Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the Ϸ20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP-or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host.RNA interference ͉ citrus tristeza virus ͉ virus synergy ͉ antiviral immunity T he discoveries of viral suppressors of RNA silencing play an important role in establishing RNA silencing as a natural antiviral response in both plant and invertebrate hosts (1). Many plant viral proteins have been identified as suppressors of RNA silencing since the initial reports in late 1998 (2-4). RNA silencing suppressors from different virus taxons are structurally diverse. However, they are typically required for long-distance virus spread and influence virulence and accumulation levels in infected plants (5-7). Progress is being made toward understanding the molecular mechanisms involved in viral suppression of RNA silencing (8-11). In RNA silencing assays, plant viral suppressors differ by their ability to suppress intracellular and͞or intercellular silencing (12-16). Intercellular silencing is mediated by the non-cell-autonomous silencing signal that can spread to destroy homologous RNAs in neighboring or distant tissues that do not contain the initial trigger, such as a silencing transgene or a replicating virus (17-19). The differential silencing suppression is best illustrated by the potyviral helper component proteinase (HC-Pro) and the cucumber mosaic virus (CMV) 2b protein. When introduced by genetic crosses into the Nicotiana tabacum line 6b5 carrying a silencing ␤-glucuronidase (GUS) transgen...

Some strains of cucumber mosaic virus (CMV) induce a bright yellow/white chlorosis in tobacco instead of the light green/dark green mosaic induced by most CMV strains. This property is controlled by RNA 3 of this tripartite virus. Recombination between cDNA clones of RNA 3 from a green mosaic strain, Fny-CMV, and a chlorotic strain, M-CMV, and inoculation of infectious transcripts of the chimeric RNAs 3, together with RNAs 1 and 2 of Fny-CMV, localized the chlorosis induction domain to a region of the coat protein gene containing two nucleotide differences. Site-directed mutagenesis of one nucleotide to change the codon for Leu129 in the M-CMV coat protein to Pro129 of Fny-CMV changed the phenotype from chlorotic to green mosaic, whereas the opposite change in phenotype was observed when the Pro129 in the Fny-CMV coat protein was altered to Ser129. Thus, the local secondary structure surrounding amino acid 129 rather than a particular amino acid per se is involved in chlorosis induction.

The differential symptom determinants of the Holmes' masked (M) and U1 strains of tobacco mosaic virus previously were mapped to the 5'-coterminal open reading frame (ORF) encoding the 126-kDa protein and the N-terminal two-thirds of the 183-kDa protein. Both proteins influence viral RNA accumulation, but the function of, and impact on, symptom formation by large domains within the 126-kDa gene, which are not conserved with sequences in analogous ORFs from other related viruses, are unknown. In the current study, cDNA clones representing each strain (i.e., MIC-TMV and U1-TMV) were mutated in these nonconserved domains to further define the nucleotides responsible for mosaic symptom induction on Nicotiana tabacum. Progeny virus of a mutant containing only eight nucleotide substitutions from the MIC-TMV sequence to the U1-TMV sequence within the 126-kDa protein ORF of MIC-TMV induced U1-TMV-like symptoms. Single or multiple substitutions among these eight nucleotides further defined residues critical for symptom modulation. Complementary substitutions in the MIC-TMV and U1-TMV sequences did not always yield progeny virus that induced complementary visual symptoms. Progeny of some mutants contained second-site spontaneous mutations at specific positions shown to influence symptom phenotype. For a subset of the stable site-directed mutants, there was no correlation between severity of systemic symptoms and chlorotic lesion size or virus accumulation in these chlorotic lesions on inoculated leaves.