Abstract
BackgroundPost transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology.ResultsWe employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b).ConclusionsWhile prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.
Highlights
Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. small interfering RNA (siRNA) contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation
We demonstrate that conjugated polymer nanoparticles (CPN) can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b)
CPNs are taken up by BY-2 protoplast but not by intact cells BY-2 protoplasts were incubated with various concentrations of CPNs (5, 10, 15, and 25 μM) for either 2 or 24 h followed by counting cells to determine the proportion of green fluorescing cells under the microscope (n = 400)
Summary
Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. SiRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Endoribonuclease-containing complexes, known as RNA-induced silencing complexes (RISCs), incorporate the miRNAs and siRNAs which act to guide the RISCs to homologous cellular mRNAs, targeting them for degradation [8,9,10]. Because most viruses are limited in their host range, additional viral vectors are being developed for small RNA delivery to diverse plant species. Another drawback of viral vectors is that they do not uniformly infect all tissues, they might spread systemically. The phenotype attributed to PTGS is mixed with the onset of virus symptoms which include mosaic pattern of disease and mild leaf curling
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