Evidence that the 75K readthrough protein of beet necrotic yellow vein virus RNA-2 is essential for transmission by the fungus Polymyxa betae

Abstract

Two mutant strains of beet necrotic yellow vein virus (BNYVV) containing deletion mutants of RNA-2 were produced during serial passage in mechanically inoculated Tetragonia expansa leaves. The mutant strains were referred to as S-0a (RNA-1 + 2a) and G-0b (RNA-1 + 2b). RNA-2a and RNA-2b were about 4.3 kb and 4.2 kb in length, respectively, whereas normal sized RNA-2 was about 4.8 kb in length. In vitro translation and immunoblot analysis showed that RNA-2, RNA-2a and RNA-2b all directed synthesis of the coat protein (Mr 22K). However, whereas wild-type RNA-2 also directed the synthesis of a coat protein readthrough protein with an Mr of 83K (predicted Mr 75K), RNA-2a and RNA-2b directed the production of readthrough proteins with MrS of 67K and 58K, respectively. This suggests that the deleted regions of RNA-2a and RNA-2b occur within the second open reading frame, which encodes a polypeptide of Mr 54K, which is translated by readthrough of the coat protein cistron. After the addition of wild-type RNA-3 and RNA-4 to all the strains, the mutant strains could not be transmitted by Polymyxa betae zoospores produced from either zoosporangia or resting spores, whereas the wild-type strains were readily transmitted. These results indicate that the 75K readthrough protein encoded by RNA-2 is essential for the transmission of BNYVV by P. betae.