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Immunolocalization of cyclotides in plant cells, tissues and organ supports their role in host defense
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy. Polish Acad Sci, W Szafer Inst Bot, 46 Lubicz St, PL-31512 Krakow, Poland..
Univ Gdansk, Fac Biol, Dept Plant Cytol & Embryol, 59 Wita Stwosza St, PL-80308 Gdansk, Poland..
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Univ Gdansk, Fac Biol, Dept Plant Cytol & Embryol, 59 Wita Stwosza St, PL-80308 Gdansk, Poland..
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2016 (English)In: Planta, ISSN 0032-0935, E-ISSN 1432-2048, Vol. 244, no 5, 1029-1040 p.Article in journal (Refereed) Published
Abstract [en]

Main conclusionThe distribution of cyclotides was visualized in plant cells, tissues and organs using immunohistochemistry. Finding of cyclotides in tissues potentially vulnerable to pathogen attacks supports their role as defense molecules. The cyclotide family of plant peptides is characterized by the cyclic cystine knot motif and its diverse biological activities. Given their insecticidal and antimicrobial properties, the role of cyclotides in planta is probably associated with host defense. Our current understanding of the cellular compartmentalization of cyclotides in the vacuole is based on indirect studies on transgenic model plants that do not express cyclotides naturally. Matrix-assisted laser desorption ionization (MALDI) imaging has also been used to study the distribution of cyclotides, but the technique's resolution was insufficient to determine their tissue or cell distribution. To avoid the limitations of these approaches, immunohistochemical visualization methods were used. Antibodies were raised in rabbits using cycloviolacin O2 (cyO2), and their specificity was determined by Western and dot blot experiments. Slides for immunohistochemical analysis were prepared from leaf, petiole and root fragments of Viola odorata and Viola uliginosa, and specimens were visualized using indirect epifluorescence microscopy. The antibodies against cyclotides were specific against selected bracelet cyclotides with high similarity (cyO2, cyO3, cyO8, cyO13) and suitable for immunohistochemistry. The tissue distribution of the cyclotides visualized in this way is consistent with their proposed role in host defense-relatively large quantities were observed in the leaf and petiole epidermis in both Viola species. Cyclotides were also found in vascular tissue in all the assessed plant organs. The vacuole storage of cyclotides was directly shown.

Place, publisher, year, edition, pages
2016. Vol. 244, no 5, 1029-1040 p.
Keyword [en]
Cyclotides, Immunohistochemistry, Host defense peptides, Viola
National Category
Medicinal Chemistry Botany
Identifiers
URN: urn:nbn:se:uu:diva-307522DOI: 10.1007/s00425-016-2562-yISI: 000385251200004PubMedID: 27394154OAI: oai:DiVA.org:uu-307522DiVA: diva2:1047540
Funder
Swedish Research Council, 621-2007-5167Swedish Foundation for Strategic Research , F06-0058
Available from: 2016-11-17 Created: 2016-11-17 Last updated: 2017-04-13Bibliographically approved
In thesis
1. Interaction of cyclotides and bacteria: A study of the cyclotide action and the bacterial reaction
Open this publication in new window or tab >>Interaction of cyclotides and bacteria: A study of the cyclotide action and the bacterial reaction
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The growing problem of antibiotic resistance and the lack of promising prospective antibiotics have forced us to search for new classes of antibiotics. Among the candidates to develop into future antibacterials are antimicrobial peptides (AMPs). These

potent, broad spectrum compounds are important components of innate immunity of organism from all kingdoms of life. One such family of mini-proteins from plants is called cyclotides, whose members are defines by cyclic backbone and a cystine knot (CCK), which confers to them extreme stability in the face of biological, chemical and physical insults.

    Some cyclotides possess Gram-negative specific antibacterial activity; the purpose of this thesis was to characterize how these molecules kill bacteria, and how bacteria would respond to treatment with cyclotides. For this purpose, Salmonella enterica and Escherichia coli mutants resistant to the cyclotides cycloviolacin O2 and cycloviolacin O19, respectively, were selected. These mutants were characterized by whole genome sequencing, genetic reconstitution, fitness measurements, and cross-resistance studies. These studies identified a number of genetic pathways for resistance development to cyclotides. These mutants displayed variable fitness profiles in laboratory growth media and in mice competition experiments, with some mutants possessing a fitness advantage in mice. Cross-resistance studies resulted in the identification of several cases of cross-resistance and collateral sensitivity between cyclotides and other AMPs/antibiotics.

     Antimicrobial effects of cyclotides were assayed in different conditions and in bacterial organisms with different surface characteristics. In addition, immunolocalization experiments were performed to explore the biological distribution of cyclotides in plants and to determine the mechanism of action of cyclotides in bacteria, respectively. Antibodies raised against cyO2 were used for this purpose. Immunohistochemical techniques applied to plant cells, tissues and organs provided the information that cyclotides were distributed in all plant organs, and were found in tissues vulnerable to pathogen attack, and that cyclotides were stored in the vacuoles of plant cells. Immunogold staining of cyclotide treated cells of S. typhimurium, showed effects of cyclotide treatment on the cell envelope components as well as cytoplasm. A higher number of cyclotide molecules was associated with the cell envelope, but a considerable fraction of them penetrated into the cytoplasm.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 229
Keyword
Cyclotides, cycloviolacin O2, cycloviolacin O3, cycloviolacin O19, antimicrobial peptide resistance, Salmonella enterica, Eschericia coli, Viola odorata
National Category
Medical and Health Sciences
Research subject
Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-318656 (URN)978-91-554-9870-2 (ISBN)
Public defence
2017-05-15, B21, Uppsala Biomedical Centre, Husarg. 3, Uppsala, 09:15 (English)
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Supervisors
Available from: 2017-04-24 Created: 2017-03-27 Last updated: 2017-05-10

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