Successful generation of anti-ToCV and TYLCV transgenic tomato plants by RNAi

Abstract

Tomato is an economically important vegetable. Tomato chlorosis virus (ToCV) and Tomato yellow leaf curl virus (TYLCV) are two major viruses that cause serious losses to tomato production. The effective method to control these two viruses is to breed antiviral species by genetic engineering techniques. In order to obtain the RNA interference (RNAi) expression vector of tomato, the coat protein (CP) genes of ToCV and TYLCV were selected in this study. The tandem sequences of the two CP genes were obtained using the recombinant PCR technique. Using Gateway cloning technology, the RNAi expression vector pRNAi-ToCV-TY including the two CP genes was constructed by attB×attP and attL×attR recombination reactions. Polymerase chain reaction and sequencing analysis confirmed that the vector was obtained successfully and contained the ToCV and TYLCV CP genes. The RNAi expression vector pRNAi-ToCV-TY was transformed into Agrobacterium strain GV3101. The RNAi vector was then used to transform tomato. The objective fragments were successfully transformed into a tomato by PCR identification. At the fourth-leaf stage, the positive transgenic plants were challenged with ToCV and TYLCV. Out of 15 transgenic plants, 33 % showed early symptoms within 4 weeks post-infection (WPI); 20 % showed delayed symptoms (5 - 7 WPI); and the remaining 47 % were symptomless even after 9 WPI. The untransformed control plants (90 %) showed severe symptoms within 2 - 4 WPI, whereas 10 % delayed symptoms.