Mutations of splice acceptor site A lead to the use of downstream AG dinucleotides as acceptor sites.

<p>(A) Splice acceptor site A was mutated both in Cabb-S and Cabb B-JI isolates. The AG dinucleotides used as acceptor sites in the wild type and mutant viruses are in bold; mutated nucleotides are underlined. Corresponding amino acids are provided below the nucleotide sequences. (B) Symptoms on turnip plants at 45 dpi. (C) Sequences at the second splice junction of three isoforms cloned from splice acceptor mutant-infected turnips (B-JI ma3 and S ma3) after RT-PCR amplification on total RNA. Exons 2 and 3 are represented by a dotted line and a solid one, respectively. (D) Immunodetection of viral proteins P1, P2, P3, P4 and P6/TAV in CaMV Cabb B-JI-, Cabb B-JI mutA-, Cabb B-JI ma3-, Cabb S- or Cabb Sma3-infected turnips plants. Total proteins from systemic leaves were analyzed by SDS-PAGE, and western blotting was performed with polyclonal antibodies. Two independent replicates are shown. (E) Electron-lucent viroplasms are not detected in ma3 mutant-infected cells. Protoplasts isolated from Cabb B-JI- (A), B-JI ma3- (B), B-JI mutA- (C), Cabb-S- (D), S-ma3-infected turnip plants (E) or from healthy plants (F) were fixed then immunolabeled with rabbit anti-P2 antibodies and mouse anti-rabbit antibodies conjugated to Alexa 488. Fluorescence images were collected by confocal laser scanning microscopy. Scale bars: 5 μm.