MUTAGENESIS OF THE AC3 OPEN READING FRAME OF AFRICAN CASSAVA MOSAIC-VIRUS DNA-A REDUCES DNA-B REPLICATION AND AMELIORATES DISEASE SYMPTOMS

Small insertions were made independently at each of four unique restriction sites on African cassava mosaic virus (ACMV) DNA A to disrupt the three overlapping complementary-sense open reading frames (ORFs) herein designated AC1, AC2 and AC3. The DNA A mutants were assayed for their infectivity by agroinoculation of monomeric constructs to Nicotiana benthamiana plants containing chromosomal insertions of ACMV DNA B. Disruption of the AC3 ORF alone resulted in a delay and amelioration of disease symptoms which correlated with reduced replication of DNA B. Normal replication of DNA A still carrying the AC3 ORF mutation was found in extracts from these plants. No ACMV DNA or symptoms were observed in corresponding inoculations with either the simultaneous disruption of the overlapping AC2 and AC3 ORFs or disruption of the AC1 ORF. Complementation by the inoculation of different mutant pairs produced a delay in disease symptoms followed by repair of mutated sites. A DNA A construct with the virus-sense AV1 (coat protein) ORF deleted was infectious producing typical ACMV disease symptoms. A similar construct with a larger deletion encompassing the complementary-sense AC3 ORF produced symptomless infections. The DNA recovered from plants revealed DNA A of normal size where the position of the deleted ORF was replaced with cloning vector DNA. Significantly reduced DNA B replication was observed for the AC3 deletion construct.