Cellular localization of Peach latent mosaic viroid in peach sections by liquid phase in situ RT‐PCR

Abstract

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 0AE2 mL 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.

Protocol: a fast and simple in situ PCR method for localising gene expression in plant tissue

“…The use of fluorescence also has additional complications compared to the more reliable and straightforward colourimetric detection (such as cost of fluorescent substrates or primers, stability of fluorescent compounds and the cost and access to fluorescence/confocal microscopes). Pesquet et al [ 18 ] used fluorescence to detect in situ PCR products in plant tissues, however the protocol was not outlined and the technique has not been used very often; problems with autofluorescence interference when using fluorescent detection of in situ PCR products have since been noted [ 37 ].…”

An Inside Look into Biological Miniatures: Molecular Mechanisms of Viroids

“…Among the various plant pathogens, viroids are the pathogenic organisms detected for the first-time using RT-PCR [ 168 ]. Subsequently, various amplification tools such as real-time RT-PCR [ 169 ], RT-PCR- Enzyme-Linked Immuno Sorbent Assay (ELISA) [ 170 ], (RT-LAMP) [ 171 ], multiplex bead-based array [ 172 ], multiplex RT-PCR [ 173 , 174 ] and in situ RT-PCR have been applied for the detection and identification of viroids [ 175 ].…”

“…Results for this method can be observed through gel electrophoresis or by fluorescent [ 181 ] and standard or multiplex RT-PCR-ELISA [ 170 , 182 ]. Boubourakas et al 2011 [ 175 ] adopted a modified RT-PCR technique using SYBR Green for in situ localization and detection of PLMVd.…”