Susana Ruiz-Ruiz,1,2 Nuria Soler,2 Jesús Sánchez-Navarro,1 Carmen Fagoaga,2 Carmelo López,1,3 Luis Navarro,2 Pedro Moreno,2 Leandro Peña,2 and Ricardo Flores1
1Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de investigaciones Científicas-Universidad Politécnica de Valencia, Spain; 2Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, Spain; and 3Instituto de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad Politécnica de Valencia, Spain
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Accepted 29 October 2012.
Citrus tristeza virus (CTV) encodes a singular protein (p23, 209 amino acids) with multiple functions, including RNA silencing suppression (RSS). Confocal laser-scanning microscopy of green fluorescent protein (GFP)-p23 agroexpressed in Nicotiana benthamiana revealed its accumulation in the nucleolus, Cajal bodies, and plasmodesmata. To dissect the nucleolar localization signal (NoLS) typically associated with basic motifs, seven truncated and 10 point-mutated versions of p23 were assayed. Deletion mutants showed that regions 50 to 86 and 100 to 157 (excluding fragment 106 to 114), both with basic motifs and the first with a zinc-finger, contain the (bipartite) NoLS. Alanine substitutions delimited this signal to three cysteines of the Zn-finger and some basic amino acids. RSS activity of p23 in N. benthamiana was abolished by essentially all mutants, indicating that it involves most p23 regions. The necrotic-inducing ability of p23 when launched in N. benthamiana from Potato virus X was only retained by deletion mutant 158-209 and one substitution mutant, showing that the Zn-finger and flanking basic motifs form part of the pathogenic determinant. Ectopic expression of p23 and some deletion mutants in transgenic Mexican lime demarcated a similar determinant, suggesting that p23 affects related pathways in citrus and N. benthamiana. Both RSS activity and pathogenicity of p23 appear related to its nucleolar localization.
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