Molecular characterization and transient expression in plants of a Mirabilis jalapa antiviral protein (MAP), and its use in functional studies

Crude protein fractions from Mirabilis jalapa of northern Iran containing Antiviral Protein (MAP) reduced the systemic accumulation a plant RNA virus in Nicotiana benthamiana. A MAP gene was amplified by RT-PCR total RNAs isolated these Iranian M. plants (MAP-Tehran) without 5´-end signal peptide sequence, to set up an experimental system that would transiently express cell cytosol agroinfiltration and simultaneously trigger response study its antiviral effects. The fragment first cloned into high-copy plasmid promoters flanking cloning site nucleotide amino acid sequences were determined compared available sequences. Two putative ribosome inhibitor domains found N-terminal C-terminal regions, respectively. MAP-Tehran transferred binary vectors for agroinfiltration. Only constructs expressed fused N- or C- halves monomeric red fluorescent used bimolecular fluorescence complementation assays obtained. In N. benthamiana leaves agroinfiltrated with constructs, signs plasmolysis became detectable under confocal microscopy at day 3 after infiltration (dai), 4 necrotic lesions visible. patches 2–3 dai, before onset death, co-expressed reporter also mechanically inoculated observed. double-alanine substitution (Glu → Ala, Arg Ala) region domain sufficed abolish all Agroinfiltration either necrosis-inducing non-inducing variants caused no significant effect on upper, non-infiltrated leaves.