S01 - Session P1 - Development of a viral vector based on the beta satellite associated with ageratum yellow vein virus

Authors: Ying-Huey Cheng *

Ageratum is a general weed in Taiwan. When ageratum co-infected with ageratum yellow vein virus (AYVV) and beta satellite, it turns the green weed into yellow. Invasion with AYVV alone just causes asymptomatic infection. The beta satellite has an adenine-rich region, a satellite conserved region and an ORF beta C1 that is essential for symptoms development. The viral vector was created by cloning direct repeat sequences of beta satellite without the C1 gene into a Ti plasmid, pCAMBIA 0380, and then subcloned to Agrobacterium tumefaciens LBA4404. When Nicotiana benthamiana and Solanum lycopersicum were co-infected with two agrobacteria harboring AYVV or beta satellite for two weeks, AYVV and beta satellite could be detected by PCR. When the viral vector carried the GFP gene, which encodes a 28.5 kDa protein, green fluorescence can be observed in the veins of the upper leaves of infected tomato. The viral vector was also used to downregulate the expression of the tomato Cyclophilin B ( CypB ) gene, which has some molecular roles as signal and chaperone molecules. The CypB hairpin containing partial sequences from CypB (260 nucleotides inverted-repeat) as a stem structure and castor bean catalase intron (190 nucleotides) as a loop, were cloned into the viral vector. The resulting plasmid was transferred to A. tumefaciens and then co-infected into tomato seedlings with AYVV as helper virus. Tomato plants inoculated with AYVV alone showed slightly down-rolled symptoms of the leaves, but smaller and irregular deformation symptoms were observed on leaves of co-inoculated plants 21 days post inoculation. According to our studies, the beta satellite vector can introduce foreign genes and stably produce proteins, or serve as a virus-induced gene silencing vector to efficiently silence target genes for functional research.