Genetic dissection of tomato bushy stunt virus p19-protein-mediated host-dependent symptom induction and systemic invasion

The plus-sense single-stranded RNA of tomato bushy stunt virus (TBSV) encod es a 19-kDa protein, which is translated from a 3' proximal open reading fr ame (p19) that is entirely nested within the cell-to-cell movement gene (p2 2). Expression of the cytosolic p19-protein induces either a systemic letha l collapse in Nicotiana benthamiana and N. clevelandii, or necrotic local l esions on resistant N. tabacum. In spinach, the p19-protein is required at high abundance for efficient systemic invasion. This study aimed to determi ne whether these seemingly different host-dependent biological activities a re governed by the same or separate regions on the 172 amino acid p19-prote in. For this purpose, codons for charged amino acids predicted to be expose d on the surface of the polypeptide and presumably available for host-speci fic interactions, were targeted for mutagenesis. A total of 12 mutants were generated, which had no deficiencies in replication or cell-to-cell moveme nt, and substitution of amino acids at the extreme N-terminal end or within the carboxyl 70 amino acids failed to cause a noticeable biological effect on plants. However, mutations dispersed between positions 43 and 85 on the N-terminal half prevented the onset of a systemic lethal necrosis on N. be nthamiana and N. clevelandii. With one exception, the same mutants elicited mostly chlorotic, rather than necrotic, local lesions on N. tabacum. Mutat ions in the central region, which substituted Arg with Gly at positions 72 or 75-78, impaired the ability of TBSV to systemically invade spinach plant s. However, substitution with Ala instead of Gly at position 72 had minimal effects on systemic spread in spinach, suggesting the possible influence o f protein structure effects. The implications are that regions on the N-ter minal portion of the p19-protein mediate interactions in a host-dependent m anner and that a central region is required for all activities either by a direct effect of the amino acids or through maintenance of structural integ rity. (C) 2000 academic Press.