Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase.

In host plants, cell-to-cell spread of tobacco mosaic virus (TMV) presumably occurs through intercellular connections, the plasmodesmata. TMV movement is mediated by a specific virus-encoded single-strand nucleic acid-binding protein, P30. The mechanism by which P30 operates is largely unknown. Here, we demonstrate that P30 expressed in transgenic plants is a phosphoprotein. We have developed an assay for in vitro phosphorylation of purified P30 by plant cell wall fractions and have localized the phosphorylation sites to amino acid residues Ser-258, Thr-261, and Ser-265. Interestingly, the P30 phosphorylation sites do not correspond to any known consensus phosphorylation sites for protein kinases. While P30 binding to single-stranded DNA (ssDNA) was shown to involve Thr-261, phosphorylation of this residue does not appear to play a role in binding activity. The protein kinase activity contained in the cell wall fractions was developmentally regulated, expressed predominantly in leaves. Within a leaf, this protein kinase activity increased with leaf maturation and correlated with the reported development of secondary plasmodesmata, sites of P30 accumulation. We suggest that phosphorylation may represent a mechanism for the host plant to sequester P30 following its localization to cell walls.