James K. Roberts

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.

Characterization of Unique Small RNA Populations from Rice Grain

Small RNAs (∼20 to 24 nucleotides) function as naturally occurring molecules critical in developmental pathways in plants and animals [1], [2]. Here we analyze small RNA populations from mature rice grain and seedlings by pyrosequencing. Using a clustering algorithm to locate regions producing small RNAs, we classified hotspots of small RNA generation within the genome. Hotspots here are defined as 1 kb regions within which small RNAs are significantly overproduced relative to the rest of the genome. Hotspots were identified to facilitate characterization of different categories of small RNA regulatory elements. Included in the hotspots, we found known members of 23 miRNA families representing 92 genes, one trans acting siRNA (ta-siRNA) gene, novel siRNA-generating coding genes and phased siRNA generating genes. Interestingly, over 20% of the small RNA population in grain came from a single foldback structure, which generated eight phased 21-nt siRNAs. This is reminiscent of a newly arising miRNA derived from duplication of progenitor genes [3], [4]. Our results provide data identifying distinct populations of small RNAs, including phased small RNAs, in mature grain to facilitate characterization of small regulatory RNA expression in monocot species.

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.

Chapter Five - Progress Towards RNAi-Mediated Insect Pest Management

Gene suppression via RNA interference (RNAi) provides an alternative strategy for insect pest management. The ingestion by insects of double-stranded RNAs targeting essential insect genes can trigger RNAi and lead to growth inhibition, developmental aberrations, reduced fecundity, and mortality. This RNAi response is particularly acute in certain coleopteran species, most notably the western corn rootworm, a devastating pest impacting corn production in the United States. The development of next-generation rootworm-protected corn hybrids includes an RNAi-based trait that provides a mode of action distinct from those of Bacillus thuringiensis insecticidal protein-based traits currently used for rootworm pest management. Unfortunately, many insect species including important lepidoptera and hemiptera pests appear largely recalcitrant in their response to environmental RNA, suggesting biological barriers that thus far limit the utility of RNAi for agricultural pest management. This review will highlight recent efforts to understand the barriers to RNA delivery in recalcitrant insect species, describe recent advances in the commercial development of insect-protected crops and biological insecticides utilizing RNAi, and discuss this strategy in the context of an integrated pest management approach.

Endogenous small RNAs in grain: Semi-quantification and sequence homology to human and animal genes

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effector molecules of RNA interference (RNAi), a highly conserved RNA-based gene suppression mechanism in plants, mammals and other eukaryotes. Endogenous RNAi-based gene suppression has been harnessed naturally and through conventional breeding to achieve desired plant phenotypes. The present study demonstrates that endogenous small RNAs, such as siRNAs and miRNAs, are abundant in soybean seeds, corn kernels, and rice grain, plant tissues that are traditionally used for food and feed. Numerous endogenous plant small RNAs were found to have perfect complementarity to human genes as well as those of other mammals. The abundance of endogenous small RNA molecules in grain from safely consumed food and feed crops such as soybean, corn, and rice and the homology of a number of these dietary small RNAs to human and animal genomes and transcriptomes establishes a history of safe consumption for dietary small RNAs.

A novel real‐time polymerase chain reaction method for high throughput quantification of small regulatory RNAs

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are important players of both transcriptional and post-transcriptional gene silencing networks. In order to investigate the functions of these small regulatory RNAs, a system with high sensitivity and specificity is desperately needed to quantitatively detect their expression levels in cells and tissues. However, their short length of 19-24 nucleotides and strong similarity between related species render most conventional expression analysis methods ineffective. Here we describe a novel primer for small RNA-specific reverse transcription and a new TaqMan technology-based real-time method for quantification of small RNAs. This method is capable of quantifying miRNA and siRNA in the femtomolar range, which is equivalent to ten copies per cell or fewer. The assay has a high dynamic range and provides linear readout of miRNA concentrations that span seven orders of magnitude and allows us to discriminate small RNAs that differ by as little as one nucleotide. Using the new method, we investigated the expression pattern of gma-miRMON1, a novel miRNA identified from soybean leaves. The results were consistent with our results obtained from Northern blot analysis of gma-miRMON1 and Affymetrix microarray analysis of the gma-miRMON1 precursor, suggesting that the new method can be used in transcription profiling.

Transformation of Cotton Production through the Use of Genetically Improved Cotton

Over time, the production of cotton has dramatically improved. Better cotton varieties are now available. Improvements in agricultural practices with the introduction of chemical insecticides, herbicides and mechanization have increased productivity and efficiency. The introductions of cotton varieties, which contain genetically engineered traits, have transformed cotton production for the better. One such trait, Bollgard® cotton, confers resistance to lepidopterous insect pests that attack cotton such as tobacco budworm, Heliothis virescens; cotton bollworm, Helicoverpa zea; and pink bollworm, Pectinophora gossypiella (Perlak et al., 2001). Introduced in 1996, Bollgard cotton has changed the way farmers approach insect control in their cotton fields. It allows growers to reduce their insecticide use (Carpenter, 2001) while improving their productivity and insect control. It is the only cotton bio-engineered trait for insect control approved in the United States.