Oligodeoxynucleotides as probes for in situ hybridization with transmission electron microscopy to specifically localize phytoplasma in plant cells.
Identifieur interne : 003794 ( Main/Exploration ); précédent : 003793; suivant : 003795Oligodeoxynucleotides as probes for in situ hybridization with transmission electron microscopy to specifically localize phytoplasma in plant cells.
Auteurs : J. Lherminier [France] ; R G Bonfiglioli ; X. Daire ; R H Symons ; E. Boudon-PadieuSource :
- Molecular and cellular probes [ 0890-8508 ] ; 1999.
Descripteurs français
- KwdFr :
- ADN bactérien (analyse), ADN des plantes (analyse), ADN ribosomique (analyse), ARN bactérien (génétique), ARN ribosomique 16S (génétique), Biotinylation, Digoxigénine, Hybridation in situ, Immunohistochimie, Inclusion de tissu, Maladies des plantes (microbiologie), Microscopie électronique (), Plantes (microbiologie), Réaction de polymérisation en chaîne, Sondes oligonucléotidiques, Tenericutes (génétique), Tenericutes (isolement et purification).
- MESH :
- analyse : ADN bactérien, ADN des plantes, ADN ribosomique.
- génétique : ARN bactérien, ARN ribosomique 16S, Tenericutes.
- isolement et purification : Tenericutes.
- microbiologie : Maladies des plantes, Plantes.
- Biotinylation, Digoxigénine, Hybridation in situ, Immunohistochimie, Inclusion de tissu, Microscopie électronique, Réaction de polymérisation en chaîne, Sondes oligonucléotidiques.
English descriptors
- KwdEn :
- Biotinylation, DNA, Bacterial (analysis), DNA, Plant (analysis), DNA, Ribosomal (analysis), Digoxigenin, Immunohistochemistry, In Situ Hybridization, Microscopy, Electron (methods), Oligonucleotide Probes, Plant Diseases (microbiology), Plants (microbiology), Polymerase Chain Reaction, RNA, Bacterial (genetics), RNA, Ribosomal, 16S (genetics), Tenericutes (genetics), Tenericutes (isolation & purification), Tissue Embedding.
- MESH :
- chemical , analysis : DNA, Bacterial, DNA, Plant, DNA, Ribosomal.
- chemical , genetics : RNA, Bacterial, RNA, Ribosomal, 16S.
- genetics : Tenericutes.
- isolation & purification : Tenericutes.
- methods : Microscopy, Electron.
- microbiology : Plant Diseases, Plants.
- Biotinylation, Digoxigenin, Immunohistochemistry, In Situ Hybridization, Oligonucleotide Probes, Polymerase Chain Reaction, Tissue Embedding.
Abstract
Phytoplasmas are plant-pathogenic mollicutes restricted to phloem. They belong to several groups in a unique phylogenetic clade. Non-related phytoplasmas may infect the same plant species, often with similar symptoms. Hence methods are needed to specifically localize phytoplasmas and to study their multiplication and movement in their hosts. Conditions for post-embedding in situ hybridization (ISH) with transmission electron microscopy using oligodeoxynucleotides as probes for labelling of phytoplasmas in plant tissues have been searched. Sections of acrylic resin-embedded tissues of phytoplasma-infected periwinkle were submitted to ISH using digoxigenin or biotin-labelled oligoprobes (22 mers). These probes were the complementary sequences of primers used in group-specific polymerase chain reaction (PCR) amplification of 16S rDNA of stolbur and elm yellows phytoplasma, respectively. Together with preliminary digestion with pepsin, different in situ denaturation conditions and formamide concentrations were tested. The grids were incubated in the hybridization mixture at 37 degreesC overnight. Detection of hybridized material was performed with gold immunocytochemistry. Specificity of labelling was checked with appropriate controls. Stringency conditions could be found to ensure specific hybridization with such short probes. A specific labelling was obtained for stolbur phytoplasma on groups of mature as well as senescent phytoplasma cells. The results show that oligonucleotides may be used as probes for phytoplasma identification in post-embedding ISH with electron microscopy.
DOI: 10.1006/mcpr.1998.0213
PubMed: 10024432
Affiliations:
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Le document en format XML
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Boudon-Padieu</name></author></analytic><series><title level="j">Molecular and cellular probes</title><idno type="ISSN">0890-8508</idno><imprint><date when="1999" type="published">1999</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Biotinylation</term><term>DNA, Bacterial (analysis)</term><term>DNA, Plant (analysis)</term><term>DNA, Ribosomal (analysis)</term><term>Digoxigenin</term><term>Immunohistochemistry</term><term>In Situ Hybridization</term><term>Microscopy, Electron (methods)</term><term>Oligonucleotide Probes</term><term>Plant Diseases (microbiology)</term><term>Plants (microbiology)</term><term>Polymerase Chain Reaction</term><term>RNA, Bacterial (genetics)</term><term>RNA, Ribosomal, 16S (genetics)</term><term>Tenericutes (genetics)</term><term>Tenericutes (isolation & purification)</term><term>Tissue Embedding</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN bactérien (analyse)</term><term>ADN des plantes (analyse)</term><term>ADN ribosomique (analyse)</term><term>ARN bactérien (génétique)</term><term>ARN ribosomique 16S (génétique)</term><term>Biotinylation</term><term>Digoxigénine</term><term>Hybridation in situ</term><term>Immunohistochimie</term><term>Inclusion de tissu</term><term>Maladies des plantes (microbiologie)</term><term>Microscopie électronique ()</term><term>Plantes (microbiologie)</term><term>Réaction de polymérisation en chaîne</term><term>Sondes oligonucléotidiques</term><term>Tenericutes (génétique)</term><term>Tenericutes (isolement et purification)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>DNA, Bacterial</term><term>DNA, Plant</term><term>DNA, Ribosomal</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>RNA, Bacterial</term><term>RNA, Ribosomal, 16S</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>ADN bactérien</term><term>ADN des plantes</term><term>ADN ribosomique</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Tenericutes</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ARN bactérien</term><term>ARN ribosomique 16S</term><term>Tenericutes</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>Tenericutes</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Tenericutes</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Microscopy, Electron</term></keywords><keywords scheme="MESH" qualifier="microbiologie" xml:lang="fr"><term>Maladies des plantes</term><term>Plantes</term></keywords><keywords scheme="MESH" qualifier="microbiology" xml:lang="en"><term>Plant Diseases</term><term>Plants</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Biotinylation</term><term>Digoxigenin</term><term>Immunohistochemistry</term><term>In Situ Hybridization</term><term>Oligonucleotide Probes</term><term>Polymerase Chain Reaction</term><term>Tissue Embedding</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Biotinylation</term><term>Digoxigénine</term><term>Hybridation in situ</term><term>Immunohistochimie</term><term>Inclusion de tissu</term><term>Microscopie électronique</term><term>Réaction de polymérisation en chaîne</term><term>Sondes oligonucléotidiques</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Phytoplasmas are plant-pathogenic mollicutes restricted to phloem. They belong to several groups in a unique phylogenetic clade. Non-related phytoplasmas may infect the same plant species, often with similar symptoms. Hence methods are needed to specifically localize phytoplasmas and to study their multiplication and movement in their hosts. Conditions for post-embedding in situ hybridization (ISH) with transmission electron microscopy using oligodeoxynucleotides as probes for labelling of phytoplasmas in plant tissues have been searched. Sections of acrylic resin-embedded tissues of phytoplasma-infected periwinkle were submitted to ISH using digoxigenin or biotin-labelled oligoprobes (22 mers). These probes were the complementary sequences of primers used in group-specific polymerase chain reaction (PCR) amplification of 16S rDNA of stolbur and elm yellows phytoplasma, respectively. Together with preliminary digestion with pepsin, different in situ denaturation conditions and formamide concentrations were tested. The grids were incubated in the hybridization mixture at 37 degreesC overnight. Detection of hybridized material was performed with gold immunocytochemistry. Specificity of labelling was checked with appropriate controls. Stringency conditions could be found to ensure specific hybridization with such short probes. A specific labelling was obtained for stolbur phytoplasma on groups of mature as well as senescent phytoplasma cells. The results show that oligonucleotides may be used as probes for phytoplasma identification in post-embedding ISH with electron microscopy.</div></front></TEI><affiliations><list><country><li>France</li></country><region><li>Bourgogne</li><li>Bourgogne-Franche-Comté</li></region></list><tree><noCountry><name sortKey="Bonfiglioli, R G" sort="Bonfiglioli, R G" uniqKey="Bonfiglioli R" first="R G" last="Bonfiglioli">R G Bonfiglioli</name><name sortKey="Boudon Padieu, E" sort="Boudon Padieu, E" uniqKey="Boudon Padieu E" first="E" last="Boudon-Padieu">E. Boudon-Padieu</name><name sortKey="Daire, X" sort="Daire, X" uniqKey="Daire X" first="X" last="Daire">X. Daire</name><name sortKey="Symons, R H" sort="Symons, R H" uniqKey="Symons R" first="R H" last="Symons">R H Symons</name></noCountry><country name="France"><region name="Bourgogne-Franche-Comté"><name sortKey="Lherminier, J" sort="Lherminier, J" uniqKey="Lherminier J" first="J" last="Lherminier">J. Lherminier</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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