High frequency induction of RNA-mediated resistance against Cucumber mosaic virus using inverted repeat constructs

Abstract

The application of RNA-mediated resistance against Cucumber mosaic virus (CMV) by using single transgene constructs generally results in only a small portion of resistant individuals. Inverted repeat constructs encoding self-complementary double-stranded RNA have been demonstrated a potential way to obtain RNA-mediated resistance at high efficiency. To test this observation as a possible method for high frequency induction of CMV resistance, Nicotiana benthamiana plants were transformed with transgenes designed to produce double strand RNA molecules of CMV RNA 2 or coat protein (CP) gene sequences. Seventy-five percent of the tested R0 plants transformed with an RNA 2-derived inverted repeat construct (1534 nt CMV sequence) showed extreme resistance to CMV, while a lower percentage of resistance (30%) was observed in R0 lines transformed with a similar construct of a shorter viral RNA 2 sequence (490 nt). The resistance level conferred by CP sequences was also efficient by using a dsRNA construct, reaching a level of 50%. Self-pollinated (S1) progenies obtained from most resistant R0 plants all showed resistance levels of 100%, perfectly correlating with the expression of transgenic siRNAs. The results indicate that the use of inverted repeat viral transgenes is a highly efficient approach to obtain CMV resistant transgenic plants. Consequently, only a handful of transgenic plants will have to be generated using such constructs for successful resistance, which enables the implementation of this protocol for crops that are difficult to transform, such as ornamental plants in which CMV is an important pathogen.