Overexpression of a Single-Chain Variable Fragment (scFv) Antibody Confers Unstable Resistance to TuMV in Chinese Cabbage

Abstract

A single-chain variable fragment (scFv) antibody is a recombinant protein composed of immunogloblin fused to a flexible linker protein. Recent studies have demonstrated that transfer of scFv to plants is effective in engineering virus resistance in plants. In this study, the utility of scFv in enhancing Turnip mosaic virus (TuMV) resistance in transgenic Chinese cabbage (Brassica rapa) was investigated. For this, a construct carrying scFv was transferred into Chinese cabbage and transgenic plant expressing scFv plants have been generated. DAS-ELISA and phenotypic data have revealed that not all transgenic plants carrying the scFv transgene were resistant to TuMV. Those transgenic lines resistant to TuMV had higher levels of scFv transcripts than transgenic lines susceptible to TuMV. Moreover, analysis of transgenic plants for methylation of the scFv transgene using the methylation-sensitive restriction enzyme HhaI has revealed that it was the non-methylated scFv transgene that was expressed in TuMV resistant lines whereas, the methylated scFv transgene was silenced, resulting in TuMV susceptibility in other transgenic lines. Bisulfate genomic sequencing, performed to further confirm methylation of the scFv transgene, has revealed that the promoter region of scFv in both resistant and susceptible transgenic was methylated but methylation was higher in susceptible plants than in resistant plants. In conclusion, overexpression of scFv confers TuMV resistance at low frequencies, and that the transgene is frequently silenced presumably due to cytotoxic effects of scFv in transgenic plants.