Construction of infectious clone of banana streak OL virus by yeast homologous recombination.

Banana streak OL virus (BSOLV) is the primary species of banana streak virus (BSV) affecting banana production in South China. Reverse genetics is a powerful method for studying viral pathogenicity and the interactions between viruses and their hosts; however, no infectious clone of BSOLV has been reported until now. In this study, the BSOLV genome was amplified in overlapping segments to assemble 1.3 times full-length sequence. The resulting construct was then cloned into the yeast-Escherichia. Coli-Agrobacterium shuttle vector pCB301 via yeast homologous recombination, yielding the infectious clone BSOLV-GZ. The complete genome of the cloned BSOLV isolate was 7, 410 bp and shares 99.81% nucleotide sequence identity with the BSOLV isolate 3-S36. The BSOLV-GZ infectious clone was introduced into Brazilian banana seedlings through Agrobacterium-mediated injection. At 30 days post-inoculation (dpi), BSOLV was detected in 19 out of 30 inoculated banana plants by RT-PCR. By 42 dpi, distinct chlorotic streaks appeared in 12 of the 19 RT-PCR-positive plants. This was confirmed by immunocapture PCR (IC-PCR) and Southern blot. Additionally, rod-shaped viruses were observed under transmission electron microscopy in the symptomatic Brazilian banana plants. These findings indicated that the banana plants inoculated with the BSOLV-GZ infectious clone exhibited typical symptoms of infection. The host range of the BSOLV-GZ infectious clone was assessed through Agrobacterium-mediated infiltration; however, none of the tested plants was detected positive by RT-PCR. This research provides a foundational basis for future studies on the activation of endogenous BSOLV and the interactions between the virus and its hosts.