Figure 1.
NIK1 phosphorylates rpL10A and relocates the cytosolic protein to the nucleus of transfected cells.
(A) Ectopic expression of NIK1 alters the nucleocytoplasmic shutting of rpL10A. YFP-L10 and NIK1-GFP were co-expressed in tobacco leaf epidermal cells, and the subcellular localization of the fluorescent fusion proteins was monitored by confocal microscopy. The frequency of co-transfected cells with rpL10A localized within the nuclei was increased. Full arrows indicate fluorescent nuclei. Scale bars are 10 µm. (B) Confocal microscopy of YFP-L18 and NIK1-GFP co-expressing epidermal cells. YFP-rpL18 and NIK1-GFP were co-expressed in tobacco leaf epidermal cells. Full arrows indicate fluorescent nucleoli. (C) In vitro phosphorylation activity of mutant NIK1s. GST fused to the C-terminal kinase domain (amino acids 298–638) of normal NIK1 (NIK1) or to mutant NIK1s (T474D and G473V/T474A) were produced in E. coli and affinity-purified using GST-Sepharose (Figure S2C). Purified GST fusions (as indicated) were incubated with equal amounts of GST-L10 or GST-QM104 in the presence of [γ-32P]ATP and separated by SDS-PAGE. The gels were stained with coomassie-blue (not shown) and visualized by autoradiography using a phosphoimager (top panel). The relative activity of autophosphorylation and phosphorylation of the L10 substrate was quantified and expressed as a percentage of the wild type kinase activity. The autophosphorylation activity was expressed as Vunits/µg enzyme/min, and substrate phosphorylation activity as Vunits/µg enzyme/µg substrate/min. (D) The efficiency of rpL10A relocalization to the nuclei correlates with kinase activity of NIK1. Tobacco leaves were co-agroinfiltrated with YFP-L10 and GFP-fusions, as indicated. The percentage of co-transfected cells containing YFP-L10 fluorescence over the nucleoplasm was registered. Values are the mean±SD of three determinations from independent experiments. In each experiment, a total of 100 to 150 cells were observed. (E) Ectopic expression of active NIK1 promotes accumulation of phosphorylated rpL10A in the nuclear extracts of co-transfected epidermal leaf cells. Nuclear extracts were prepared from YFP-rpL10A–transfected leaves (lanes 1 and 5), as well as from leaves co-transfected with YFP-rpL10A and NIK1-GFP (lanes 2 and 6), YFP-rpL10A and G473V/T474A-GFP (lanes 3 and 7), or YFP-rpL10A and T474D-GFP (lanes 4 and 8), separated by SDS-PAGE and immunoblotted using a GFP antibody. In lanes 5, 6, 7, and 8, the nuclear extracts were treated with alkaline phosphatase prior to electrophoresis.
Figure 2.
NIK1 associates detectably with rpL10A in vivo.
Tobacco leaves were agroinoculated carrying the DNA constructs as indicated on the top of the lanes. About 72 hours post-transfection, protein extracts were prepared from protoplasts of agroinoculated leaves and used for isolation of protein complexes with anti-GFP serum and protein A-Sepharose. Immunocomplexes were separated by SDS-PAGE and probed with either GFP antibody (A) or an anti-rpL10A serum (B).
Figure 3.
rpL10A knockout lines display enhanced susceptibility phenotype to geminivirus infection as nik1 null alleles.
(A) Detection of viral DNA in infected lines. Ecotype Col-0, nik1, and rpl10a lines at the seven-leaf stage were infected with an attenuated form of CaLCuV by biolistic delivery of tandemly repeated viral DNA-A and DNA-B. Total DNA was isolated from infected plants at 7 DPI, and viral DNA was detected with DNA-B-specific primers. IN refers to CaLCuV-inoculated plants and UN to mock-inoculated plants. + indicates control plasmid DNA as template. The positions of DNA standard markers are shown on the left in kbp. The gel shows a representative sampling of Col-0 and rpL10A infected plants. (B) Course of infection in rpl10a, nik1, and Col-O lines. Values represent the percentages of systemically infected plants at different days postinoculation (DPI). The data are the means of three independent experiments. In each experiment, 20 plants of each line were inoculated with 2 µg of tandemly repeated DNA-A plus DNA-B per plant. (C) Infection rates in rpl10a and in nik1 KO lines. The infection rate is expressed as number of DPI required to get 50% infected plants (DPI50%). Values for DPI50% are the mean±standard deviation from five replicas.
Figure 4.
Geminivirus infection interferes with the NIK1-mediated nuclear localization of rpL10A.
Tandemly repeated TGMV DNA-A and DNA-B were introduced into tobacco leaves by biolistic inoculation. Five days postinoculation, the infected leaves were co-agroinfiltrated with the combinations: YFP-L10 (A), YFP-L18 (B), L10-GFP and YFP-NSP (C), YFP-L10 and L18-GFP (D), YFP-L10 and NIK1-GFP (E). Full arrows indicate fluorescent nuclei, and traced arrows stomata. Scale bars are 10 µm. Bright is the corresponding transmitted light image of tobacco leaf epidermal cells.
Figure 5.
Overexpression of NIK1 in tomato delays virus infection and impacts development.
(A) NIK transcript accumulation in transgenic lines. Semi-quantitative RT–PCR was performed with cDNA prepared from WT seedlings, 35S:NIK1-4 (N4) or 35S:NIK1-6 (N6) transgenic lines with gene-specific primers, as indicated. Control reactions were conducted with polyA+ RNA from WT and transgenic cell lines without reverse transcriptase (C−) and with plasmid DNA (C+). (B) Symptoms associated with ToYSV infection in WT and 35S:NIK1-4 (35S-AtNIK1) T1 transgenic lines. WT plants and 35S:NIK1-4 (N4) T1 lines at the six-leaf stage were infected with ToYSV by biolistic delivery of tandemly repeated viral DNA-A and DNA-B. UN indicated plants that were bombarded with tungsten particle without viral DNA and IN shows infected plants at 28 days postinoculation. (C) Course of infection in WT and 35S-NIK1-4 lines. WT plants, T1 35S:NIK1-4 (N4), and T1 35S-NIK1-6 (N6) transgenic lines at the six-leaf stage were infected with ToYSV by biolistic delivery of tandemly repeated viral DNA-A and DNA-B. Values represent the percentages of systemically infected plants at different days postinoculation (DPI). The data are the means of three independent experiments. In each experiment, 20 plants of each line were inoculated with 2 µg of tandemly repeated DNA-A plus DNA-B per plant. (D) Growth rate of T0 transgenic (35S-AtNIK1) plants in comparison with untransformed, wild type plants. The growth rate of in-vitro-grown seedlings was measured as a function of the height of the plant. To evaluate growth rate, T0 primary transformants 35S-NIK1-4 (N4) and 35S-NIK1-6 (N6) as well as in-vitro–grown wild type (WT) lines were each replicated into ten uniformly sized clones that were allowed to regenerate for one week when height measurements were initiated (Day 0). The height was recorded again after 15 days of growth. (E) Developmental performance of T0 35S-AtNIK1 uninfected transgenic lines and WT plants (WT), 30 days after their transfer to the greenhouse.
Figure 6.
Model for NIK1-mediated signaling pathway.
Stress-induced oligomerization of the extracellular domain of NIK1 brings the intracellular kinase domains into proximity and allows them to transphosphorylate and activate one another. Upon activation, NIK1 phosphorylates rpL10A, promoting its translocation to the nucleus where it may mount a defense strategy that prevents virus proliferation of spread. Conversely, binding of NSP to the NIK1 kinase domain (A-Loop) inhibits autophosphorylation of NIK1 and, thus, prevents receptor kinase activation and signaling.