Regulation of NIK1 Activation Figure 9. Antagonistic roles of Thr-469 and Thr-474 phosphorylation sites in NIK-mediated defense signaling. Ecotype Columbia (Col- 0), nik1 lines or nik1 transgenic lines ectopically expressing wild type NIK1 (NIK1*), NIK1G473V/T474A double mutant kinase gene (mNIK-A and mNIK-B independently transformed lines), T474A or T469A transgenes were inoculated with CaLCuV by biolistic delivery. (A). Symptoms associated with CaLCuV infection in transformed knockout lines. On the top, the indicated plants were bombarded with tungsten particles without viral DNA. The bottom panels show infected plants at 14 days post-inoculation (DPI). The panels show representative samples of Col-0, nik1 and mNIK-A plants. (B). Detection of viral DNA in infected lines. Total DNA was isolated from infected plants at 7 DPI and detected with viral DNA-specific primers and transgene-specific primers in the same reaction. IN refers to CaLCuV-inoculated plants and UN to mock-inoculated plants. + indicates control plasmid DNA as template. The gel shows representative samples of Col-0 and mNIK-A plants. Each lane represents individual plants. The upper band (1.4 kb) is the amplified fragment from the transgene and the lower band (0.5 kb) is the viral DNA fragment. (C). Course of infection in nik1 transgenic lines ectopically expressing NIK1 (NIK*), NIK1G473V/T474A double mutant kinase gene (mNIK-A and mNIK-B), T469A or T474A transgenes. The values represent the percentages of systemically infected plants at different DPI50% and are given as mean of three determinations from independent experiments. (D). Infection rates in nik1 transgenic lines. The infection rate is expressed as number of DPI required to get 50% infected plants (DPI 50%). The data are means of four independent experiments. doi:10.1371/journal.pone.0005781.g009 mutants T469A and T474A to examine whether redirection of rpL10 to the nucleus would couple NIK activation to the defense response. The subcellular localization of rpL10 in co-transfected leaf cells was assayed by confocal microscopy (Figure S2, merged field) and the frequency of cells with nuclear-localized rpL10 was recorded (Figure 10). Ectopically expressed rpL10 in transfected cells is predominantly localized in the cytoplasm and only a small fraction (5%) of transfected cells contains nuclear rpL10. Co- expression with NIK1, however, altered the nucleocytoplasmic shuttling of rpL10 because the cell frequency with nucleus- localized YFP-rpL10 was significantly increased to 50.664% of co-transfected leaf cells. As expected [21], the hyperactive T474D mutant enhanced translocation of rpL10 to the nucleus (6868% of transfected cells with nuclear rpL10). While the inactive G473V/ T474A mutant [21] or the defective T474A mutant failed to redirect rpL10 to the nuclei of co-transfected cells, the T469A mutant was almost as effective as wild type NIK1 in mediating rpL10 nuclear translocation as it increased the frequency of co- transfected cells with nuclear rpL10 to 35613%. Although T469A did not enhance translocation of rpL10 to the nucleus as would be expected from its increased in vitro substrate phosphorylation activity when compared with wild type NIK1, these results showed that mutation at Thr-469 did not impair NIK-mediated nuclear relocation of rpL10, confirming in vivo that the Thr-469 and Thr- 474 phosphorylation sites play distinct roles in NIK activation. They also demonstrated that mutations at phosphorylation sites within the A-loop of NIK1 that antagonistically regulate kinase function acted similarly in conferring tolerance to virus infection and nuclear relocalization of rpL10. This positive correlation is consistent with the notion that the regulated nucleocytoplasmic shuttling of rpL10 links the antiviral response to receptor activation. Discussion The NIK receptor has been hypothesized to mediate an antiviral defense response through a reversible phosphorylation strategy that both initiates a signaling pathway as well as PLoS ONE | www.plosone.org 10 June 2009 | Volume 4 | Issue 6 | e5781