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Tools & Techniques: dsRNA Delivery; miRNA Construction, Prediction in Plants


NEW YORK (GenomeWeb) – Further expanding the RNAi and microRNA toolkits for plant research, groups from institutes in Japan, China, and the US this month published details about new approaches for the delivery and detection of these gene-silencing molecules.

dsRNA delivery

In the first report, a team of Japanese scientists reported a new method to complex synthetic dsRNA with a carrier peptide capable of triggering transient and localized RNAi in specific tissues and organs.

According to the group, this delivery system can potentially be used with any plant or gene without the need for specialized equipment or protoplast preparations. Additionally, it is specific enough to induce target downregulation in a single floral bud or an immature leaf, which avoids plant lethality and enables the functional analysis of essential genes.

The dsRNA delivery approach stems from previous efforts by the group to develop plasmid DNA delivery system that incorporated carrier peptides containing a few functional domains as new gene carriers for plant and animal cells, the team wrote this month in Plant Biotechnology Journal.

Following successful experiments with that system in Arabidopsis thaliana and Nicotiana benthamiana leaves, the investigators turned their attention to dsRNA delivery, selecting a fusion peptide that combined a copolymer of histidine and lysine with a cell-penetrating peptide dubbed Bp100.

Bp100 was originally designed as an antimicrobial peptide against plant pathogens, as well as to destabilize cell walls and plasma membranes. Meanwhile, the histidine/lysine polycation is known to interact with the negatively charged dsRNA via ionic interactions to form complexes.

To demonstrate their system, the scientists showed it could silence both exogenous and endogenous genes — yellow fluorescent protein (YFP) in a transgenic Arabidopsis plant expressing the reporter and the chalcone synthase gene, which is responsible for the anthocyanin biosynthesis pathway — in single plant leaves. To prove the system's utility with practical plants, the leaves of a transgenic poplar expressing YFP was treated with the complex to silence the gene's expression.

Target gene knockdown was observed within nine hours of treatment.

Although infiltration with the peptide-dsRNA complexes was achieved using a syringe, the researchers noted in the paper that it is possible to introduce the RNAi molecules into a wide range of large leaves by immersing the plants in a solution containing the complexes under vacuum conditions. To extend the duration of gene knockdown, multiple infiltrations may be conducted, they added.

miRNA construction

Focusing on another route to plant gene silencing, a group of Chinese investigators has developed a novel method for the construction of artificial miRNAs in plants.

Previously, the creation of synthetic miRNAs is based on an miRNA precursor and requires three pairs of primers and four systems of PCR reactions, the scientists wrote in Biotechnology Letters. New methods for miRNA vector construction have been reported, but are time-consuming and have a high inefficiency of recombination.

Most of these existing methods also involve the modification of flanking stem sequences of endogenous precursors to create restriction enzyme sites for entering miRNA sequences. Although previous reports suggest that the flanking sequences are vital to endogenous miRNA production, the researchers wrote in the paper, "We contend that it may not be optimal to mutate [artificial] miRNA endogenous precursors."

For their approach, the researchers took advantage of the Golden Gate cloning method, which uses a type II restriction enzyme that cleaves DNA outside of its recognition sequence, and showed that it could generate artificial miRNAs in one restricting-ligation step in five minutes.

The resultant miRNAs were then expressed in tobacco and rice, and silenced their targets as expected.

miRNA prediction

To overcome many of the problems with existing miRNA prediction tools, two researchers from Michigan State University have developed software that uses expression patterns of miRNA and the criteria for plant miRNA annotation to accurately predict the non-coding RNAs from one or more small RNA-seq data samples of the same species.

Although there are other methods for plant miRNA annotation based on RNA-seq data, these typically have high rates of false positives, long run times, and can be difficult to install and use, the team wrote in Bioinformatics. Many of these also only work for genomes already in their databases, hampering the analysis of new genomes.

To that end, the two created miR-Prefer (miRNA Prediction from Small RNA-Seq Data), which they said requires minimal informatics expertise, having low dependency on other programs, does not require a user to compile or install a pipeline, and includes a checkpoint feature that allows an unfinished job to be stopped and resumed later.

When tested against other next-generation-based miRNA prediction tools using two datasets from Arabidopsis, miR-Prefer proved highly sensitive and had a low false-positive rate. Additionally, it was significantly speedier and used less data than competing tools.