Comparing p20’ s RNA silencing suppressing activity among five phylogenetic groups of Citrus Tristeza virus Natália T. Marques & Ângela A. Costa & Débora Lopes & Gonçalo Silva & Gustavo Nolasco Accepted: 8 September 2011 / Published online: 18 January 2012 # KNPV 2011 Abstract The p20 protein encoded by the Citrus Tristeza Virus (CTV) was previously identified as a RNA silencing suppressor. In this study, we analyzed the p20’ s suppressing activity from five phylogenetic groups of CTV, using the co-infiltration assay of Green fluorescence protein (GFP) gene and the suppressor gene in 16C line Nicotiana benthamiana plants. Green fluorescence, GFP mRNA relative levels and GFP specific siRNAS were compared showing in most cases, only slight differences. Contrary to previous studies, the p20 suppressor was not able to impede neither short range nor systemic spreading of RNA silencing. The suppressor from the phylogenetic group 4 revealed a much reduced activity when compared with the others. At present we still don’t know whether this property is a characteristic of this group or an atypical feature due to a unique point mutation. The differences in the symptom type and intensity originated by isolates belonging to the phylogenetic groups assayed could not be related to differences to the p20 suppressor ’ s activity. Keywords RNA silencing suppressor . VSR . p21- like . GFP . Closterovirus Introduction One of the RNA silencing mechanism functions is to protect plant cells against viruses. The RNA silencing mechanism involves the cleavage of incoming double- stranded RNA regions and the formation of 21–24 nucleotide (nt) short interfering RNAs (siRNAs), which guide, in a sequence specific manner, the degradation of cognate RNAs. A signal moves from the cells, undergo- ing the initial silencing, to the neighbouring cells as well as to distant cells through the phloem, priming the RNA silencing in naive cells (Baulcombe 2004; Brodersen and Voinnet 2006). To counteract this defence, viruses produce RNA silencing suppressors (VSR), which allows them to block the silencing mechanism at several points (Li and Ding 2006). VSR activity has, in several cases, been related to viral symptoms (Li and Ding 2006; Diaz-Pendon and Ding 2008). Citrus tristeza virus ((CTV; family Closteroviri- dae, genus Closterovirus) has a single-stranded, positive-sense RNA genome of approximately 19.3 Kb (Karasev et al. 1995). This phloem-limited virus is the causal agent of tristeza, the most damaging disease of Eur J Plant Pathol (2012) 133:229–235 DOI 10.1007/s10658-011-9877-0 N. T. Marques : Â. A. Costa : D. Lopes : G. Silva : G. Nolasco Center for Biodiversity, Functional and Integrative Genomics, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal G. Nolasco (*) Faculdade de Ciências e Tecnologia, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal e-mail: gnolasco@ualg.pt