Development of the ALSV-mediated Gene Evaluation System for Prunus

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 Prunus using the ALSV viral vector system. Partial sequences of phytoene desaturase (PDS) and PmDAM6 (Prunus mume Dormancy Associated MADS-box 6 gene) of Japanese apricot (Prunus mume) were cloned and ligated separately 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 double CaMV35S promoter sequences. The partial PmPDS and PmDAM6 sequence were separately ligated in frame with the coding sequences for the movement protein and the capsid protein Vp25 flanking the cloning site. The resultant pBICAL2-PmPDS and pBICAL2-PmDAM6 were introduced separately into a disarmed Agrobacterium strain EHA105. The pBICAL1 constructed based on the RNA1 of ALSV was also introduced into EHA105. To amplify and produce recombinant ALSV particles, leaves of Nicotiana benthamiana and Chenopodium quinoa were infected with pBICAL1/EHA105 and pBICAL2-PmPDS/EHA105 or pBICAL2-PmDAM6/EHA105 at the same time. The amplified ALSV in N. benthamiana and C. quinoa was isolated and used to infect peach (P. persica) and Japanese apricot (P. mume) seedlings. We will discuss a possible use of VIGS-based gene evaluation system for Prunus fruit tree species.