cold treated at 4uC for 2 days in the dark and then exposed to white light. Seedlings were grown at 22uC on plates containing Murashige-Skoog medium for 3 weeks and then transferred to soil. Plants were grown in a growth chamber at 22uC under long-day conditions (16 h light/8 h dark). nik1 KO lines were transformed with pK7F-NIK1 [18], pK7F-NIK1G473VT474A, pK7F- NIK1T469A or pK7F-NIK1T474A using the floral dip method [51]. Transgene incorporation was monitored by PCR using a forward 35S promoter-specific primer, MC36 (59-tccttcgcaa- gacccttcctc-39), and a reverse NIK1-specific primer, LRAt- NIK1RG (59-agaaagctgggtcgagtcataagagatttcgatg- 39). Transgene expression was confirmed by real time PCR with the primers AtNIK1-Fwd and AtNIK1-Rvs. For quantitation of gene expression, we used actin as a control gene and the primers AtACTIN-Fwd and AtACTIN-Rvs, as described above. We selected two independently transformed lines expressing the NIK1G473VT474A transgene (mNIK-A and mNIK-B), one nik1 line expressing NIK1 (NIK1*), T474A or T469A transgene for the infection assays. Accumulation of NIK mutant proteins in planta Nicotiana tabacunn leaves were agroinoculated with pK7F-NIK1 [18], pK7F-NIK1G473V/T474A, pK7F-NIK1T469A, pK7F- NIK1T474A and pK7F-NIK1T474D using Agrobacterium tumefa- ciens strain GV3101, as previously described [28]. About 72 hours pos-agroinfiltration, 1-cm2 leaf explants were excised and fluorescence patterns were examined in epidermal cells using a Zeiss inverted LSM510 META laser scanning microscope with an argon laser and a helium n´eon laser with 406 or 606 oil immersion objective. For imaging GFP, the 488 nm excitation line and the 500 to 530 nm band pass filter were used. The pinhole was usually set to give a 1 to 1.5 mm optical slice. Post-acquisition image processing was done using the LSM 5 Browser software (Carl- Zeiss) and Adobe Photoshop (Adobe Systems). CaLCuV inoculation and analysis of infected plants Arabidopsis thaliana plants at the seven-leaf stage were inoculated with plasmids containing partial tandem repeats of CaLCuV DNA-A and DNA-B by biolistic delivery [52] and the course of infection was monitored as described previously [18,30]. We used an attenuated form of the virus, in which the coat protein ORF in CaLCuV DNA-A was interrupted by introducing a stop codon at amino acid position 47. The inoculated plants were transferred to growth chamber and examined for symptom development (leaf necrosis, chlorosis, leaf epinasty, leaf curly, young leaf death and stunted growth). Total nucleic acid was extracted from systemi- cally infected leaves and viral DNA was detected by PCR with DNA-A or DNA-B begomovirus-specific primers [53]. In multiplex PCRs the stably incorporated transgene was also examined using the transgene-specific primers (MC36 and LRAtNIK1RG) in combination with DNA-B CaLCuV-specific primers (566CLCVBFBR1v, 59-ggcgtggggtatcttactc-39 and 1253CLCVBRBR1c, 59-gacatagcatcggacatcc-39). In each experi- ment, 20 plants of each line (Col-0, nik1, nik-1 expressing NIK1 mutant proteins) were inoculated with 2 mg of tandemly repeated DNA-A plus DNA-B per plant. The course of infection was registered with data from three independent experiments. DP150% (days post- inoculation to reach 50% of infected plants) was obtained with data from four independent experiments. Subcellular localization of proteins For subcellular localization of proteins, Nicotiana tabacum leaves were agroinoculated with pK7F-L10 or pYFP-L10 in combination Regulation of NIK1 Activation with pK7F-NIK1, pK7F-NIK1T474D, pK7F-NIK1T474A, pK7F-NIK1T469A or pK7F-NIK1G473V/T474A using Agrobac- terium tumefaciens strain GV3101. Nicotiana tabacum plants were grown in a greenhouse with natural day length illumination. For experimental use, plants (about three week after germination) were transferred to a growth chamber at 21uC with a 16-hour light and 8-hour dark cycle. Agrobacterium-mediated transient expression in tobacco leaf epidermal cells was conducted as previously described [21]. About 72 hours post-agroinfiltration, 1-cm2 leaf explants were excised and GFP and YFP fluorescence patterns were examined in epidermal cells with 406 or 606 oil immersion objective and a Zeiss inverted LSM510 META laser scanning microscope equipped with an argon laser and a helium laser as excitation source. For imaging GFP, the 458–488 nm excitation line and the 500 to 530 nm band pass filter were used. Excitation of YFP was at 514–560 nm and YFP emission was detected by using a 560–600 nm filter. Controls were performed to ensure clear separation of GFP and YFP signals. The pinhole was usually set to give a 1 to 1.5 mm optical slice. In each independent experiment, a total of 100 to 150 cells were observed and the number of cells with nucleus-localized rpL10 was recorded. Supporting Information Figure S1 SDS-PAGE of E. coli- produced GST fusions. GST-fused to the C-terminal kinase domain of normal NIK1 (GST-KDNIK1) or to mutant NIK1s, as indicated, were produced in E. coli, affinity-purified, separated by SDS/PAGE and stained with coomassie brilliant blue. GST-L10 corresponds to a ribosomal protein L10 (rpL10) fused to GST and GST-NSP is a CaLCuV NSP fusion. Molecular mass markers (kDa) are shown on the left. Found at: doi:10.1371/journal.pone.0005781.s001 (1.05 MB TIF) Figure S2 Effect of ectopic expression of NIK1 and A-loop mutants on nucleocytoplasmic shuttling of rpL10A. NIK1- GFP+YFP-L10, T469A-GFP+YFP-L10, T474D-GFP+YFP-L10 or T474A-GFP+YFP-L10 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 (merged field) with rpL10A localized within the nuclei was obtained. In each experiment, a total of 100 to 150 cells were observed. Full arrows indicate fluorescent nuclei. Scale bars are 10 mm Found at: doi:10.1371/journal.pone.0005781.s002 (5.15 MB TIF) Table S1 Primers for mutagenesis within the kinase domain of NIK1 Found at: doi:10.1371/journal.pone.0005781.s003 (0.04 MB DOC) Acknowledgments We are grateful to Dr Rebecca Boston and Dr Niki Robertson (North Carolina State Universtiy, USA) for critically reading the manuscript. We thank Anete Valente (Universidade Federal de Vic¸osa, Brazil) for the help with real time PCR and the UFV microscopy core facility for the use of the LSCM. Author Contributions Conceived and designed the experiments: AAS CMC HJOR EPBF. Performed the experiments: AAS CMC LHF HJOR. Analyzed the data: AAS CMC HJOR EPBF. Contributed reagents/materials/analysis tools: EPBF. Wrote the paper: AAS EPBF. PLoS ONE | www.plosone.org 14 June 2009 | Volume 4 | Issue 6 | e5781