Gregory R. Heck

Control of coleopteran insect pests through RNA interference

Commercial biotechnology solutions for controlling lepidopteran and coleopteran insect pests on crops depend on the expression of Bacillus thuringiensis insecticidal proteins, most of which permeabilize the membranes of gut epithelial cells of susceptible insects. However, insect control strategies involving a different mode of action would be valuable for managing the emergence of insect resistance. Toward this end, we demonstrate that ingestion of double-stranded (ds)RNAs supplied in an artificial diet triggers RNA interference in several coleopteran species, most notably the western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte. This may result in larval stunting and mortality. Transgenic corn plants engineered to express WCR dsRNAs show a significant reduction in WCR feeding damage in a growth chamber assay, suggesting that the RNAi pathway can be exploited to control insect pests via in planta expression of a dsRNA.

Investigations into glyphosate-resistant horseweed (Conyza canadensis): retention, uptake, translocation, and metabolism

Abstract The mechanism of glyphosate resistance in horseweed was investigated. Eleven biotypes of putative sensitive (S) and resistant (R) horseweed were obtained from regions across the United States and examined for foliar retention, absorption, translocation, and metabolism of glyphosate. Initial studies used spray application of 14C-glyphosate to simulate field application. When S and R biotypes were compared in the absence of toxicity at a sublethal dose, we observed comparable retention and absorption but reduced root translocation in the R biotypes. S and R biotypes from Delaware were further examined at field use rates and results confirmed similar retention and absorption but reduced root translocation in the R biotypes. Application of 14C-glyphosate to a single leaf demonstrated reduced export out of the treated leaf and lower glyphosate import into other leaves, the roots, and the crown in R relative to S biotypes. Examination of the treated leaf by autoradiography showed that glyphosate loading into the apoplast and phloem was delayed and reduced in the R biotype. Our results consistently showed a strong correlation between impaired glyphosate translocation and resistance. Tissues from both S and R biotypes showed elevated levels of shikimate suggesting that 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) remained sensitive to glyphosate. Analysis of tissue shikimate levels demonstrated reduced efficiency in EPSPS inhibition in the R biotypes. Our results suggest that resistance is likely due to altered cellular distribution that impaired phloem loading and plastidic import of glyphosate resulting in reduced overall translocation as well as inhibition of EPSPS. Nomenclature: Glyphosate; horseweed, Conyza canadensis (L.) Cronq. ERICA.

Characterizing the Mechanism of Action of Double-Stranded RNA Activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)

RNA interference (RNAi) has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) larvae via oral delivery of synthetic double-stranded RNA (dsRNA) in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7) as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si) RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality) comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.

Lack of detectable oral bioavailability of plant microRNAs after feeding in mice

965 chow preparation where we observed the expected distribution and abundance of rice miRNAs. Oryza sativa (rice) osa-miR168a was among the most abundant miRNAs (Supplementary Table 1) in both ricecontaining chow and rice grain, consistent with previous reports9,12. Diet composition had no impact on food consumption (Supplementary Fig. 1). Following completion of the feeding regimen, small RNAs were sequenced from mouse liver and plasma samples using the Illumina (San Diego) HiSeq system. Details of the experimental protocols can be found in Supplementary Methods. We observed the expected endogenous miRNA profile and miRNA length distribution in mouse plasma and liver and rice samples, indicating consistent quality of the small RNA sequencing procedure (Supplementary Fig. 2a,b). Analysis of small RNAs from plasma and liver of mice fed on balanced rice chow and rice chow did not reveal measurable uptake of any rice grain miRNAs, including osa-miR168a. Of >10 million total sequence reads per library, fewer than ten reads identical to known rice miRNAs per library were noted in five out of eight samples from mice fed on rice-containing chow and four out of five samples from mice fed on synthetic chow (Table 1). Synthetic chow did not contain any grain or forage from plants (all plant-derived ingredients were highly purified isolates, for example, cornstarch and soybean oil); therefore, these low number of rice miRNAmappable reads could be explained by sequence errors or by cross-contamination. Mapping of mouse small RNA data to all annotated rice miRNAs in miRBase v19 identified a low number of mouse small RNA reads identical to several rice miRNA sequences (Table 1). Even so, most of the rice-like sequences were identical to the miR414 sequence (Supplementary Table 1), which is not detectable in rice grain12. In addition, these plant-like sequences were present in similar quantities in all mouse To the Editor: Human therapeutics based on nucleic acid targeting rely on sequence-specific interactions between effector and target molecules to achieve beneficial effects through specific modification to the expression of targeted genes. A variety of such compounds are being tested in laboratories and in clinical trials to treat a range of genetic and acquired diseases. Efficient, safe and cost-effective delivery of nucleic acid–based drug candidates is required to enable therapeutic levels of targeted gene regulation and overall success of this exciting new class of therapeutics. For several types of compounds in this class, effective drug delivery relies on injection of formulated nucleic acids at the site of action or into the bloodstream. Oral delivery would excel as a treatment strategy as it could offer convenient and patient-friendly features, however, progress in this approach has been hampered by substantial challenges associated with biological barriers that limit oral activity of nucleic acid therapeutics (e.g., stability within and uptake of nucleic acids from the mammalian gastrointestinal tract, nucleases and membrane barriers)1. Considerable effort has been applied to improve the stability and uptake efficiency of orally administered nucleic acids by introducing chemical modifications and formulating with excipients; however, limited success has been reported thus far2,3. The naturally occurring RNA interference (RNAi) response has been extensively reported after feeding double-stranded RNA (dsRNA) in some invertebrates, such as the model organism Caenorhabditis elegans4 and some agricultural pests5,6 (e.g., corn rootworm and cotton bollworm). Yet, despite responsiveness to ingested dsRNA, a recent survey revealed substantial variation in sensitivity to dsRNA in other Caenorhabditis nematodes7 and other invertebrate species8. In addition, despite major efforts in academic and pharmaceutical laboratories to activate the RNA silencing pathway in response to ingested RNA, the phenomenon had not been reported in mammals until a recent publication by Zhang et al.9 in Cell Research. This report described the uptake of plant-derived microRNAs (miRNA) into the serum, liver and a few other tissues in mice following consumption of rice, as well as apparent gene regulatory activity in the liver. The observation provided a potentially groundbreaking new possibility that RNAbased therapies could be delivered to mammals through oral administration and at the same time opened a discussion on the evolutionary impact of environmental dietary nucleic acid effects across broad phylogenies. A recently reported survey of a large number of animal small RNA datasets from public sources has not revealed evidence for any major plant-derived miRNA accumulation in animal samples10. Given the number of questions evoked by these analyses, the limited success with oral RNA delivery for pharmaceutical development, the history of safe consumption for dietary small RNAs11 and lack of evidence for uptake of plant-derived dietary small RNAs, we felt further evaluation of miRNA uptake and the potential for cross-kingdom gene regulation in animals was warranted to assess the prevalence, impact and robustness of the phenomenon. To address this question, we conducted a well-controlled mouse feeding study with rice-containing chow diets or with a purified synthetic chow devoid of plant grain or forage. After a two-week acclimation on synthetic chow (modified AIN93-G), animals were fasted for 12 h and then placed on synthetic chow; a nutritionally balanced, rice-containing chow (modified AIN93-G with 40.8% rice); or rice-based chow (75% rice), for 1, 3 and 7 days (Supplementary Methods). These groups are referred to herein as synthetic chow, balanced rice chow and rice chow, respectively. To confirm rice miRNA availability in feeding material, we first sequenced rice small RNAs from ricecontaining chow and rice grains used for Lack of detectable oral bioavailability of plant microRNAs after feeding in mice correspondence

Regulation of Gene Expression in Plants through miRNA Inactivation

Eukaryotic organisms possess a complex RNA-directed gene expression regulatory network allowing the production of unique gene expression patterns. A recent addition to the repertoire of RNA-based gene regulation is miRNA target decoys, endogenous RNA that can negatively regulate miRNA activity. miRNA decoys have been shown to be a valuable tool for understanding the function of several miRNA families in plants and invertebrates. Engineering and precise manipulation of an endogenous RNA regulatory network through modification of miRNA activity also affords a significant opportunity to achieve a desired outcome of enhanced plant development or response to environmental stresses. Here we report that expression of miRNA decoys as single or heteromeric non-cleavable microRNA (miRNA) sites embedded in either non-protein-coding or within the 3′ untranslated region of protein-coding transcripts can regulate the expression of one or more miRNA targets. By altering the sequence of the miRNA decoy sites, we were able to attenuate miRNA inactivation, which allowed for fine regulation of native miRNA targets and the production of a desirable range of plant phenotypes. Thus, our results demonstrate miRNA decoys are a flexible and robust tool, not only for studying miRNA function, but also for targeted engineering of gene expression in plants. Computational analysis of the Arabidopsis transcriptome revealed a number of potential miRNA decoys, suggesting that endogenous decoys may have an important role in natural modulation of expression in plants.

Environmental RNAi in herbivorous insects

Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism.

Functional Genomics Analysis of Horseweed (Conyza canadensis) with Special Reference to the Evolution of Non-Target-Site Glyphosate Resistance

Abstract The evolution of glyphosate resistance in weedy species places an environmentally benign herbicide in peril. The first report of a dicot plant with evolved glyphosate resistance was horseweed, which occurred in 2001. Since then, several species have evolved glyphosate resistance and genomic information about nontarget resistance mechanisms in any of them ranges from none to little. Here, we report a study combining iGentifier transcriptome analysis, cDNA sequencing, and a heterologous microarray analysis to explore potential molecular and transcriptomic mechanisms of nontarget glyphosate resistance of horseweed. The results indicate that similar molecular mechanisms might exist for nontarget herbicide resistance across multiple resistant plants from different locations, even though resistance among these resistant plants likely evolved independently and available evidence suggests resistance has evolved at least four separate times. In addition, both the microarray and sequence analyses identified non–target-site resistance candidate genes for follow-on functional genomics analysis. Nomenclature: Glyphosate, horseweed, Conyza canadensis (L.) Cronq. ERICA

Cotton Plants Expressing a Hemipteran-Active Bacillus thuringiensis Crystal Protein Impact the Development and Survival of Lygus hesperus (Hemiptera: Miridae) Nymphs

ABSTRACT The plant bugs Lygus hesperus Knight (Hemiptera: Miridae) and L. lineolaris (Palisot de Beauvois) have emerged as economic pests of cotton in the United States. These hemipteran species are refractory to the insect control traits found in genetically modified commercial varieties of cotton. In this article, we report the isolation and characterization of a 35 kDa crystal protein from Bacillus thuringiensis, designated TIC807, which causes reduced mass gain and mortality of L. hesperus and L. lineolaris nymphs when presented in an artificial diet feeding assay. Cotton plants expressing the TIC807 protein were observed to impact the survival and development of L. hesperus nymphs in a concentration-dependent manner. These results, demonstrating in planta activity of a Lygus insecticidal protein, represent an important milestone in the development of cotton varieties protected from Lygus feeding damage.

Quantification of transgene-derived double-stranded RNA in plants using the QuantiGene nucleic acid detection platform.

The expanding use of RNA interference (RNAi) in agricultural biotechnology necessitates tools for characterizing and quantifying double-stranded RNA (dsRNA)-containing transcripts that are expressed in transgenic plants. We sought to detect and quantify such transcripts in transgenic maize lines engineered to control western corn rootworm (Diabrotica virgifera virgifera LeConte) via overexpression of an inverted repeat sequence bearing a portion of the putative corn rootworm orthologue of yeast Snf7 (DvSnf7), an essential component of insect cell receptor sorting. A quantitative assay was developed to detect DvSnf7 sense strand-containing dsRNA transcripts that is based on the QuantiGene Plex 2.0 RNA assay platform from Affymetrix. The QuantiGene assay utilizes cooperative binding of multiple oligonucleotide probes with specificity for the target sequence resulting in exceptionally high assay specificity. Successful implementation of this assay required heat denaturation in the presence of the oligonucleotide probes prior to hybridization, presumably to dissociate primary transcripts carrying the duplex dsRNA structure. The dsRNA assay was validated using a strategy analogous to the rigorous enzyme-linked immunosorbent assay evaluations that are typically performed for foreign proteins expressed in transgenic plants. Validation studies indicated that the assay is sensitive (to 10 pg of dsRNA/g of fresh tissue), highly reproducible, and linear over ∼2.5 logs. The assay was validated using purified RNA from multiple maize tissue types, and studies indicate that the assay is also quantitative in crude tissue lysates. To the best of our knowledge, this is the first report of a non-polymerase chain reaction-based quantitative assay for dsRNA-containing transcripts, based on the use of the QuantiGene technology platform, and will broadly facilitate characterization of dsRNA in biological and environmental samples.

Computational sequence analysis of predicted long dsRNA transcriptomes of major crops reveals sequence complementarity with human genes

Long double-stranded RNAs (long dsRNAs) are precursors for the effector molecules of sequence-specific RNA-based gene silencing in eukaryotes. Plant cells can contain numerous endogenous long dsRNAs. This study demonstrates that such endogenous long dsRNAs in plants have sequence complementarity to human genes. Many of these complementary long dsRNAs have perfect sequence complementarity of at least 21 nucleotides to human genes; enough complementarity to potentially trigger gene silencing in targeted human cells if delivered in functional form. However, the number and diversity of long dsRNA molecules in plant tissue from crops such as lettuce, tomato, corn, soy and rice with complementarity to human genes that have a long history of safe consumption supports a conclusion that long dsRNAs do not present a significant dietary risk.