PVX-tolerant potato development using a nucleic acid-hydrolyzing recombinant antibody.

3D8 scFv, a catalytic recombinant antibody developed in the MRL mouse, exhibits nucleic acid-hydrolyzing activity. Previous studies have demonstrated that tobacco plants harboring 3D8 scFv antibodies showed broad-spectrum resistance to infection by both DNA and RNA viruses. In this study, potatoes were transformed with the 3D8 scFv gene and screened by potato virus X (PVX) challenge. Starting with the T0 and T1 potato lines, PVX-tolerant T1 potatoes were identified in the field and characterized by ELISA and RT-PCR analysis. T2 potatoes were propagated for T3 generation and additional virus challenges in the field, and 44% of the 3D8 scFv T3 transgenic potatoes grown in GMO fields were found to be tolerant to PVX infection. Tubers from PVX-tolerant T3 lines were 60% bigger and 24% heavier, compared with tubers from PVX-susceptible transgenic lines and wild-type potatoes. Three-step virus challenge experiments and molecular characterization techniques were used for plants grown in growth chambers or fields to identify 3D8 scFv-transgenic, PVX-tolerant potatoes. These studies also revealed that the viral tolerance enabled by 3D8 scFv persisted during asexual propagation.

Differential identification of three species of Curtovirus using loop-mediated isothermal amplification.

Rapid and sensitive detection methods for three species of Curtovirus were developed using a loop-mediated isothermal amplification (LAMP) technique. A universal primer set for detecting the three main species of Curtovirus at the same time, and three kinds of species-specific primer sets were designed and used for LAMP reactions. Results from the LAMP reactions were visualized both by color changes after adding SYBR Green I staining dye and by DNA laddering on agarose gel electrophoresis. The optimal conditions for the curtovirus LAMP reaction were confirmed at 60°C for the universal primers and at 62°C for the three species-specific primer sets. Amplification of curtoviruses by LAMP reaction was ten-fold more sensitive than that by polymerase chain reaction. Primers designed for curtovirus detection in this study did not anneal to or amplify DNA from other DNA or RNA viruses (tomato yellow leaf curl virus, tomato spotted wilt virus, and potato virus Y). Taken together, the primer sets and reaction conditions developed in this study show that the LAMP technique could be a useful tool to detect the three species of Curtovirus simultaneously and distinguish them in the laboratory and the field.