Rice Dwarf Virus Non-structural Protein Pns11 Localized In Nucleolus And Interacted With The Major Nucleolar Protein Fibrillarin

Rice dwarf virus (RDV), which belongs to the genus Phytoreovirus in the family Reoviridae, is one of the most widespread and disastrous rice-infecting viruses causing great yield reduction in southeast Asian countries, such as Japan, Korea, Philippines and Nepal. Rice seedlings of the moderately resistant variety YX-2292 were collected at 22 days post inoculation (dpi) for transcriptome analysis (using the GeneChip Rice Genome Array of Affymetrix). The results showed that a total of 856 genes were RDV-responsive, in which 838 genes were upregulated and 18 downregulated, by applying SAM (Significant Analysis of Microarray) software with the criteria of fold changes>1.5; false discovery rate (q-value) <0.05 to screen significantly differentially expressed genes. Interestingly, these studies showed that a great number of nucleolar and ribosomal genes was affected. Among the 856 RDV-responsive genes, only 581 have gene annotations. To gain further insight into the functions of RDV-responsive genes, these genes were classified into 14 categories: including unclassified for genes with no annotated function (24.44%), stress/defense related genes (13.77%), transcription factor (9.98%), signal transduction (9.29%), metabolism (9.29%), protein fate (8.43%), protein synthesis (6.20%), transposon/ retrotransposon protein / RNA silencing pathway genes/possible genome stress induced genes (5.51%), cell wall function (5.16%), phytohormone related(2.41%), secretory pathway (2.07%), transporter/chanel (1.38%), cytoskeleton or cytosjeleton associated (1.03%) and cell cycle related (1.03%).Tansient expression after fusion with eGFP showed that Pns6, the movement protein of RDV localized mainly in cell membrane and formed punctate sites reminiscent of plasmodesma. Pns10, a silencing suppressor of RDV, has a diffuse pattern of localization, which resembles free eGFP to localize in cytoplasmic. Pns11 was shown to be a dual targeted protein, which could be found in both the chloroplasts and the nuclei. The nuclear localization of Pns11 was further studied. The results showed that Pns11 colocalized with fibrillarin, which is often usd as a marker gene of the nucleolus. This suggested that Pns11 was a nucleolus localized protein. Futher studies showed that a nuclear location signal located in Pns11 was required for the nucleolar localization of Pns11. Deletion mutants of Pns11 were created and their localization characterized. The results indicated that the basic amino acids of Pns11 NLS play an essential role in Pns11 location. The 8 basic amino acids"RK----------KKGGKK", 132-144 site, and the C-terminal six basic amino acids "-RKSKRK"in the Pns11 NLS are the Pns11 nucleolus localization signals (NoLS), which is necessary for Pns11 nucleolus location.To further study the relationships between Pns11 and the nucleolus or nucleolus related genes. Possible interactions between the rice and tobacco fibrillarin and RDV Pns11 were explored by using yeast two hybrid experiments and BiFC.The results of BiFC suggested that Pns11 might weakly interact with fibrillarin. Fibrillarin of Nicotiana benthamiana was silenced by using VIGS. The fibrillarin-silenced N. benthamiana showed phenotypes including loss of apical dominance reduced plant size and dwarfism, delayed development distortion and albinism of newly developed leaves. These, to some extent, resemble symptoms caused by RDV, suggesting that RDV might induce the development of symptomes through the alteration of nucleolar genes expression or through interaction with nucleolar components. The fibrillarin-silenced N. benthamiana was used to further characterize Pns11. The results showed that the absence of fibrillarin had no effects on the nucleular localization of Pns11. However, the silencing suppressor acctivities of Pns11 might depend on the presence of fibrillarin. It is likely that Pns11 may interact with fibrillarin or some other genes downstream of fibrillarin.Rice gall dwarf virus (RGDV) and RDV both belong to the genus Phytoreovirus. RGDV Pns12 has 22% sequence identities with RDV Pns11. In this study, we showed that Pns12 of RGDV exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c. Pns12 of RGDV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Expression of Pns12 also enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, these results suggested that RGDV Pns12 might function as a virus suppressor of RNA silencing (VSR) that target an upstream step of the dsRNA formation in the RNA silencing pathway. Further, we showed that Pns12 localized mainly in nucleus and nucleolus in tobacco epidermal cells, which is similar to RDV Pns11.