered solutions (32P-activity (32P-dsRNA nor 32P-containing products retained by PAGE, we conclude that 32P-dsRNA was likely degraded into LMW 32P-containing molecules that were too small to be retained in the gel (e.g., short oligomers (<15 bp), monomers, or inorganic phosphate; detailed explanation in To further characterize solution-phase dsRNA degradation, we performed another 24 h incubation experiment in which we filtered the soil solutions before adding theP-dsRNA. When these solutions were filtered again after incubation, 79 ± 8% of theP-activity remained in the final filtrate ( Figure 1 E). Therefore, the removal of cells and particles from solution prior to incubation increased the fraction ofP-activity that remained dissolved. In a solution that was only filtered prior to incubation, the formation of the HMW products was reduced by ∼75% relative to the unfiltered solution ( Figure 1 F). Though dissolvedP-activity remained in the filtrate and few HMW products formed in preincubation filtered solutions, we did not detect intact dissolvedP-dsRNA in the preincubation filtered solution or its filtrate ( Figure 1 F). The absence ofP-dsRNA suggested that it had been degraded by extracellular hydrolases that were not removed in the preincubation filtration step. However, unlike small hydrolysis products produced fromP-ssRNA hydrolysis by RNase T1 ( Figure 1 B), noP-containing hydrolysis products were retained on the PAGE gel in the preincubation filtered solutions ( Figure 1 F). Because the filtrate contained dissolvedP-activity ( Figure 1 E) but not intactP-dsRNA norP-containing products retained by PAGE, we conclude thatP-dsRNA was likely degraded into LMWP-containing molecules that were too small to be retained in the gel (e.g., short oligomers (<15 bp), monomers, or inorganic phosphate; detailed explanation in Supporting Information ).

32P from dsRNA into HMW products (as opposed to resulting from 32P-containing molecules adsorbed to larger particles also removed by filtration), we incubated 32P-dsRNA in unfiltered soil solutions obtained from both native and X-ray preirradiated soils (soil 2.2 in 32P-dsRNA in the soil solutions for 3 h, more 32P-dsRNA remained intact in the solutions obtained from X-ray preirradiated soils than from the native soils (i.e., 30 and 40% more in soils 2.2 and 2.3, respectively). This finding indicates that the stability of dsRNA increased when the number of viable microorganisms decreased. While intact 32P-dsRNA was not detected in any of the solutions after incubation for 24 h (despite 32P-activity remaining in solution, 32P-containing dsRNA and/or its degradation products. To support the involvement of microorganisms in the incorporation ofP from dsRNA into HMW products (as opposed to resulting fromP-containing molecules adsorbed to larger particles also removed by filtration), we incubatedP-dsRNA in unfiltered soil solutions obtained from both native and X-ray preirradiated soils (soil 2.2 in Figures 1 G, H; soil 2.3 in Figure S10 ). We verified that X-ray preirradiation of the two soils decreased the number of viable microorganisms of the respective soil solutions ( Figures S3–S5 ). At the same time, solutions from the preirradiated soils contained nonviable microorganisms ( Figures S4, S5 ) and other particulates. Solutions from X-ray preirradiated soils were expected to also retain some enzymatic activity. (47,48) After incubation ofP-dsRNA in the soil solutions for 3 h, moreP-dsRNA remained intact in the solutions obtained from X-ray preirradiated soils than from the native soils (i.e., 30 and 40% more in soils 2.2 and 2.3, respectively). This finding indicates that the stability of dsRNA increased when the number of viable microorganisms decreased. While intactP-dsRNA was not detected in any of the solutions after incubation for 24 h (despiteP-activity remaining in solution, Figure S11 ), the formation of HMW products was suppressed by >90% in solutions with fewer viable microorganisms from X-ray preirradiated soils as compared to solutions from native soils ( Figure 1 H). These results support the formation of HMW products due to microbial utilization ofP-containing dsRNA and/or its degradation products.

These results collected by dsRNA incubation in solutions provide strong evidence for degradation of dissolved dsRNA, even in solutions with decreased microbial activity obtained by filtration or X-ray soil preirradiation. While a reduction in microbial activity only slightly decreased dsRNA loss, it largely decreased the formation of 32P-containing HMW products. Taken together, these results are consistent with degradation of dissolved dsRNA in soil solutions by extracellular microbial hydrolases and—in systems containing viable microorganisms—microbial uptake and utilization of the dsRNA or its hydrolysis products.