Effect of dicer-like proteins2 and 4 and RNA-dependent RNA polymerase1 as RNA silencing components on cyclic mosaic symptom development in tobacco infected with the Cucumber mosaic virus

Abstract

The Nicotiana tabacum genome contains four Dicer-like proteins (DCLs) and six RNA-dependent RNA polymerase (RDR) homologues involved in the RNA silencing mechanism employed against viral infection. DCL1 synthesizes 18-21 nt-long microRNA, whereas DCL2, DCL3 and DCL4 produce 22 nt, 24 nt and 21 nt-long siRNA, respectively, in the RNA silencing process. This study aimed to clarify which components among these are involved in changes in the amount of virus and the development of symptoms in Cucumber mosaic virus (CMV)-infected tobacco. Infected transgenic tobacco lines with a single down-regulation of DCL2, DCL4, RDR1 or a double down-regulation of both DCL2 and 4 were analyzed. The amounts of viral RNA in young developing leaves in transgenic tobacco lines were examined by Northern blot analysis. Most transgenic plants inoculated with CMV Pepo, a virulent strain, exhibited cyclic mosaic symptoms. The amount of viral RNA in single down-regulated lines varied based on leaf position in a similar manner to that noted in non-transgenic tobacco, while that of the double down-regulated line did not. Furthermore, the expression of RNA-silencing-related genes during high and low CMV infection did not differ among the transgenic plants. These results suggested that (i) changes in the amounts of the virus in the developing leaves of all the single down-regulated lines were associated with cyclic symptom expression in fully expanded leaves, and (ii) the lower expression of DCL2, DCL4 and RDR1 may be sufficient to establish cyclic symptom development.

Keywords

• dicer-like proteins
• RNA-dependent RNA polymerase
• RNA silencing
• Cucumber mosaic virus
• mosaic symptom