Transformation of Nicotiana benthamiana with different BWYV (Beet western yellows virus) sequences to test for virus resistance

Abstract

Nicotiana benthamiana plants were transformed by means of Agrobacterium tumefaciens carrying various constructs to test for resistance for BWYV (Beet western yellows virus), the constructs used included either the viral replicase (ORF1/2), one of two smaller sequences involving the 5¢ and 3¢ ends (5¢3¢S and 5¢3¢AS) of the viral genome of BWYV. According to different criteria such as ELISA, PCR, growth under kanamycin selection at 200 mg l )1 and phenotype some lines were chosen as candidates to be tested for resistance against BWYV. Five lines of each construct were randomly selected. These lines were analysed by Northern blot for expression of the transgene of interest and/or the nptII gene. Greenhouse resistance tests were performed in 15 transgenic N. benthamiana lines (5 per construct) and in two controls. The transgenic plantlets were transferred to the greenhouse and 20 plants from each line were inoculated with BWYV using the natural vector Myzus persicae, while other 20 were kept as uninoculated control. At 4, 6 and 8 weeks post-infection (wpi) a BWYV ELISA of the inoculated plants was carried out and the height of each plant was measured, while the weight was determined at the end of the experiment. None of the transgenic lines tested showed resistance to the virus.Abbreviations: 5¢3¢S -5¢3¢ sense; 5¢3¢AS -5¢3¢ antisense; BWYV -beet western yellows virus; CP -coat protein; dpi -days post infection; Km -kanamycin; ORF -open reading frame; PEBV -pea early browning virus; PLRV -potato leafroll virus; PMMV -pepper mild mosaic virus; wpi -weeks' postinfection