Endogenous tassel-specific small RNAs-mediated RNA interference enables a novel glyphosate-inducible male sterility system for commercial production of hybrid seed in Zea mays L.

Hybrid crops produce higher yields than their inbred parents due to heterosis. For high purity of hybrid seeds, it is critical to eliminate self-pollination. Manual or mechanical removal of male parts (such as detasseling in maize) is labor-intensive, fuel and time-consuming, and can cause physical damage to female plants, resulting in significant seed yield reductions. Many male-sterility systems either require a maintainer for male-sterile line propagation or are often affected by environmental factors. Roundup® Hybridization System (RHS) utilizes glyphosate to induce male sterility, which effectively eliminates the need for maintainer lines and removal of male parts for commercial hybrid seed production. The first-generation RHS (RHS1) is based on low expression of a glyphosate-insensitive 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in pollen. This report presents the second-generation RHS (RHS2) technology built on RNA interference (RNAi) combined with CP4 EPSPS. It utilizes maize endogenous male tissue-specific small interfering RNAs (mts-siRNAs) to trigger cleavage of the CP4 EPSPS mRNA specifically in tassels, resulting in glyphosate-sensitive male cells due to lack of the CP4 EPSPS protein. Male sterility is then induced by glyphosate application at the stages critical for pollen development, and the male-sterile plants are used as the female parent to produce hybrid seed. The endogenous mts-siRNAs are conserved across maize germplasms, and the inducible male sterility was replicated in representative germplasms through introgression of a CP4 EPSPS transgene containing the mts-siRNA target sequence. This technology combines the relative simplicity and convenience of a systemic herbicide spray methodology with targeted protein expression to create an inducible male sterility system for industrial production of row crop hybrid seeds in an environmentally-independent manner.

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.

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.

Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte).

The sequence specificity of the endogenous RNA interference pathway allows targeted suppression of genes essential for insect survival and enables the development of durable and efficacious insecticidal products having a low likelihood to adversely impact non-target organisms. The spectrum of insecticidal activity of a 240 nucleotide (nt) dsRNA targeting the Snf7 ortholog in Western Corn Rootworm (WCR; Diabrotica virgifera virgifera) was characterized by selecting and testing insects based upon their phylogenetic relatedness to WCR. Insect species, representing 10 families and 4 Orders, were evaluated in subchronic or chronic diet bioassays that measured potential lethal and sublethal effects. When a specific species could not be tested in diet bioassays, the ortholog to the WCR Snf7 gene (DvSnf7) was cloned and corresponding dsRNAs were tested against WCR and Colorado potato beetle (Leptinotarsa decemlineata); model systems known to be sensitive to ingested dsRNA. Bioassay results demonstrate that the spectrum of activity for DvSnf7 is narrow and activity is only evident in a subset of beetles within the Galerucinae subfamily of Chrysomelidae (>90% identity with WCR Snf7 240 nt). This approach allowed for evaluating the relationship between minimum shared nt sequence length and activity. A shared sequence length of ≥ 21 nt was required for efficacy against WCR (containing 221 potential 21-nt matches) and all active orthologs contained at least three 21 nt matches. These results also suggest that WCR resistance to DvSnf7 dsRNA due to single nucleotide polymorphisms in the target sequence of 240 nt is highly unlikely.

Analysis of plant-derived miRNAs in animal small RNA datasets.

BACKGROUND: Plants contain significant quantities of small RNAs (sRNAs) derived from various sRNA biogenesis pathways. Many of these sRNAs play regulatory roles in plants. Previous analysis revealed that numerous sRNAs in corn, rice and soybean seeds have high sequence similarity to animal genes. However, exogenous RNA is considered to be unstable within the gastrointestinal tract of many animals, thus limiting potential for any adverse effects from consumption of dietary RNA. A recent paper reported that putative plant miRNAs were detected in animal plasma and serum, presumably acquired through ingestion, and may have a functional impact in the consuming organisms. RESULTS: To address the question of how common this phenomenon could be, we searched for plant miRNAs sequences in public sRNA datasets from various tissues of mammals, chicken and insects. Our analyses revealed that plant miRNAs were present in the animal sRNA datasets, and significantly miR168 was extremely over-represented. Furthermore, all or nearly all (>96%) miR168 sequences were monocot derived for most datasets, including datasets for two insects reared on dicot plants in their respective experiments. To investigate if plant-derived miRNAs, including miR168, could accumulate and move systemically in insects, we conducted insect feeding studies for three insects including corn rootworm, which has been shown to be responsive to plant-produced long double-stranded RNAs. CONCLUSIONS: Our analyses suggest that the observed plant miRNAs in animal sRNA datasets can originate in the process of sequencing, and that accumulation of plant miRNAs via dietary exposure is not universal in animals.

RNA decoys: an emerging component of plant regulatory networks?

The role of non-coding RNAs (ncRNAs), both short and long ncRNAs, in the regulation of gene expression has become evident in recent years. Non-coding RNA-based regulation is achieved through a variety of mechanisms; some are relatively well-characterized, while others are much less understood. MicroRNAs (miRNAs), a class of endogenous small RNAs, function as master regulators of gene expression in eukaryotic organisms. A notable, recently discovered role for long ncRNAs is that of miRNA decoys, also referred to as target mimics or sponges, in which long ncRNAs carry a short stretch of sequence sharing homology to miRNA-binding sites in endogenous targets. As a consequence, miRNA decoys are able to sequester and inactivate miRNA function. Engineered miRNA decoys are also efficacious and useful tools for studying gene function. We recently demonstrated that the potential of miRNA decoys to inactivate miRNAs in the model plants Arabidopsis thaliana and Nicotiana benthamiana is dependent on the level of sequence complementarity to miRNAs of interest. The flexibility of the miRNA decoy approach in sequence-dependent miRNA inactivation, backbone choice, ability to simultaneously inactivate multiple miRNAs, and more importantly, to achieve a desirable level of miRNA inactivation, makes it a potentially useful tool for crop improvement. This research addendum reports the functional extension of miRNA decoys from model plants to crops. Furthermore, endogenous miRNA decoys, first described in plants, have been proposed to play a significant role in regulating the transcriptome in eukaryotes. Using computational analysis, we have identified numerous endogenous sequences with potential miRNA decoy activity for conserved miRNAs in several plant species. Our data suggest that endogenous miRNA decoys can be widespread in plants and may be a component of the global gene expression regulatory network in plants.

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.

An intron sense suppression construct targeting soybean FAD2-1 requires a double-stranded RNA-producing inverted repeat T-DNA insert.

We demonstrate that the transformation of soybean (Glycine max) with sense suppression constructs using intron sequences from the fatty acid oleyl Delta12 desaturase gene FAD2-1A leads to efficient and specific reduction of FAD2-1 transcripts in developing seeds, increased oleic acid, and decreased polyunsaturated fatty acids. The related FAD2-2 transcripts are only marginally affected. Despite screening a large number of independent transformants, no single-copy efficacious transformants could be found. Invariably, all the least complex transgenic loci have two T-DNA copies in an inverted repeat configuration, centered at the right borders. We show that this T-DNA configuration produces an inverted repeat transcript and that small interfering RNAs accumulate against the target sequence.

Introgression of a Tombusvirus Resistance Locus from Nicotiana edwardsonii var. Columbia to N. clevelandii.

ABSTRACT A new variety of Nicotiana, N. edwardsonii var. Columbia, was evaluated for its capacity to serve as a new source for virus resistance genes. Columbia was developed from a hybridization between N. glutinosa and N. clevelandii, the same parents used for the formation of the original N. edwardsonii. However, in contrast to the original N. edwardsonii, crosses between Columbia and either of its parents are fertile. Thus, the inheritance of virus resistance genes present in N. glutinosa could be characterized by using Columbia as a bridge plant in crosses with the susceptible parent, N. clevelandii. To determine how virus resistance genes would segregate in interspecific crosses between Columbia and N. clevelandii, we followed the fate of the N gene, a single dominant gene that specifies resistance to Tobacco mosaic virus (TMV). Our genetic evidence indicated that the entire chromosome containing the N gene was introgressed into N. clevelandii to create an addition line, designated N. clevelandii line 19. Although line 19 was homozygous for resistance to TMV, it remained susceptible to Tomato bushy stunt virus (TBSV) and Cauliflower mosaic virus (CaMV) strain W260, indicating that resistance to these viruses must reside on other N. glutinosa chromosomes. We also developed a second addition line, N. clevelandii line 36, which was homozygous for resistance to TBSV. Line 36 was susceptible to TMV and CaMV strain W260, but was resistant to other tombusviruses, including Cucumber necrosis virus, Cymbidium ringspot virus, Lettuce necrotic stunt virus, and Carnation Italian ringspot virus.

Temporal expression of PR-1 and enhanced mature plant resistance to virus infection is controlled by a single dominant gene in a new Nicotiana hybrid.

A new variety of Nicotiana edwardsonii, designated N. edwardsonii cv. Columbia, expresses pathogenesis-related (PR) proteins in a temporal manner 45 to 49 days postplanting and also exhibits enhanced resistance to Tobacco mosaic virus, Tobacco necrosis virus, and Tomato bushy stunt virus. In contrast, PR proteins were not expressed in the original N. edwardsonii variety at comparable ages but were induced after onset of a hypersensitive response to viral infection. The temporal induction of PR proteins in 'Columbia' was correlated with increases in salicylic acid and glycosylated salicylic acid. Earlier studies noted that some Nicotiana hybrids derived from interspecific crosses constitutively express PR proteins, but the genetic basis of this phenomenon had not been investigated, likely because many interspecific Nicotiana crosses are sterile. However, the close genetic relationship between N. edwardsonii and 'Columbia' indicated that a hybrid between these two plants might be fertile, and this proved to be true. Genetic crosses between 'Columbia' and N. edwardsonii demonstrated that a single, dominant gene conditioned temporal expression of PR proteins and enhanced resistance. This gene was designated TPR1 (for temporal expression of PR proteins).