Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores

Abstract

Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals, in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat, Tat(2), M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat(2) (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores, respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31% of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat(2)-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal laser microscopy was carried out to recommend a ratio of 4:1 Tat(2)-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat(2) can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing simple, time saving, cost effective plant genetic engineering technologies.

Cell‐penetrating peptides: Nanocarrier for macromolecule delivery in living cells

“…The second step involves macropinocytosis for uptake of CPP-cargo complex, and the final step is dependent on parameters such as pH causing disruption of micropinosome resulting in release of the CPP-cargo complex into the cytosol. Our studies on Tat-cargo complex in plant cells do not rule out possibility of simultaneous involvement of various mechanisms for cellular internalization (3,62). This observation is further corroborated by the studies carried by research groups working with mammalian cell lines (64,95).…”

“…Our group has been interest in doubled haploids of man-made cereal triticale because of their applicability in various areas of genetic engineering. Our group is the first one to study CPP-mediated protein transduction and nucleic acid transfection in plant microspores (62). As described in detail later, our results demonstrate notable amenability of microspores toward CPPs and their cargo complex uptake during the transduction and transfection studies.…”

“…In plant system, cytotoxic effects were not observed with CPPs alone or in complex with their cargoes, at the concentration investigated in somatic embryos or microspores (3,62). Viability of the microspores immediately after treatment with CPPs or their cargoes was also similar to the control microspores (65-70%).…”

A molecular method for the delivery of small molecules and proteins across the cell wall of algae using molecular transporters

“…Despite this progress with mammalian cells, little is known about the ability of GR-MoTrs to enter nonmammalian cells, especially those organisms of research and commercial significance that possess a cell wall. Only a few studies of GR-MoTrs with plant cells have been reported (18)(19)(20)(21)(22) and, to our knowledge, there is only a single investigation of GR-MoTrs with algae (23). In the latter study, Chlorella vulgaris, a species of green algae with a cellulosic cell wall, was found to be impermeable to a GFP-nona-(L)-arginine fusion protein.…”

Haploids and doubled haploids in Brassica spp. for genetic and genomic research

“…Transformation efficiency was based on the number of GUSpositive cells. A recent publication on a novel transformation system in triticale involved the use of cell-penetrating peptides (Chugh et al 2009), which have the capability to translocate across cell membranes, and have been used in mammalian systems to move molecules into the cell. It is thought that peptide uptake occurs through pores in the cell membrane.…”