Figure 1.

Phosphorylation of P20. (A) In vitro phosphorylation. Purified rP20 was co-incubated in protein extracts of BaMV and satBaMV co-infected N. benthamiana leaves in the absence or presence of 100 mM EDTA and [γ-³²P]ATP or [γ-³²P]GTP and analyzed by 12.5% SDS–PAGE and autoradiography. Reaction mixture lacking rP20 or leaf protein extract served as controls. (C) In vivo phosphorylation. N. benthamiana protoplasts were mock-inoculated, inoculated with BaMV viral RNA alone or co-inoculated with BaMV RNA and satBaMV RNA transcripts by electroporation and cultured with [³³P]orthophosphate. After 20 h inoculation, protoplasts were harvested, lysed, total proteins were immunoprecipitated with anti-P20 serum, analyzed by 12.5% SDS–PAGE and detected by autoradiography. (B and D), Immunodetection of P20 phosphorylated in vitro and in vivo with anti-P20 serum.