Figure 1.

The βC1 protein of the Radish leaf curl betasatellite (RaLCB) interacts with the nuclear‐encoded chloroplast protein; PsbP. (a) Yeast strain AH109 cells were co‐transformed with different combinations of yeast two‐hybrid constructs as mentioned. The co‐transformed AH109 cells were grown on either synthetic dropout medium SD‐Leu‐Trp or selection medium SD‐Leu‐Trp‐His supplemented with 5 mM 3‐amino‐1,2,4‐triazole as serial dilution from cultures OD₆₀₀ of 1.0. Yeast cells co‐transformed with AD plus BD or AD‐T_Ag plus BD‐P₅₃ act as negative and positive controls, respectively. (b) An equal amount of purified GST/GST‐βC1 protein was used to pull‐down MBP/MBP‐PsbP protein. Immunoblotting was carried out using either anti‐GST or anti‐MBP antibodies. Coomassie‐stained gel served to monitor the amount of protein present in input and pull‐down. (c) Different combinations of Agrobacterium cells harbouring bimolecular fluorescence complementation (BiFC) constructs were co‐infiltrated into the leaves of 3 to 4 weeks old N. benthamiana plants as mentioned. The lower epidermal cells of co‐infiltrated leaves were visualized under a confocal microscope. The reconstituted Venus protein fluorescence was observed in the periphery of the epidermal cell of leaves co‐infiltrated with Agrobacterium harbouring both pVYNE‐βC1 and pVYCE(R)‐PsbP constructs. Nuclei of the epidermal cells were stained with 4, 6‐diamidino‐2‐phenylindole (DAPI). Scale bar represents 20 μM. (d) Schematic representation of the C‐terminal deletion constructs of PsbP protein used for yeast two‐hybrid assay and BiFC assay. (e) Yeast two‐hybrid assay carried out with βC1 and different domains of PsbP. (f) BiFC assay was performed on N. benthamiana plants leaves infiltrated with Agrobacterium carrying plasmid combinations as mentioned. Scale bar represents 20 μm.