Fast-track applications: The potential for direct delivery of proteins and nucleic acids to plant cells for the discovery of gene function
© Roberts; licensee BioMed Central Ltd. 2005
Received: 21 November 2005
Accepted: 15 December 2005
Published: 15 December 2005
In animal systems, several methods exist for the direct delivery of nucleic acids and proteins into cells for functional analysis. Until recently, these methods have not been applied to plant systems. Now, however, several preliminary reports suggest that both nucleic acids and proteins can also be delivered into plant cells by very simple, direct application. This promises to open the way for high-throughput screening for gene function in a range of plant species.
The development of assays that permit high-throughput screening for biological function is an essential goal if we are to fully exploit genome sequence information in plants. Such assays might include over-expression or gene silencing, or the determination of cellular and subcellular localisation of mRNAs and proteins. The majority of techniques that currently exist to perform such assays rely on the production of transgenic plants, or vector-based transient transformation assays. Such methods are necessarily labour intensive and time-consuming, limiting the ability of most researchers to carry out genuine 'functional genomics' projects. However, several recent publications describe systems that permit the direct delivery of nucleic acids and proteins into plant cells in a functional state, providing the potential for rapid functional assays.
Delivery of macromolecules into animal cells
For many years, researchers using animal cell systems have used synthetic nucleic acids to manipulate gene expression. For example, the use of antisense oligodeoxynucleotides to suppress gene expression was first reported over a quarter of a century ago . Single- and double-stranded DNA and RNA molecules can be introduced into mammalian cells by simple direct application to the culture media, or assisted by various transfection reagents, resulting in antisense or siRNA-mediated suppression of gene expression. A range of different modified nucleic acids that bring different characteristics in terms of stability and binding to target sequences are now used, such as morpholinos, locked nucleic acids, peptide nucleic acids, etc. . In many cases these are being developed as potential therapeutic agents .
More recently, proteins and other macromolecules have been delivered into cells by linking them to so-called protein transduction domains (PTDs). These are short peptide sequences that when added to the N-terminus of a recombinant protein, or conjugated to other molecules, can carry those molecules directly into cells (reviewed in ). The best known are found in the HIV-1 transcriptional activator Tat, and the Drosophila transcription factor, Antennapedia . PTDs are generally short, polybasic peptide sequences, and artificial polycationic peptides, such as polyarginine are also effective. Importantly, the uptake of molecules tagged with these peptides does not require specific receptors, endocytosis or active transport. The ability of PTDs to carry molecules across membranes is believed to be the result of the physical characteristics of their interactions with lipid bilayers, suggesting that they should work in any system.
In the past, it has generally been assumed that such delivery systems would not work in plant cells, due to the presence of the cell wall and the difficulty of delivery to multicellular, differentiated tissues. However, work in several laboratories has recently shown that in fact, both proteins and nucleic acids can be efficiently delivered into plant cells in a functional form.
Delivery of macromolecules into plant cells
Peptide transduction domains have also been used to deliver proteins into plant cells. Again, the technique employed was remarkably simple and effective. Chang et al., , produced recombinant GFP proteins in E. coli, either alone or tagged with the Tat PTD or a 9mer polyarginine peptide (R9). When these purified proteins were applied to roots of onion or tomato plants, fluorescence rapidly became visible within the nuclei and cytoplasm of cells treated with Tat-GFP and R9-GFP, but not un-tagged GFP. Uptake of PTD-tagged GFP was detectable within 1 min of application, and was maximal in 5 min. Remarkably, cells throughout the root showed fluorescence – not just those in contact with the protein solution. As in animal systems, uptake was not affected by low temperature or inhibitors of endocytosis. GFP fluorescence was maintained for at least 2 days following a 5 min application, suggesting that PTDs are able to deliver proteins that can remain functional for a significant period of time.
Applications of oligonucleotide and protein delivery into intact tissues.
Antisense gene silencing
Post-transcriptional gene silencing
Peptide nucleic acids
Inhibition of gene expression by chromosomal interactions
Sub-cellular localisation of tagged proteins
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