| Cucumber mosaic virus (CMV) is an economically important pathogen, and is increasingly becoming a model virus for study of virus and host interaction. CMV genome compromises of three single stranded sense RNAs, encoding five viral proteins. Amongst,2b protein encoded by the subgenomic RNA4A of genome RNA2is a multi-functional protein. CMV2b protein efficiently suppresses small RNA-mediated RNA silencing and salicylic acid mediated defense against CMV. Additionally, CMV2b protein plays an important role in CMV virulence and long-distance movement of CMV in plant. It targets into nucleus due to possession of either one (Subgroup II strains) or two (Subgroup I) nuclear localization signals (NLS). By date, the molecular basis of targeting CMV2b into nucleus is still largely unknown.Transportation of nucleoprotein into nucleus mainly depends on Importin (Karyopherin) alpha/beta pathway. Arabidopsis thaliana as a model plant encodes9putative importin alpha proteins (IMPal-9). We questioned whether CMV2b protein targets into nucleus by binding with Importin alpha protein(s)? To answer this question, first of all, we successfully amplified coding sequences of Arabidopsis IMPal-9by regular RT-PCR. Subsequently, interaction of CMV2b with all of Importin alpha proteins was not detected using yeat two hybrid experiment. To confirm potential interaction of CMV2b with importin alpha proteins, we tested interaction in planta using bimolecular fluorescence complementary assays (BiFC). Interestingly, results showed that CMV2b interacted with IMPa2, IMand IMPa6. Afterwards, single mutants Fny2bNLS1-6A-YFPC and Fny2bNLS2-4A-YFPC and double mutant Fny2bNLS1,2-6A,4A-YFPC were constructed by replacement of either NLS1or NLS2, or both with alanine residues using overlapping PCR technique. Then, interactions of these three2b mutants with IMPa2, IMPa3and IMPa6were tested using BiFC. Result of fluorescence observation showed that no fluorescence was detected exception of weak fluorescence present from co-expression of Fny2bNLS2-4A-YFPC and IMPa3. This demonstrates that NLS1and NLS2are dispensable for CMV2b binding with IMPa2and IMPa6, and IMPa3binds CMV2b by recognizing NLS1. Then, we co-expressed GFP-2b with IMPa2, IMPa3or IMPa6through agroinfiltration. Observation of GFP fluorescence showed that transient expression of IMPa2markedly enhanced nuclear accumulation of CMV2b protean; furthermore, their co-expression led to production of necrosis on the agroinfiltrated leaves. Afterwards, effect of importin alpha proteins on viral symptoms was analyzed using Arabidopsis mutant plants. In this experiment, the CMV mutant Fny-CMV2bNLS expressing the2b protein predominantly nuclear enrichment was used to infect plants, and infection of the mutant virus caused severe necrosis on systemic leaves of wild-type Arabidopsis plants. Observation of viral symptoms indicated that necrosis was obviously alleviated on Arabidopsis mutant impa2plants, compared with wild-type Arabidopsis plants. All these results suggest that nuclear localization of CMV2b protein is associated with binding to the IMPa2protein. Finally, using GFP fusion, subcellular distributions of IMPa2, IMPa3and IMPa6were visualized. Results showed that compared with control GUS-GFP protein predominantly present in cytoplasm, all these three Importin proteins accumulated in nuclear to a distinct extent. IMPa2mainly enriched in nucleus.Taken together, in this work, we primarily evidenced that the2b protein from subgroup I strain interacted with Arabidopsis IMPa2, IMPa3and IMPa6, and IMPa2mediated nuclear accumulation of CMV2b. |
