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 center dot 2 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.