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

  • Genetic Transformation and Hybridization
  • Published:
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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, Tat2, M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat2 (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 Tat2-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 Tat2-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat2 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.

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Abbreviations

CPP:

Cell-penetrating peptide

EIPA:

5-(N-ethyl-N-isopropyl)-amiloride

FITC:

Fluoro-iso-thiocyanate

pVEC:

Peptide vascular endothelial-cadherin

RT:

Room temperature

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Acknowledgments

The authors acknowledge Alberta Peptide Institute (API), Canada, for custom synthesis of the peptides. We thank Dr. Fran Leggett (Lethbridge Research Centre) for confocal laser imaging and size determination of the peptide–DNA complex. Support provided by Doug Bray (University of Lethbridge) for the use of confocal microscope facility at the Canadian Centre for Behavioural Neuroscience (CCBN, University of Lethbridge) is duly acknowledged. We thank Dr Ray Wu (Cornell University, USA) for plasmid pAct-1GUS. AC acknowledges Natural Science and Engineering Research Council of Canada (NSERC) for the award of Visiting Fellowship. Authors are grateful to the Matching Investment Initiative (MII) program and Alberta Agriculture Research Institute (AARI) for providing financial support for the study.

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  1. Agriculture and Agri-Food Canada, Lethbridge Research Centre, 5403-1st Avenue South, P.O. Box 3000, Lethbridge, AB, T1J 4B1, Canada

    Archana Chugh, Eric Amundsen & François Eudes

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  1. Archana Chugh
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  2. Eric Amundsen
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  3. François Eudes
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Correspondence to François Eudes.

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Communicated by H. Ebinuma.

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Chugh, A., Amundsen, E. & Eudes, F. Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores. Plant Cell Rep 28, 801–810 (2009). https://doi.org/10.1007/s00299-009-0692-4

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  • DOI: https://doi.org/10.1007/s00299-009-0692-4

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