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