Apple latent spherical virus (ALSV) vectors have been shown to effectively induce stable virus-induced gene silencing (VIGS) in a wide range of plant species including Rosaceous fruit tree species, such as apple and pear. In this study, we attempted to develop a VIGS-based gene evaluation system for fruit tree species in Prunus, using the ALSV viral vector system. Partial sequence of PHYTOENE DESATURASE (PDS) of apricot (Prunus armeniaca) was cloned and ligated into the T-DNA region of a binary vector pBICAL2. The T-DNA region of pBICAL2, designed based on the RNA2 of ALSV, contains a single ORF for the ALSV polyprotein under the control of CaMV35S promoter sequence. The partial ParPDS sequence was ligated in frame with the coding sequences for the movement protein and the capsid protein Vp25 flanking the cloning site. The resultant pBICAL2-ParPDS was introduced into a disarmed Agrobacterium strain EHA105. The pBICAL1, a binary plasmid for the expression of RNA1 of ALSV in plants, was also introduced into EHA105. To amplify and produce recombinant ALSV particles, leaves of Nicotiana benthamiana were infected with pBICAL1/EHA105 and pBICAL2-ParPDS/EHA105 at the same time. The amplified ParPDS-ALSV in N. benthamiana was isolated and infected to the cotyledons of Prunus seeds right after germination by a particle bombardment. Uniform discoloration of the upper leaves, a typical knock-down phenotype of PDS, was observed several weeks after inoculation in a certain range of fruit tree species in Prunus.
