Regulation of NIK1 Activation Figure 7. Substrate phosphorylation activity of NIK1 and mutants on rpL10. Purified GST fusions (as indicated) were incubated with equal amounts of GST-L10 in the presence of [c-32P]ATP and separated by SDS-PAGE (A). The gels were stained with Coomassie-brilliant blue (bottom) and visualized by autoradiography using a phosphoimager (top). Protein loading was normalized by densitometric scanning of the Coomassie-stained gel. The relative activity of autophosphorylation (B) and phosphorylation of the substrate (C) were quantified and expressed as Vunits/mg enzyme/ min and Vunits/mg enzyme/mg substrate/min, respectively. doi:10.1371/journal.pone.0005781.g007 expressing the G473V/T474A mutant protein (mNIK-A and mNIK-B), and transformed lines expressing T469A or T474A mutant proteins (Figure 8A). In addition, we also demonstrated by transient expression in tobacco leaves that the mutant receptors fused to GFP accumulated to detectable levels in transfected plants, as they displayed the same fluorescence pattern as a GFP- fused wild type kinase (Figure 8B). Immunoblots showed that NIK1 and mutant proteins fused to GFP accumulated to similar levels when transiently expressed in tobacco leaves [data not shown, 21]. These results confirmed that the mutations in the kinase domain of NIK1 did not affect the capacity of the protein to be associated with the cell surface and to accumulate stably in transformed plants. The transgenic lines expressing the intact NIK1 (NIK*), the double mutant protein (mNIK-A and mNIK-B), T469A and T474A mutants were biolistically inoculated with an attenuated form of CaLCuV [18]. All inoculated lines developed typical symptoms of CaLCuV infection although with different intensity (Figure 9A and data not shown). The accumulation of viral DNA was detected in all symptomatic plants by PCR with transgene-specific and viral DNA-specific multiplex primers (Figure 9B). Ectopic expression of wild type NIK1 in nik1 KO lines restored the wild type phenotype with respect to susceptibility to geminivirus infection, as the transgenic lines developed attenuated symptoms (mild stunting with epinasty and moderate chlorosis) and displayed similar infection rates as Col-0 (Figure 9C). In other independent experiments, the infectivity data, expressed as DPI50% (days post-inoculation to reach 50% of infected plants), further confirmed that NIK* and Col-0 displayed the same PLoS ONE | www.plosone.org 8 June 2009 | Volume 4 | Issue 6 | e5781