Rorippa indica HSPRO2 expression in transgenic Brassica juncea induces tolerance against mustard aphid Lipaphis erysimi

We have previously established that RiHSPRO2, a nematode resistance protein-like homolog from wild crucifer Rorippa indica (L.) Hiern is a potent candidate to control mustard aphid Lipaphis erysimi. The present study further exploits this protein through structure prediction, biosafety assessment and transgenic study. The RiHSPRO2 protein showed an abundance of alpha helices intervened with loops in the homology-based three-dimensional model. No allergenic moiety was found in its amino acid sequence based on homology search in Protein Data Base. The secondary structure of RiHSPRO2 was unstable at temperatures above 50 degrees C. In vitro pepsin digestion assay revealed the protein to be digested in pepsin supplemented Simulated Gastric Fluid (SGF) within 2minutes. The protein is proved to be biologically safe as per the FAO/WHO guidelines. An efficient Agrobacterium-mediated Brassica juncea transformation involving direct organogenesis with mean transformation frequency of 5.06 +/- 0.28% is reported. RiHSPRO2, under the influence of constitutive promoter CaMV35S, was transformed into susceptible B. juncea cv. B85. Southern hybridization confirmed stable integration of the transgene and Western blotting confirmed consistent expression of RiHSPRO2 in the genetically modified Brassica lines. The transgene segregated following Mendelian 3:1 ratio in the successive generation. Detached leaf aphid bioassay and in planta aphid bioassay in transgenic B. juncea lines revealed a reduction in aphid survivability by 45% and a decrease in aphid fecundity by 45.6%.