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Plant Pathology
Boubourakas, I.N., Laboratory of Plant Pathology, Department of Crop Science, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
Voloudakis, A.E., Laboratory of Plant Breeding and Biometry, Department of Crop Science, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
Fasseas, K., Laboratory of Electron Microscopy, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
Resnick, N., Agricultural Research Organization, Institute of Plant Science, Department of Ornamental Horticulture, Bet Dagan 50250, Israel
Koltai, H., Agricultural Research Organization, Institute of Plant Science, Department of Ornamental Horticulture, Bet Dagan 50250, Israel
Kyriakopoulou, P.E., Laboratory of Plant Pathology, Department of Crop Science, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
An in situ localization method for Peach latent mosaic viroid (PLMVd) that is highly sensitive, has low background signal, has a short specimen processing time and is simple and inexpensive compared to other similar methods, is described. The method is based on SYBR Green reverse transcription-polymerase chain reaction (RT-PCR) amplification of pepsin, DNase I pre-treated and FAA-fixed peach leaf sections. The leaves used were derived from healthy and PLMVd infected peach plants, including a plant infected by a Peach Calico variant of the viroid. All steps of the assay, except for the signal detection, were carried out in liquid phase in 0·2mL PCR tubes instead of on microscope slides, as usually used. Epifluorescence microscopy to detect PLMVd or rbcL (the positive control gene) amplified products revealed a bright signal in the leaf part corresponding to the palisade parenchyma, with sub-cellular localization of the signals in the chloroplasts, the organelles where PLMVd is known to replicate and accumulate. Although the method proved to be effective for the green peach PLMVd infected tissues, a yellow-green background fluorescent signal, sometimes more intense due to overexposure, was observed, presumably due to chlorophyll auto-fluorescence. In contrast, no auto-fluorescence signal was observed in the calico infected albino tissues. This is the first report of using liquid phase in situ RT-PCR for the cellular localization of a plant pathogen. © 2010 The Authors. Plant Pathology © 2010 BSPP.
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Cellular localization of Peach latent mosaic viroid in peach sections by liquid phase in situ RT-PCR
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Boubourakas, I.N., Laboratory of Plant Pathology, Department of Crop Science, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
Voloudakis, A.E., Laboratory of Plant Breeding and Biometry, Department of Crop Science, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
Fasseas, K., Laboratory of Electron Microscopy, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
Resnick, N., Agricultural Research Organization, Institute of Plant Science, Department of Ornamental Horticulture, Bet Dagan 50250, Israel
Koltai, H., Agricultural Research Organization, Institute of Plant Science, Department of Ornamental Horticulture, Bet Dagan 50250, Israel
Kyriakopoulou, P.E., Laboratory of Plant Pathology, Department of Crop Science, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
Cellular localization of Peach latent mosaic viroid in peach sections by liquid phase in situ RT-PCR
An in situ localization method for Peach latent mosaic viroid (PLMVd) that is highly sensitive, has low background signal, has a short specimen processing time and is simple and inexpensive compared to other similar methods, is described. The method is based on SYBR Green reverse transcription-polymerase chain reaction (RT-PCR) amplification of pepsin, DNase I pre-treated and FAA-fixed peach leaf sections. The leaves used were derived from healthy and PLMVd infected peach plants, including a plant infected by a Peach Calico variant of the viroid. All steps of the assay, except for the signal detection, were carried out in liquid phase in 0·2mL PCR tubes instead of on microscope slides, as usually used. Epifluorescence microscopy to detect PLMVd or rbcL (the positive control gene) amplified products revealed a bright signal in the leaf part corresponding to the palisade parenchyma, with sub-cellular localization of the signals in the chloroplasts, the organelles where PLMVd is known to replicate and accumulate. Although the method proved to be effective for the green peach PLMVd infected tissues, a yellow-green background fluorescent signal, sometimes more intense due to overexposure, was observed, presumably due to chlorophyll auto-fluorescence. In contrast, no auto-fluorescence signal was observed in the calico infected albino tissues. This is the first report of using liquid phase in situ RT-PCR for the cellular localization of a plant pathogen. © 2010 The Authors. Plant Pathology © 2010 BSPP.
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