Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species.

MAIN CONCLUSION: 22 nt siRNAs applied to leaves induce production of transitive sRNAs for targeted genes and can enhance local silencing. Systemic silencing was only observed for a GFP transgene. RNA interference (RNAi) is a gene silencing mechanism important in regulating gene expression during plant development, response to the environment and defense. Better understanding of the molecular mechanisms of this pathway may lead to future strategies to improve crop traits of value. An abrasion method to deliver siRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for three endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GENOMES UNCOUPLED4 (GUN4). However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also produced transitive siRNAs in response to 22 nt siRNAs. 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for three of the genes. These special properties of 22 nt siRNAs were also observed for the CHL-H gene in A. cruentus. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

Carbon Dots for Efficient Small Interfering RNA Delivery and Gene Silencing in Plants.

The initiation of RNA interference (RNAi) by topically applied small interfering RNA has potential applications for plant functional genomics, crop improvement and crop protection, but the primary obstacle for the development of this technology is the efficient delivery of RNAi effectors into the cell. The plant cell wall is a particularly challenging barrier for the delivery of macromolecules because many of the transfection agents that are commonly used with animal cells produce nanocomplexes that are significantly larger than the size exclusion limit of the cell wall. Here, we illustrate the use of a class of very small nanoparticles, called carbon dots, for delivering small interfering RNA into the model plants Nicotiana benthamiana and tomato (Solanum lycopersicum). Low-pressure spray application of these formulations with a spreading surfactant resulted in strong silencing of GFP transgenes in both species. The delivery efficacy of carbon dot formulations was also demonstrated by the silencing of endogenous genes that encode two subunits of magnesium chelatase, an enzyme necessary for chlorophyll synthesis. The strong visible phenotypes observed with the carbon dot-facilitated delivery were validated by measuring significant reductions in the target gene transcript and/or protein levels. Methods for the delivery of RNAi effectors into plants, such as the carbon dot formulations described here, could become valuable tools for gene silencing in plants with practical applications in plant functional genomics and agriculture.

Beyond identity: Understanding the contribution of the 5' nucleotide of the antisense strand to RNAi activity.

In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene's mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5' nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5' nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5' nt identities and different pairing states between the 5' antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5' nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5' thermodynamic rules governing topical siRNA efficacy across plants and animals.

Systemic GFP silencing is associated with high transgene expression in Nicotiana benthamiana.

Gene silencing in plants using topical dsRNA is a new approach that has the potential to be a sustainable component of the agricultural production systems of the future. However, more research is needed to enable this technology as an economical and efficacious supplement to current crop protection practices. Systemic gene silencing is one key enabling aspect. The objective of this research was to better understand topically-induced, systemic transgene silencing in Nicotiana benthamiana. A previous report details sequencing of the integration site of the Green Fluorescent Protein (GFP) transgene in the well-known N. benthamiana GFP16C event. This investigation revealed an inadvertent co-integration of part of a bacterial transposase in this line. To determine the effect of this transgene configuration on systemic silencing, new GFP transgenic lines with or without the transposase sequences were produced. GFP expression levels in the 19 single-copy events and three hemizygous GFP16C lines produced for this study ranged from 50-72% of the homozygous GFP16C line. GFP expression was equivalent to GFP16C in a two-copy event. Local GFP silencing was observed in all transgenic and GFP16C hemizygous lines after topical application of carbon dot-based formulations containing a GFP targeting dsRNA. The GFP16C-like systemic silencing phenotype was only observed in the two-copy line. The partial transposase had no impact on transgene expression level, local GFP silencing, small RNA abundance and distribution, or systemic GFP silencing in the transgenic lines. We conclude that high transgene expression level is a key enabler of topically-induced, systemic transgene silencing in N. benthamiana.

Barriers to Efficient Foliar Uptake of dsRNA and Molecular Barriers to dsRNA Activity in Plant Cells.

Foliar application of dsRNA to elicit an RNA interference (RNAi) response is currently under consideration as a crop protection strategy. To access the RNAi machinery of a plant, foliarly applied dsRNAs must traverse the plant cuticle, avoid nuclease degradation, and penetrate the cell wall and plasma membrane. Application methods and co-formulants have been identified by Bayer Crop Science researchers and others that can help bypass barriers to dsRNA uptake in plants leading to an RNAi response in greenhouse grown, young plants and cell cultures. However, these advances in dsRNA delivery have yet to yield systemic RNAi silencing of an endogenous gene target required for product concepts such as weed control. Systemic RNAi silencing in plants has only been observed with the GFP transgene in Nicotiana benthamiana. Because biologically meaningful whole plant RNAi has not been observed for endogenous gene products in N. benthamiana or in other plant species tested, under growing conditions including field production, the regulatory risk assessment of foliarly applied dsRNA-based products should not consider exposure scenarios that include systemic response to small RNAs in treated plants.

Estimation of the nuclear DNA content of gossypium species.

BACKGROUND AND AIMS: Gossypium is an economically important, globally distributed taxon comprising more than 50 species. DNA content estimates from about half of the species indicate over a 3-fold variation exists. However, the nine DNA content estimates for G. hirsutum reveal over a 2-fold difference for this species alone. Recent reports have shown that several plant compounds can bias DNA content estimates obtained by commonly used methods. The purpose of this research was to examine the standardization procedures used for DNA content determinations with flow cytometry as applied to Gossypium, and generate revised DNA content estimates for all available Gossypium species using best-standard practices. METHODS: Flow cytometry was used to measure fluorescence of isolated Gossypium nuclei stained with propidium iodide. Fluorescence values were converted to DNA content estimates based on the nuclear fluorescence of standard genotypes of barley, corn and rice. Various combinations of nuclei preparations relative to the standards were evaluated for their influence on the estimates. KEY RESULTS: Both external standardization and internal standardization with Oryza sativa 'IR36' yielded statistically similar DNA content estimates for Gossypium. Internal standardization with Hordeum vulgare 'Sultan' resulted in a high estimate of DNA content. Nuclear DNA content estimates were generated for 37 Gossypium species using external standardization. Estimates of ancestral genome sizes reveal that both increases and decreases in nuclear DNA content have occurred. Variation in intraspecific and intragenomic DNA content was low, and the allopolyploid AD-genome size was nearly the additive of its progenitor genomes. CONCLUSIONS: Due to unknown factors, internal standardization with H. vulgare 'Sultan' may not be appropriate for DNA content determinations of Gossypium. The current DNA content estimates support accepted cytogenetic divisions of the genus. Gossypium is a genus that exhibits genome constancy both through speciation within genomic groups and allopolyploidization.

DNA content and expression of genes related to cell cycling in developing Gossypium hirsutum (Malvaceae) fibers.

The cell cycle in cotton (Gossypium hirsutum) fibers is poorly understood. The objective of this study was to evaluate the cell cycle status and DNA content in developing cotton fibers. The DNA content and cell cycle distribution in fiber and hypocotyl cells were determined by flow cytometry. Expression levels of minichrosomal maintenance protein (mcm), cyclin B, and a retinoblastoma-like protein (rb) genes were determined with real-time PCR in fibers and dividing and nondividing tissues. No endoreduplication occurred, nor did genome size or percentage of G1-phase nuclei differ between hypocotyls and fibers. Approximately 13 and 17% of fiber nuclei were in the S phase in 14 days after anthesis (d) fibers and 25 d fibers, respectively. The mcm and cyclin B were expressed at higher levels in fibers than in mature leaves, but expression levels in fibers were less than 15% of meristematic tissues. Rb was expressed in fibers at levels less than 50% of mature leaves or meristematic tissues. Based on an apparent increase in S-phase cells as fibers mature and the low level of expression of genes associated with cell cycle progression, we conclude that S-phase arrest occurs in developing cotton fiber.