Pathogenicity of Alternanthera mosaic virus is affected by determinants in RNA-dependent RNA polymerase and by reduced efficacy of silencing suppression in a movement-competent TGB1

Four biologically active cDNA clones were derived from the Alternanthera mosaic virus (AltMV; genus Potexvirus) isolate, AltMV-SP, which differ in symptoms in infected Nicotiana benthamiana plants. Two clones induced necrosis and plant death; a mixture of all four clones induced milder symptoms than AltMV-SP. Replication of all clones was enhanced by a minimum of fourfold at 15 6C. A mixture of clones 4-7 (severe) and 3-1 (mild) was indistinguishable from AltMV-SP, but the ratio of 4-7 to 3-1 differed at 25 and 15 6C. RNA copy numbers of mixed infections were always below those of 4-7 alone. Determinants of symptom severity were identified in both Pol and TGB1; the mildest (4-1) and most severe (3-7) clones differed at three residues in the ‘core’ Pol domain [R(1110)P, K(1121)R, R(1255)K] and one [S(1535)P] in the C-terminal Pol domain of RNA-dependent RNA polymerase, and one in TGB1 [P(88)L]. Pol [P1110,R1121,K1255]+TGB1L(88)] always induced systemic necrosis at 15 6C. Gene exchanges of Pol and TGB1 each affected replication and symptom expression, with TGB1P(88) significantly reducing silencing suppression. The difference in silencing suppression between TGB1P(88) and TGB1L(88) was confirmed by an agroinfiltration assay. Further, co-expression of TGB1P(88) and TGB1L(88) resulted in interference in the suppression of silencing by TGB1L(88). Yeast two-hybrid analysis confirmed that TGB1P(88) and TGB1L(88) interact. These results identify a TGB1 residue that significantly affects replication and silencing suppression, but maintains full movement functions.