Regulation of NIK1 Activation Figure 8. Ectopic expression of mutated NIK1 genes. (A). Expression of intact and mutated NIK1 genes in nik1 KO lines. Knockout lines were stably transformed with wild type NIK1 (NIK1*), G473V/T474A (mNIK1-A and mNIK1-B independently transformed lines), T469A or T474A mutant constructs and transgene expression was analyzed by real time RT-PCR. Col-0 corresponds to the expression of endogenous NIK1 in wild type background. Values are given as mean6SD from three replicates. (B). Subcellular localization of NIK1-GFP and NIK mutant-GFP fusion proteins in Nicotiana tabacum leaves. Confocal fluorescence images from epidermal cells of Nicotiana tabacum leaves 72 hours after agroinoculation with NIK1- GFP, G473V/T474A-GFP, T474A-GFP, T474AD-GFP or T469A-GFP, under the control of the 35S promoter. doi:10.1371/journal.pone.0005781.g008 efficiency of virus infection (Figure 9D). In contrast, ectopic expression of the G473A/T474A inactive kinase in KO lines (mNIK-A and mNIK-B) did not complement the nik1 loss-of- function defect and the independent transgenic lines kept a similarly enhanced susceptibility phenotype as the nik1 line (Figures 9C and 9D). Conversely, the mutant T469A with a high substrate phosphorylation activity complemented the nik1 loss-of- function defect as efficiently as the NIK1 transgene (NIK*) and the T469A transformed line displayed an infection rate similar to NIK1* (Figure 9C), with DPI50% similar to Col-0 and NIK1* (Figure 9D). Ectopic expression of T474A defective kinase did not fully complement the nik1 loss-of-function phenotype and an enhanced susceptibility phenotype in T474A lines was apparent from 12 days post-inoculation as compared to that of NIK* and T469A lines (Figure 9C). DPI50% resulting from T474A expression was slightly higher than that of the inactive kinase (mNIK-A and mNIK-B) lines, but yet significantly lower than that of NIK* and T469A lines (Figure 9D). These results establish that NIK1 functions as an authentic defense receptor as it requires activation to mediate an antiviral signaling. Furthermore, they confirmed in vivo that phosphorylation at position Thr-474 is essential for full NIK1 kinase activation and defense signaling as opposed to phosphorylation at position Thr-469 that is likely to repress substrate phosphorylation activity. Thr-474 and Thr-469 residues sustain their antagonistic roles in NIK-mediated nuclear relocalization of rpL10 The T474D mutant was not included in our complementation assay because we could not recover T474D expressing trans- formed lines. However, the relevance of this mutation has been recently addressed in vivo by the demonstration that the T474D mutant exhibits an enhanced capacity to phosphorylate and relocate rpL10 from the cytoplasm to the nucleus when transiently expressed in tobacco infiltrated leaves [21]. NIK-mediated nuclear relocalization of rpL10 is thought to trigger a defense response that impairs virus proliferation or spread. We used the antagonistic PLoS ONE | www.plosone.org 9 June 2009 | Volume 4 | Issue 6 | e5781