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Cellular Location of Prune dwarf virus in Almond Sections by In Situ Reverse Transcription-Polymerase Chain Reaction

First, second, and fourth authors: Instituto de Biologia Experimental e Tecnológica / Instituto de Tecnologia Química e Biológica, Quinta do Marquês, 2784-505 Oeiras, Portugal; third author: Unidade de Ciências e Tecnologias Agrárias da Universidade do Algarve, Campus de Gambelas, 8000 Faro, Portugal; and fourth author: Dep. Biologia Vegetal, Fac. Ciências de Lisboa, Campo Grande 1749-016 Lisboa, Portugal

In situ reverse transcription-polymerase chain reaction (RT-PCR) was used in young leaves (from trees and in vitro shoots) and flower buds of almond (Prunus dulcis), a stone fruit, for cellular location of Prune dwarf virus (PDV, a member of the genus Ilarvirus). Sections obtained from samples fixed in formaldehyde and embedded in paraffin were refixed in formaldehyde to increase tissue preservation in the RT-PCR steps. The coat protein gene of PDV was used as the target to produce a cDNA copy that was amplified by PCR and visualized using a direct detection method with digoxigenin-labeled nucleotides. Protein digestion, PCR, and detection strategies were optimized for increased tissue preservation and signal intensity. PDV was found in infected samples within the vascular tissue of young leaves and flower buds as well as in the mesophyll in developing floral organs and in the generative and vegetative cells of pollen grains. PDV signals were observed in a ring surrounding the nucleus and spread in the cytoplasm. The results obtained are discussed in terms of the technique optimization and PDV distribution in tissues and transmission through pollen. The optimized protocol of in situ RT-PCR is a powerful technique to reveal low-abundant RNA species. Therefore, it is appropriate to study cell and subcellular distribution of RNA viruses in woody species.