Environmental fate of RNA interference pesticides: Adsorption and

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Environmental Processes

Environmental fate of RNA interference pesticides: Adsorption and degradation of double-stranded RNA molecules in agricultural soils Kimberly M. Parker, Verónica Barragán Borrero, Daniël M. van Leeuwen, Mark A. Lever, Bogdan Mateescu, and Michael Sander Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b05576 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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Environmental Science & Technology

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Environmental fate of RNA interference pesticides: Adsorption and degradation of double-

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stranded RNA molecules in agricultural soils

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Kimberly M. Parker,*a,b Verónica Barragán Borrero,c Daniël M. van Leeuwen,c Mark A.

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Lever,b Bogdan Mateescu,c Michael Sander*b

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a Department

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of Energy, Environmental & Chemical Engineering,

Washington University in St. Louis, St. Louis, Missouri 63130, United States b Institute c

of Biogeochemistry and Pollutant Dynamics, ETH Zurich, 8092 Zurich, Switzerland

Institute of Molecular Plant Biology, Department of Biology, ETH Zurich, 8092 Zurich,

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Switzerland

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*(K.M.P.) Email: [email protected], phone: (314)935-3461; fax: (314)935-7211.

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*(M.S.) Email: [email protected]; +41(0)44 632 8314.

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Words: 5,342.

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Figures: 4 (2,100 word-equivalents).

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Total word-equivalents: 7,442.

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ACS Paragon Plus Environment

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Abstract.

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Double-stranded RNA (dsRNA) pesticides are a new generation of crop protectants that

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interfere with protein expression in targeted pest insects by a cellular mechanism called RNA

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interference (RNAi). The ecological risk assessment of these emerging pesticides necessitates an

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understanding of the fate of dsRNA molecules in receiving environments, among which

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agricultural soils are most important. We herein present an experimental approach using

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phosphorous-32 (32P)-radiolabeled dsRNA that allows studying key fate processes of dsRNA in

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soils with unprecedented sensitivity. This approach resolves previous analytical challenges in

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quantifying unlabeled dsRNA and its degradation products in soils. We demonstrate that

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dsRNA and its degradation products are quantifiable at concentrations as low as a few nanogram

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dsRNA per g soil by both Cerenkov counting (to quantify total 32P-activity) and by polyacrylamide

29

gel electrophoresis (PAGE) followed by phosphorimaging (to detect intact 32P-dsRNA and its 32P-

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containing degradation products). We show that dsRNA molecules added to soil suspensions

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undergo adsorption to soil particle surfaces, degradation in solution, and potential uptake by soil

32

microorganisms. The results of this work on dsRNA adsorption and degradation advance a

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process-based understanding of the fate of dsRNA in soils and will inform ecological risk

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assessments of emerging dsRNA pesticides.


32P-

2

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Introduction.

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RNA interference (RNAi) has recently been implemented in agricultural biotechnology to

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protect crops against pests.1-8 RNAi is a conserved cellular mechanism in eukaryotic organisms

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that regulates gene expression at a post-transcriptional stage by disrupting the translation of

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messenger RNAs (mRNAs) into proteins.9,10 In RNAi-based crop protection, pesticidal double-

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stranded RNA (dsRNA) is taken up by the target pest (e.g., through ingestion) and subsequently

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directs the degradation of specific, targeted pest mRNA.4,5,7,11 Degradation of this mRNA prevents

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the synthesis of essential proteins in the pest, resulting in reduced pest growth or leading to pest

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mortality.5,12-15 RNAi biotechnology has been shown to be effective against an array of agricultural

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pests including insects, nematodes, viruses and fungi.8 The first commercially-available RNAi-

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based product targeting an insect pest is a genetically engineered maize that expresses a dsRNA

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pesticide within the plant’s tissue (e.g., as a so-called “plant-incorporated protectant”).1,16

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However, RNAi-based crop protection can also be achieved by applying exogenous dsRNA

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pesticides to crops.17,18

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Agricultural use of RNAi biotechnology is expected to result in the release of pesticidal

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dsRNA to agricultural soils and other secondary receiving environments.19 Consequently, dsRNA

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pesticides must undergo ecological risk assessment (ERA) to evaluate potential ecological hazards

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to non-target organisms that may be exposed to the dsRNA.20,21 However, ERAs are currently

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challenged by a poor understanding of dsRNA fate and stability in soils, resulting in large

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uncertainties in estimated dsRNA pesticide concentrations in soils and hence exposure levels of

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non-target organisms.19 Consequently, process-oriented research on dsRNA fate in soils has been

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requested by the Scientific Advisory Panels to the U.S. Environmental Protection Agency,22-24 as

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well as other U.S. and European regulatory agencies.25,26


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To date, there are only a few published studies assessing dsRNA fate in soils and sediments

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using laboratory incubations.27-29 These studies universally determined dsRNA dissipation (i.e.,

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concentration decrease of intact dsRNA over time) using hybridization assays.30 While these

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studies demonstrated decreasing dissolved dsRNA concentrations over the timescale of days, their

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experimental approach required relatively high dsRNA concentrations (i.e., g/mL or g/g-soil

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levels)27-29 and did not allow the contributions of dsRNA adsorption to particle surfaces and of

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dsRNA (bio)degradation to the overall concentration decrease to be assessed. As both adsorption

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and degradation are expected to control dsRNA fate in receiving environments19 based existing

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knowledge on the fate of DNA and single-stranded RNA (ssRNA),31-35 a clear delineation of

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adsorption and degradation of dsRNA in soils at environmentally relevant dsRNA concentrations

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(likely ng/g-soil or lower levels, as calculated in the Supporting Information (SI)) is critical for

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ERAs.19 Specifically, analytical approaches are needed to quantify and differentiate both dissolved

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and adsorbed dsRNA molecules, as well as products of dsRNA degradation and incorporation into

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biomolecules.

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The goal of this work was to investigate adsorption and degradation of dsRNA molecules

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in agricultural soils. To this end, we developed an approach using 32-phosphorous (32P)-labeling

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of dsRNA in combination with radiochemical analysis techniques. Previously, DNA fate has been

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investigated using tritium labeling (3H).34-36 As opposed to working with unlabeled dsRNA

76

molecules, the use of the

77

quantification of both intact dsRNA and products of dsRNA degradation and also enables

78

balancing of

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and associated with soil particulates. The half-life of

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investigate adsorption and degradation of 32P-dsRNA in soils, as both processes are expected to

32P-activity

32P-labeled

added as

dsRNA (hereafter

32P-dsRNA

32P-dsRNA)

via quantification of 32P

enables highly sensitive

32P-activity

both in solution

(14.3 days) is sufficiently long to


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occur over hour to day timescales.27-29 In this study, we investigated adsorption and degradation

82

of freely dissolved

83

processes investigated herein apply to dsRNA pesticides that are exogenously applied to crops

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(e.g., via spray application)17 and that are released from the tissue of RNAi crops into soils. In case

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of the latter, we note that the scope of our study did not allow also addressing potential processes

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that affect dsRNA molecules within RNAi crop tissue prior to their release into the soil. We first

87

assessed degradation of

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(Scheme 1A). In a second set of experiments, we investigated concurrent degradation and

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adsorption of

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implications of the experimental results for the fate of dsRNA molecules in agricultural soils in

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the broader context of ERA of dsRNA pesticides.

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Materials and Methods.

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Synthesis of 32P-dsRNA molecules.

32P-dsRNA

molecules in soil solutions and suspensions. We expect that the

32P-dsRNA

32P-dsRNA

in solutions prepared from an agricultural soil suspension

added to soil suspensions (Scheme 1B). Finally, we discuss the

94

Materials and supplies used in this study are described in the SI. We synthesized a 32P-

95

dsRNA molecule with 261 base pairs (bp), similar in size to emerging dsRNA pesticides.5,13,37 The

96

synthesis method is detailed in the SI. In brief, the DNA templates were first amplified by

97

polymerase chain reaction (PCR) using forward primers containing the T7 promoter sequence.

98

Next, sense and antisense ssRNA molecules were produced by in vitro transcription of the DNA

99

templates using T7 RNA polymerase. The produced molecules were confirmed to be the correct

100

size (Figure S1). Both sense and antisense ssRNA strands were 32P-labeled by introducing [alpha-

101

32P]uridine

102

during the in vitro transcription reaction. Finally, the sense and antisense 32P-ssRNA strands were

103

mixed in equimolar concentrations and annealed to produce the 32P-dsRNA molecule. The 32P-

triphosphate (UTP) (800 Ci/mmol; such that 1% of the total UTP was


32P-labeled)

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dsRNA was stored frozen until its use in experiments. At the start of each experiment, the nominal

105

32P-dsRNA

106

presence of intact 32P-dsRNA was verified across experiments by analysis of controls (e.g., shown

107

in Figure 1A,B, as well as additional figures in the SI) prepared using the same 32P-dsRNA stock

108

interspersed among the experiments.

109

Soil preparation and characterization.

concentration was verified by measuring 32P activity aliquoted into each sample. The

110

A total of six diverse agricultural standard soils were obtained from the

111

Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer (LUFA Speyer) (Table S2).

112

The soils included one loamy sand (soil 2.1), three sandy loams (soils 2.2, 2.3, and 5M), one loam

113

(soil 2.4), and one clay (soil 6S). Each soil was homogenized by sieving (2 mm) and stored in the

114

dark at 4 ºC until use. Subsamples of the six soils were treated with X-ray irradiation (dose = 43.5

115

kGy) by Synergy Health Däniken AG to decrease microbial activity. Effectiveness of this

116

treatment was confirmed by decreased soil basal respiration and abundance of viable

117

microorganisms in the irradiated soils relative to the untreated soils (Figure S2-S5).

118

Incubation of 32P-dsRNA in soil solutions and suspensions.

119

In the first set of experiments (Scheme 1A), we incubated 32P-dsRNA in soil solutions. Soil

120

solutions were obtained by placing soil samples (0.20 g) in nuclease-free microcentrifuge tubes

121

(selected due to negligible 32P-dsRNA adsorption to tube walls, Figure S6), followed by adding

122

0.4 mL incubation buffers to each tube. The incubation buffers contained sodium chloride (10

123

mM) and either 4-morpholineethanesulfonic acid (MES, pKa= 6.15; used for soils 2.1, 2.2, and

124

2.3) or 4-morpholinepropanesulfonic acid (MOPS, pKa= 7.20; used for soils 2.4, 5M, and 6S)

125

(both 5 mM) and were adjusted to the pH of the soils (4.9-7.4, Table S2). The identity and

126

concentration (5 mM) of the buffer were selected to result in stable pH buffering during the


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127

incubation at the natural pH values reported for the respective soils. We chose organic pH-buffers

128

over phosphate which would have competed with dsRNA for adsorption sites on mineral

129

surfaces.38,39 Following initial vortex mixing, soil samples were incubated for 24 hr under

130

horizontal shaking (500 rpm; 22 ºC). At the end of the incubation, the samples were centrifuged

131

for 5 minutes (10,000 RCF; 4 ºC). The supernatant solution (0.325 mL) was then pipette-

132

transferred to a new microcentrifuge tube. To this solution, we added

133

concentration = 30 ng/mL), followed by incubation for 0.5, 3 or 24 hr under horizontal shaking

134

(500 rpm; 22 ºC). Finally, the 32P-dsRNA and its degradation products in the soil solution were

135

analyzed as described below.

136

In the second set of experiments (Scheme 1B), we incubated

32P-dsRNA

32P-dsRNA

(final

in soil

137

suspensions. We added 0.4 mL incubation buffer containing 30 ng/mL

138

sample (0.20 g). After initial mixing, samples were incubated (0.3-30 hr) under horizontal shaking

139

(500 rpm; 22 ºC). At the end of the incubation, the samples were centrifuged for 5 minutes (10,000

140

RCF; 4 ºC). The supernatant solution (0.325 mL) was transferred to a new microcentrifuge tube

141

for analysis. Next, from the soil pellet, dsRNA was extracted into 0.55 mL extraction buffer (220

142

mM borate, pH 9.4; 7 mM EDTA; 3 mM sodium hexametaphosphate, to competitively displace

143

dsRNA from particle surfaces). This extraction buffer composition was adapted from buffers

144

effective in extracting DNA and RNA from sediments,40 while minimizing artefactual dsRNA

145

degradation (i.e., base-catalyzed hydrolysis of phosphodiester bonds41) and lysis of

146

microorganisms. After resuspending the soil pellet in the extraction buffer, the vials were shaken

147

for 1 hr in the dark (500 rpm; 4 ºC) and then centrifuged for 5 minutes (10,000 RCF; 4 ºC). The

148

supernatant solution (0.550 mL) was transferred to a new microcentrifuge tube for analysis.


32P-dsRNA

to each soil

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149

All buffers were prepared with sterile-filtered water in pre-baked glassware and autoclaved

150

once per day for three consecutive days to avoid microbial and nuclease contaminations (details

151

in SI).

152

Quantification of 32P-activity in soil solutions and pellets.

153

The 32P-activities in both soil solutions and pellets were quantified by Cerenkov counting

154

using a Packard Tri-Carb liquid scintillation counter. Each sample was analyzed twice. Results

155

were averaged and corrected for signal suppression by the sample matrix and 32P radioactive decay

156

(Figure S7; Table S3). We determined the distribution of

157

pellet after accounting for the volume of solution that we could not remove from the pellet when

158

pipette-transferring the supernatant to a new microcentrifuge tube. We calculated the 32P-activity

159

in the residual solution by assuming

160

supernatant and the residual solution.

161

Size analysis of 32P-dsRNA and degradation products.

162

32P-activity

32P-activity

between the solution and

per unit volume was equivalent between the

We used polyacrylamide gel electrophoresis (PAGE) coupled to phosphorimaging to semi-

163

quantitatively analyze dissolved

164

degradation and incorporation into biomolecules. For this analysis, samples (5 µL) were mixed

165

with 2x loading dye (5 µL, 95% formamide), heated (95˚C; 5 min), and then loaded on denaturing

166

PAGE gels (14% polyacrylamide-8 M urea). Denaturing PAGE gels42 were selected so that RNA

167

molecules were separated only by length. Gels were run at 100 V for 2 hours, dried, and exposed

168

overnight on a storage phosphor screen (GE). The phosphor screen was scanned on a Typhoon

169

Phosphorimager. The image intensity as a function of migration distance into the gel was

170

quantified using ImageJ Fiji software after background subtraction. Using 32P-dsRNA standards,

171

we determined that the image intensity increased with increasing 32P-activity according to a non-

32P-dsRNA

molecules and

32P-containing


products of dsRNA

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linear function (Figure S8). We showed that matrix components in the soil supernatant solutions

173

did not affect dsRNA migration or intensity, but that matrix components in the soil extracts

174

increased the dsRNA migration distance and decreased measured 32P intensities (Figure S9).

175

Statistical analysis.

176

Results represent means and standard deviations (indicated by error bars), unless otherwise

177

stated. Statistical analyses were performed using Prism 7 for Mac OS X (GraphPad Software, Inc.).

178

Statistical significance was determined using an unpaired Student’s t-test with Welch’s correction

179

(ns = not significant, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001).

180

Results and Discussion.

181

Incubation of 32P-dsRNA in soil solutions.

182

To assess degradation and microbial utilization of dissolved 32P-dsRNA, while at the same

183

time minimizing

184

solutions collected as the supernatant of centrifuged soil suspensions (Scheme 1A). Centrifugation

185

removed most, but not all, particulates: the solution contained a small number of soil particles and

186

active microorganisms (Figure S3-5). Because abiotic dsRNA hydrolysis is slow under the

187

experimental pH and temperature conditions (i.e., on a timescale of years),41 we anticipated

188

decreasing dissolved

189

occurring processes: (i) dsRNA hydrolysis by extracellular hydrolases and (ii) microbial uptake

190

and utilization of dsRNA and its hydrolysis products. We note that while hydrolases either specific

191

to or competent towards dsRNA have been identified,43-45 their abundance and activity in soils is

192

not documented.

32P-dsRNA

adsorption to soil particles, we first incubated

32P-dsRNA

32P-dsRNA

in soil

concentrations during incubation would result from two co-

193

We first verified that PAGE coupled to phosphorimaging allows investigating hydrolysis

194

reactions by detecting both loss of the intact molecule and formation of lower molecular weight


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(LMW) fragments. To this end, in a control experiment, we treated 32P-ssRNA with the ssRNA-

196

specific46 RNase T1 hydrolase to generate smaller ssRNA fragments. We note that we monitored

197

ssRNA hydrolysis by RNase T1 because a dsRNA-specific hydrolase was not available to us.

198

RNase T1 treatment decreased the amounts of intact ssRNA and produced LMW fragments

199

(Figure 1A, B). The total 32P counts in the gel (i.e., intensity values integrated over the gel length)

200

in the ssRNA sample degraded by RNase T1 was the same as in the untreated ssRNA sample. We

201

therefore concluded that most 32P-containing hydrolysis products were retained in the gel under

202

the selected run conditions.

203

As expected, the ssRNA-specific RNase T1 did not degrade dsRNA (Figure 1A, B).

204

However, incubation of dsRNA in the soil solution resulted in decreasing dissolved concentrations

205

of intact dsRNA (Figure 1C, D), indicating that dsRNA degradation may occur in the soil solution.

206

While intact 32P-dsRNA was still present after 0.5 hr of incubation, it was no longer detectable

207

after 24 hr incubation. Total 32P activity retained in the gel decreased by 45 (8)% from 0.5 to 24

208

hr. In comparison to ssRNA hydrolysis by RNase T1 (Figure 1B), incubation of dsRNA in the soil

209

solution resulted in few detectable LMW hydrolysis products, demonstrated by low intensities at

210

longer migration distances (Figure 1D). Instead, a sharp peak in 32P-activity was detected at a short

211

migration distance (< 0.1 cm into the gel). The intensity at this location approximately doubled

212

during incubation from 0.5 to 24 hr. Based on the short migration distance, we hypothesized that

213

these higher molecular weight (HMW)

214

microorganisms synthesized from 32P-dsRNA and/or its hydrolysis products. To provide support

215

that these products resulted from microbial uptake and utilization, we conducted a series of

216

experiments in which microbial activity in the solutions was reduced either by filter-sterilization

217

or by X-ray pre-treatment of the soils, as described in the following paragraphs.

32P-containing

molecules were biomolecules that


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After incubating 32P-dsRNA in soil solutions for 0.5 or 24 hr, we passed aliquots of the soil

219

solutions through 0.22 µm syringe filters and quantified dissolved 32P-activity in the filtrate (Figure

220

1E). Filtration removed microbial cells (Figure S3) and larger particles from the solutions. After

221

incubation for 0.5 hr, the 32P-activity in the filtrate corresponded to 79 ± 3% of the initial total 32P-

222

activity added to the solution as 32P-dsRNA; therefore, most of the added 32P remained dissolved

223

over the 0.5 hr incubation. The minor loss of dissolved 32P-activity observed at this early timepoint

224

may have resulted from either adsorption of

225

transfer of 32P-containing molecules to cells or particles. In contrast, after 24 hr incubation, no 32P-

226

activity was quantifiable in the filtrate, demonstrating that the solution was completely depleted of

227

dissolved

228

HMW products (nor other products) were detected by PAGE analysis of the filtrate (Figure 1F).

229

These findings imply that incubation of 32P-dsRNA from 0.5 to 24 hr resulted in substantial transfer

230

of the 32P-activity into cells or onto suspended particles that were removed by filtration.

32P-dsRNA

and

32P-containing

32P-containing

molecules to the filter apparatus or

degradation products. Consistently, no

32P-containing

231

To further characterize solution-phase dsRNA degradation, we performed another 24 hr

232

incubation experiments in which we filtered the soil solutions before adding the 32P-dsRNA. When

233

these solutions were filtered again after incubation, 79 ± 8% of the 32P-activity remained in the

234

final filtrate (Figure 1E). Therefore, the removal of cells and particles from solution prior to

235

incubation increased the fraction of

236

only filtered prior to incubation, the formation of the HMW products was reduced by ~75% relative

237

to the unfiltered solution (Figure 1F). Though dissolved 32P-activity remained in the filtrate and

238

few HMW products formed in pre-incubation filtered solutions, we did not detect intact dissolved

239

32P-dsRNA

240

dsRNA suggested that it had been degraded by extracellular hydrolases that were not removed in

32P-activity

that remained dissolved. In a solution that was

in the pre-incubation filtered solution or its filtrate (Figure 1F). The absence of 32P-


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the pre-incubation filtration step. However, unlike small hydrolysis products produced from 32P-

242

ssRNA hydrolysis by RNase T1 (Figure 1B), no 32P-containing hydrolysis products were retained

243

on the PAGE gel in the pre-incubation filtered solutions (Figure 1F). Because the filtrate contained

244

dissolved 32P-activity (Figure 1E) but not intact 32P-dsRNA nor 32P-containing products retained

245

by PAGE, we conclude that 32P-dsRNA was likely degraded into LMW 32P-containing molecules

246

that were too small to be retained in the gel (e.g., short oligomers (< 15 bp), monomers, or

247

inorganic phosphate; detailed explanation in SI).

248

To support the involvement of microorganisms in the incorporation of

32P

from dsRNA

249

into HMW products (as opposed to resulting from

250

particles also removed by filtration), we incubated 32P-dsRNA in unfiltered soil solutions obtained

251

from both native and X-ray pre-treated soils (soil 2.2 in Figure 1G, H; soil 2.3 in Figure S10). We

252

verified that X-ray pre-treatment of the two soils decreased the number of viable microorganisms

253

of the respective soil solutions (Figure S3-S5). At the same time, solutions from the treated soils

254

contained nonviable microorganisms (Figure S4, S5) and other particulates. Solutions from X-ray

255

treated soils were expected to also retain some enzymatic activity.47,48 After incubation of

256

dsRNA in the soil solutions for 3 hr, more 32P-dsRNA remained intact in the solutions obtained

257

from X-ray treated soils than from the untreated soils (i.e., 30 and 40% more in soils 2.2 and 2.3,

258

respectively). This finding indicates that the stability of dsRNA increased when the number of

259

viable microorganisms decreased. While intact 32P-dsRNA was not detected in any of the solutions

260

after incubation for 24 hr (despite 32P-activity remaining in solution, Figure S11), the formation of

261

HMW products was suppressed by >90% in solutions with fewer viable microorganisms from X-

262

ray treated soils as compared to solutions from untreated soils (Figure 1H). These results support

32P-containing


molecules adsorbed to larger

32P-

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263

the formation of HMW products due to microbial utilization of 32P-containing dsRNA and/or its

264

degradation products.

265

These results collected by dsRNA incubation in solutions provide strong evidence for

266

degradation of dissolved dsRNA, even in solutions with decreased microbial activity obtained by

267

filtration or X-ray soil treatment. While a reduction in microbial activity only slightly decreased

268

dsRNA loss, it largely decreased the formation of 32P-containing HMW products. Taken together,

269

these results are consistent with degradation of dissolved dsRNA in soil solutions by extracellular

270

microbial hydrolases and – if viable microorganisms are present – microbial uptake and utilization

271

of the dsRNA or its hydrolysis products.

272

Distribution of 32P-activity between solution and soil particulates in soil suspensions.

273

In comparison to soil solutions, dsRNA in soil suspensions is subjected to additional

274

processes due to the presence of soil particles, including degradation and utilization of dsRNA by

275

particle-associated microorganisms and adsorption to particle surfaces. In a first set of soil

276

suspension experiments, we assessed the timescale over which 32P-activity, added as 32P-dsRNA

277

to the solution phase, became associated with suspended particulates of soil 2.4 (selected because

278

of its high basal respiration rate, Figure S2). To this end, we incubated

279

suspensions for 0.3-30 hr, followed by centrifugation and quantification of 32P-activity in both the

280

supernatant solution (i.e., 32Paq) and in the soil pellet (i.e., 32Psoil) (Figure 2A). Over the first 6 hr,

281

32P

282

activity remained in solution. The decrease in 32Paq during the incubation was matched by an equal

283

increase in 32Psoil. The sum of 32Paq and 32Psoil (i.e., 32Ptotal) remained approximately constant over

284

the entire incubation and was in good agreement with the initial 32P-activity added as 32P-dsRNA.

285

The use of 32P-dsRNA thus allows closing the balance on added 32P-activity in soil suspensions.

aq

32P-dsRNA

in soil

decreased at an approximately constant rate. After 12 hr of incubation, 12 hr, most (>85%) 32P-activity was both soil-associated and non-

32 extracted

Psoil

formed initially after 0.3 hr of incubation reflected adsorbed

32 non - extracted

Psoil

that formed after this initial phase was largely

32P

Based on the results obtained with soil 2.4, we selected incubation times of 3 hr (when

298

32P

299

experiments. We incubated 32P-dsRNA for 3 and 24 hr in suspensions prepared with five additional

300

soils with varying physicochemical properties (Table S2) and subsequently quantified 32Paq and

301

32P

302

and 85% of 32Ptotal – indicating that soil properties greatly affected the rates at which 32P-containing

303

molecules associated with soil particulates. For all tested soils except 2.3, 32Paq further decreased

304

from 3 to 24 hr of incubation (p < 0.05). In soil 2.3, 32Paq had already decreased to only ~5% of

305

32P

306

incubation. In all soils and at both timepoints, the balance on 32P-activity label was closed (i.e.,

307

decreases in 32Paq over time were offset by increases in 32Psoil). Among soils, 32Pextracted accounted soil

308

for 10-60% of 32Ptotal. With the exception of soil 2.1, 32Pextracted remained approximately constant soil

soil

soil

total

increased with time) and 24 hr (when

32P

soil

had plateaued) for further incubation

(Figure S12). After 3 hr incubation, 32Paq for the tested soils ranged widely – between 5%

within 3 hr of incubation. For all soils, 32Paq had decreased to < 20% of 32Ptotal after 24 hr of


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Page 15 of 31


32 non - extracted

309

or slightly decreased when the incubation time increased from 3 to 24 hr, while

310

increased from 5-40% of 32Ptotal after 3 hr of incubation to 25-85% of 32Ptotal after 24 hr incubation.

311

We hypothesized that differences in the distribution of

32P

Psoil

among the six soils resulted

312

from different rates and extents of processes that relate to certain soil-specific factors (e.g., pH,

313

soil texture, microbial activity) (Table S2, Figure S2). We tested for correlations of

314

, and Pextracted soil

315

physicochemical properties of these soils (Table S4). None of the

316

― extracted after 24 hr was higher in samples with high with soil pH. However, the amount of 32Pnon soil

317

fine content ( 0.05; * p < 0.05; ** p < 0.01; ***

618

p < 0.001, **** p < 0.0001).


29


HMW A 14000 products intact

12000

14000 products intact 12000

8000 6000 No treatment

0 -1

0

1

4000

2 3 4 Distance (cm)

intact

3000 Intensity

6000

X-ray treated No treatment

2000 5

0 -1

6

extract from soil 2.2 tincubation = 3 hr X-ray treated

2000 No treatment

D

0

4000

1

2 3 4 Distance (cm)

intact

3000 Intensity

2000

0 -1

0

1

2 3 4 Distance (cm)

5

0 -1

F

HMW products intact solution from soil 2.3

0

1

2 3 4 Distance (cm)

HMW products intact

8000

5

6

solution from soil 2.3 tincubation = 24 hr

2000

No treatment

0 -1

0

4000

1

2 3 4 Distance (cm) intact

3000

Intensity

X-ray treated

4000

5


No treatment

1000

No treatment

0 -1

6

2000

0 -1

X-ray treated

2000

H

0

1

4000

2 3 4 Distance (cm)

intact

3000 Intensity

Intensity

6000

4000

Intensity

extract from soil 2.2 tincubation = 24 hr

No treatment

6

6000

619

6

X-ray treated

2000

tincubation = 3 hr

G

5

1000

1000

E 8000

8000 4000

4000

C


10000

X-ray treated

Intensity

Intensity

10000

HMW

B


Page 30 of 31

5

6


2000

No treatment

1000

0

1

2 3 4 Distance (cm)

5

6

0 -1

0

1

2 3 4 Distance (cm)

5

6

620

Figure 3. PAGE analysis of supernatant solution and extracts collected from soil suspensions (0.20

621

g soil in 0.40 mL incubation solution containing 10 mM NaCl and 5 mM buffer at native soil pH)


30

Page 31 of 31


622

incubated with 32P-dsRNA (12 ng). Intact dsRNA and higher molecular weight (HMW) products

623

are indicated. The soil suspensions were prepared using soil 2.2 (A-D) and soil 2.3 (E-H) with or

624

without treatment by X-ray irradiation (43.5 kGy). The supernatant solution was obtained by

625

centrifugation and analyzed after incubation for 3 hr (A, E) or 24 hr (B, F), The extract was

626

obtained from the soil pellet (extraction buffer: pH 9.4; 220 mM borate, 7 mM EDTA, 3 mM

627

hexametaphosphate) from the same samples (incubation period of 3 hr (C, G) or 24 hr (D, H).

628

Results shown are from triplicate experiments.


31

Environmental fate of RNA interference pesticides: Adsorption and

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