Applications of CPPs in Genome Editing of Plants.

Cell penetrating peptides (CPPs) are short peptides that are able to translocate themselves and their cargo into cells. The progressive and continuous application of CPPs in various fields of basic and applied research shows that they are efficient delivery vectors for an assortment of biomolecules, including nucleic acids and proteins. This feature makes CPPs an excellent tool for modification of plant genomes through transgenesis and genome editing. In this review, we present the progress during the last three decades in application of CPPs for delivery of DNA, RNA, and proteins into plant cells and tissues. Moreover, we highlight the exploiting of CPPs as advantageous and beneficial tool for plant genome editing via delivery of nuclease proteins, and provide a practical example of genome alternation through CPP-delivered nucleases. Finally, the current exploitation of peptides in organelle-specific DNA delivery and modification of organellar genomes is discussed.

Tetrabutylphosphonium Bromide Reduces Size and Polydispersity Index of Tat2:siRNA Nano-Complexes for Triticale RNAi.

Cell-penetrating peptides (CPPs) are short 8-30 amino-acid oligopeptides that act as effective transducers of macromolecular cargo, particularly nucleic acids. They have been implemented in delivering dsDNA, ssDNA, and dsRNA into animal and plant cells. CPPs and nucleic acids form nano-complexes that are often 100-300 nm in size but still effectively transit the cell membrane of animal cells, but are less effective with plant cells due to the plant cell wall. To overcome this obstacle, nano-complexes of the CPP Tat2 and various lengths of nucleic acid (21-mer siRNA duplex (dsRNA) to ~5.5 kb circular plasmid) were evaluated for size using dynamic light scattering (DLS), under conditions of increasing ionic strength (Ic) and addition of phase transfer catalyst salts (tetrabutylammonium bromide-TBAB and tetrabutylphosphonium bromide-TBPB) and sugars (maltose-mannitol solution). It was found that the combination of 21-mer siRNA:Tat2 complexes with TBPB produced small 10-20 nm diameter nano-complexes with a polydispersity index (PDI) of ~0.1. Furthermore, it was found that for each length of nucleic acid that a linear mathematical relationship existed between the theoretical volume of the nano-complex and the nucleic acid length. Next, nano-complex formulation was tested for its ability to carry small interfering RNA molecules into plant cells and to trigger silencing of phytoene desaturase (PDS) in Triticale leaves. RT-qPCR showed 75% suppression of PDS, demonstrating that TBPB acts as an adjuvant in effecting the entry and efficacy of siRNA in young Triticale plants.

Applications of CPPs in Genome Modulation of Plants.

Cell-penetrating peptides (CPPs) are a class of short peptides that are known to translocate inside living cells through the cell membrane. Many CPPs show an ability to bind and deliver macromolecular cargoes such as DNA, RNA and protein into living cells, making them excellent transfection and transduction agents with low cytotoxicity. While their use is well established in mammalian cell systems, they have also been explored in the last decade as transfection agents in plant cells. Their efficacy has been demonstrated in both monocot and dicot clades as well as a variety of tissues and cell cultures, from leaves to protoplasts. Factors affecting CPP and CPP-cargo uptake have been addressed with specific attention to the plant cell wall and classes of CPPs utilized in plant cell systems. It has been shown that internalization of most free peptides in plant cells has been dominated by direct translocation across the cell membrane, while CPP-macromolecular cargo complexes and conjugates were translocated via macropinocytosis. Moreover, functionalization of CPPs resulted in generation of peptides with specialized cargo delivery attributes, e.g., for specific subcellular targeting. Thus, the use of CPPs in plants presents a promising method for plant transgenesis as well as genome regulation and modification.

A novel method of transgene delivery into triticale plants using the Agrobacterium transferred DNA-derived nano-complex.

Genetic transformation of monocotyledonous plants still presents a challenge for plant biologists and biotechnologists because monocots are difficult to transform with Agrobacterium tumefaciens, whereas other transgenesis methods, such as gold particle-mediated transformation, result in poor transgene expression because of integration of truncated DNA molecules. We developed a method of transgene delivery into monocots. This method relies on the use of an in vitro-prepared nano-complex consisting of transferred DNA, virulence protein D2, and recombination protein A delivered to triticale microspores with the help of a Tat2 cell-penetrating peptide. We showed that this approach allowed for single transgene copy integration events and prevented degradation of delivered DNA, thus leading to the integration of intact copies of the transgene into the genome of triticale plants. This resulted in transgene expression in all transgenic plants regenerated from microspores transfected with the full transferred DNA/protein complex. This approach can easily substitute the bombardment technique currently used for monocots and will be highly valuable for plant biology and biotechnology.

The stimulatory effect of CaCl(2), NaCl and NH(4)NO(3) salts on the ssDNA-binding activity of RecA depends on nucleotide cofactor and buffer pH.

The single-stranded DNA binding activity of the Escherichia coli RecA protein is crucial for homologous recombination to occur. This and other biochemical activities of ssDNA binding proteins may be affected by various factors. In this study, we analyzed the effect of CaCl(2), NaCl and NH(4)NO(3) salts in combination with the pH and nucleotide cofactor effect on the ssDNA-binding activity of RecA. The studies revealed that, in addition to the inhibitory effect, these salts exert also a stimulatory effect on RecA. These effects occur only under very strict conditions, and the presence or absence and the type of nucleotide cofactor play here a major role. It was observed that in contrast to ATP, ATPγS prevented the inhibitory effect of NaCl and NH(4)NO(3), even at very high salt concentration. These results indicate that ATPγS most likely stabilizes the structure of RecA required for DNA binding, making it resistant to high salt concentrations.

Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation.

BACKGROUND: PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome. SCOPE: This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution. CONCLUSIONS: Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.

Plant transgenesis.

Epigenetic effects such as gene silencing and variable expression are unintended consequences of plant transformation, a problem that is present in the transformation of all plant species. There is not yet a reliable way to prevent epigenetic silencing; however, the probability of epigenetic effects may be reduced by choosing an appropriate method of transgene introduction into a plant cell. Most methods used in plant biotechnology, such as direct gene transfer and particle bombardment, result in the introduction of multiple DNA molecules and, as a consequence, multi-copy multi-locus insertion patterns. These multiple insertions may lead to variations in transgene expression, epigenetic silencing being the most extreme. In contrast, Agrobacterium-mediated plant transformation procedures rarely cause such unintended effects. In this chapter, we present advantages and disadvantages of the Agrobacterium-mediated plant transformation method as well as protocols for transformation of Arabidopsis generative tissues and tobacco seedlings as the most classical techniques in these model plants, i.e., vacuum infiltration of explants and floral dip methods. Moreover, epigenetic effects of transgenes such as silencing related to the position and insertion effects as well as effects of the regeneration procedure causing somaclonal variation will be briefly discussed.

Identification and functional analysis of PCNA1 and PCNA-like1 genes of Phaseolus coccineus.

Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication and in many other processes in eukaryotic cells. Genetic analysis of Phaseolus coccineus showed the presence of at least two PCNA-like genes in the runner bean genome. Two PCNA genes have previously been found in a few plant species including Arabidopsis, tobacco, and maize. In these species, genes were nearly identical. Two cDNAs of P. coccineus PCNA (PcPCNA1 and PcPCNA-like1) have been identified that differ distinctly from each other. Interestingly, both the genetic organization of PcPCNA1 and PcPCNA-like1 genes and their expression patterns were similar, but these were the only similarities between these genes and their products. The identity between PcPCNA1 and PcPCNA-like1 at the amino acid level was only 54%, with PcPCNA-like1 lacking motifs that are crucial for the activity typical of PCNA. Consequently, these two proteins showed different properties. PcPCNA1 behaved like a typical PCNA protein: it formed a homotrimer and stimulated the activity of human DNA polymerase delta. In addition, PcPCNA1 interacted with a p21 peptide and was recognized by an anti-human PCNA monoclonal antibody PC10. By contrast, PcPCNA-like1 was detected as a monomer and was unable to stimulate the DNA polymerase delta activity. PcPCNA-like1 also could not interact with p21 and was not recognized by the PC10 antibody. Our results suggest that PcPCNA-like1 either is unable to function alone and therefore might be a component of the heterotrimeric PCNA ring or may have other, yet unknown functions. Alternatively, the PcPCNA-like1 gene may represent a pseudogene.

Molecular cloning of Phaseolus vulgaris cDNA encoding proliferating cell nuclear antigen.

A cDNA fragment encoding a common bean (Phaseolus vulgaris) proliferating cell nuclear antigen (PCNA) was isolated using rapid amplification of cDNA 3' end (3' RACE) method, cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 798 nucleotides encoding a protein of 265 amino acids. Alignment of the common bean PCNA predicted sequence shows its high degree of identity with PCNA from other plant species. Analysis of PCNA content in the germinating embryos of common bean showed a decrease in the protein level after 60h of germination. Moreover, PCNA was not detected in the tested plant organs (root, stem, leaf and flower). The presence of PCNA in the germinating seeds and its absence from mature plants suggests that this protein plays a crucial role during early stages of plant development.

Identification of Arabidopsis rat mutants.

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7.

We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1). Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals. The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals. We show that AtTRN1 binds to similar domains in hnRNP-like proteins from plants. AtTRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms. Like all nuclear transport receptors of the importin-beta family, AtTRN1 binds to the regulatory GTPase Ran from Arabidopsis. We demonstrated that the amino terminus of AtTRN1 is necessary for this interaction. Recombinant AtTRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays. We identified three plant substrate proteins that interact with AtTRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis, AtGRP7 and AtGRP8. Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal. In addition, fluorescently labelled AtGRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis AtTRN1 and human TRN1. These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants.