Phosphorylation regulates the subcellular localization of Cucumber Mosaic Virus 2b protein

Abstract

The 2b protein of Cucumber mosaic virus has a role in nearly all steps of the viral cycle including cell-to-cell movement, symptom induction and suppression of antiviral RNA silencing. Previous studies demonstrated the presence of 2b protein in the nucleus and in cytoplasm as well. Phosphorylation site of 2b protein is conserved in all CMV isolates, including proposed constitute motifs for casein kinase II and cyclin-dependent kinase 2. To discern the impact of 2b protein phosphorylation, we created eight different mutants to mimic the non-phosporylated (serine to alanine) as well as the phosphorylated state (serine to aspartic acid) of the protein. We compared these mutants to the wild-type (Rs-CMV) virus in terms of symptom induction, gene silencing suppressor activity as well as in cellular localization. Here, in this study we confirmed the phosphorylation of 2b protein in vivo, both in infected N. benthamiana and in infiltrated patches. Mutants containing aspartic acid in the phosphorylation site accumulated only in the cytoplasm indicating that phosphorylated 2b protein could not enter the nucleus. We identified a conserved dual phosphorylation switch in CMV 2b protein, which equilibrates the shuttling of the 2b protein between the nucleus and the cytoplasm, and regulates the suppressor activity of the 2b protein.

Figure 1

In vivo phosphorylation of Cucumber mosaic virus 2b protein. Western blot analysis of infected (10 dpi) and agroinfiltrated N. benthamiana plants with Rs-2bHis using penta-his and phosphoserine antibodies. Coomassie staining was used to monitor the equivalence of protein loading.

Figure 2

Symptoms (A) and accumulation of CMV and the different 2b protein mutants (B) in N. benthamiana plants. Infections with Rs-SPS/40-42/APA, Rs-SPS/40-42/DPD were asymptomatic during the monitored period (six weeks). Infection with Rs-SPS/40-42/SPD and Rs-SPS/40-42/DPS induced mild symptoms: mild leaf distortion, mild systemic mosaic, and mild stunting (A). Rs-SPS/40-42/APS, Rs-SPS/40-42/SAS and Rs-SPS/40-42/SPA exhibited severe symptoms, including systemic mosaic, leaf distortion, and chlorosis. However, symptoms induced by these mutants were never as severe as those induced by Rs-CMV. CMV accumulation in systemically infected leaves analysed by northern hybridization at 7 dpi. The viral RNA accumulation was in accordance with the strength of the symptoms. The radiolabelled probe was specific for Rs-CMV RNA 3. Ethidium bromide-stained rRNA from the same volume of the sample is shown below each lane.

Figure 3

Analysis of suppression of transgene-induced silencing of Rs2b and the different mutants. A binary vector expressing the GFP reporter gene was co-infiltrated into Nicotiana benthamiana leaves with an empty binary vector or with binary vectors expressing 2b protein or 2b/40-42/AAA, 2b/40-42/APS, 2b/40-42/SPA, 2b/40-42/APA, 2b/40-42/SAS, 2b/40-42/DPS, 2b/40-42/SPD and 2b/40-42/DPS 2b protein construct (A). Mutants 2b/40-42/SAS, 2b/40-42/APS and 2b/40-42/SPA retained partial suppressor activity. Mutants 2b/40-42/DPS, 2b/40-42/SPD, 2b/40-42/DPD, 2b/40-42/APA and 2b/40-42/AAA failed to suppress GFP activity. The accumulation level of GFP RNA was quantitatively measured by qRT-PCR in the infiltrated leaves (B). Parallel detection of the fluorescence of GFP proteins on SDS-PAGE by illuminating the gel with UV lamp, immunoblot analyses of accumulation the His-tagged 2b protein mutants in agroinfiltrated patches by penta-his antibody detection and silver staining to monitor the equivalence of protein loading (C).

Figure 4

Subcellular localization of Rs-2b and its mutants detected by BiFC in Nicotiana benthamiana plants. N. benthamiana plants were co-agroinfiltrated with transiently expressed 2b proteins fused to either the N or C terminus of yellow fluorescent protein (YFP). Rs-CMV was clearly detectable in cytoplasm as well as in nuclei (A,B). No fluorescence was observed by confocal microscopic imaging in cells within leaf patches co-infiltrated with A. tumefaciens cells expressing 2b/40-42/AAA. In the case of mutant 2b/40-42/DPD fluorescence was detectable only in cytoplasm, not in the nucleus, nor in the nucleolus (A,B). The bar represents 50 µm (A) or 25 µm.

Figure 5

Subcellular localization of Rs2b-EGFP, 2b/40-42/APA-EGFP and 2b/40-42/DPD-EGFP in N. benthamiana plants. N. benthamiana plants were infiltrated with transiently expressed Rs2b-EGFP, 2b/40-42/APA-EGFP and 2b/40-42/DPD-EGFP fusion proteins. Rs2b-EGFP and 2b/40-42/APA-EGFP were clearly detectable in cytoplasm as well as in nuclei. The mutant 2b/40-42/DPD was detected in cytoplasm. The bar represents 50 µm.

Figure 6

Western blot analysis of the Rs2b and the different mutants from cell nuclei purified from infiltrated N. benthamiana leaves. His-tagged 2b proteins were detected with penta-his antibody and silver staining was used to monitor the equivalence of protein loading. Mutants SPS/40-42/AAA, SPS/40-42/APA, and single mutants SPS/40-42/APS, SPS/40-42/SPA and SPS/40-42/SAS were detectable from purified nucleoli with penta-his antibody. The mutants 2b/40-42/DPS, 2b/40-42/SPD and 2b/40-42/DPD were not detectable in nuclei. DAPI-staining was used for visualisation of the purified nuclei (appearing blue) with fluorescence microscope using UV-light. The bar represents 10 µm.

Figure 7

Analysis of the native and the mutant CMV 2b (SS/40,42/DD) tetramer MD simulations. The initial structures were taken as references when calculating RMSD values (A). The native siRNA-2b tetramer ribonucleoprotein complex reached a stable equilibrium state (black line), while the mutant complex became unstable (red line). Starting and ending structures of the MD simulation are in cartoon representation (B). The sites of residue 40 and 42 of the 2b chains are encircled.

Figure 8

Residues 40 and 42 of the CMV 2b protein have key stabilizing role in the siRNA binding. The S40 and the S42 residues in the native 2b tetramer formed stable H-bonds with the phosphate oxygen atoms of the cytosine-14 (C14_PO) and/or guanosine-13 (C13_PO) (A,C). Sometimes, for only very short time these serine side chains swing toward the solvent. The D40 and the D42 residues in the mutant 2b tetramer disrupted the siRNA binding (B,D). CMV 2b protein chain A and the siRNA1 are in cartoon representation (C,D). The studied residues are in licorice representation.