kinase activity. In support of this, MALDI-TOF/TOF experi- ments conducted on the phosphorylated form of the tryptic peptide 459LLDHQDSHVTTAVR472 demonstrated that Thr-469 can be phosphorylated. However, our data do not rule out the possibility that the putative relief of structural constraints was due to the introduction of alanine at the structurally restrictive position. The confirmation that this conserved threonine residue is phosphorylated in vivo is necessary to distinguish between these possibilities. The previous assignment of NIK as a defense gene was based on circumstantial evidence that linked the enhanced susceptibility phenotype to geminivirus infection to the loss of NIK function. Here we performed complementation assays in nik1 alleles, which demonstrated that NIK is an authentic defense signal transducer as it requires kinase activation to mediate an adaptive response to geminivirus infection. In fact, while the ectopic expression of NIK1 in the allele background restored the wild type phenotype by decreasing virus susceptibility, the expression of the inactive G473V/ T474A kinase did not reverse the nik1 enhanced susceptibility phenotype, which was associated with a high infection rate and development of severe symptoms upon infection. By examining the effects of the phosphorylation site mutations on NIK-mediated nuclear relocalization of rpL10, we found a perfect correlation on the ability of the mutations to affect tolerance and rpL10 nuclear relocation. The T474A mutant failed to restore the NIK1-mediated tolerance against geminivirus and to redirect rpL10 to nuclei of co- transfected cells. In contrast, mutation at Thr-469 did not impair NIK-mediated nuclear relocation of rpL10 or tolerance against geminivirus. Collectively, these results further substantiate the notion that relocation of rpL10 to the nucleus links the antiviral response to receptor activation. Furthermore, they confirmed in vivo the antagonistic role of the Thr-469 and Thr-474 phosphorylation sites in the regulation of NIK activation. They also suggest that the NIK1 residue Thr-474 is functionally equivalent to the corresponding BAK1 residue Thr-455 and BRI1 residue Thr-1049, which aligned at the same position in their respective A-loop and have been shown to be required for kinase function and signaling in planta [34,45]. There is no precedent in the literature for negative regulation of kinase activity through phosphorylation of conserved sites within the A-loop of plant RLKs. Although phosphorylation of threonine residues within the A-loop has been demonstrated in vitro for SERK1 and in vivo for BRI1 and BAK1, the functionally relevant phosphorylated residues regulate positively kinase activity [33,34,45]. Our results may have uncovered a novel scheme for kinase regulation in which autophosphorylation of Thr-469 within the NIK1 A-loop would control negatively the phosphorylation of rpL10 and hence would allow the kinase to control more efficiently the extent of the response in a sustained signaling pathway. Whether this inhibitory mechanism is specific for the rpL10 substrate, providing NIK1 with the capacity to differentially phosphorylate pathway components remains to be determined. In the case of BAK1, the activation-loop residue Thr-450, which is equivalent to NIK1 Thr-469, has been shown to play independent and separate roles in BR signaling and flagellin signaling [34]. Based on the present findings and common features of LRR- RLK, a mechanistic model for a NIK-mediated defense signaling pathway and its interaction with the geminivirus NSP can be proposed. In this model, upon unidentified stimuli, the LRR extracellular domain undergoes oligomerization that, in turn, brings the intracellular kinase domains into close proximity, allowing them to transphosphorylate and to activate one another. The active kinase recruits and phosphorylates the downstream component rpL10 to propagate the defense-signaling cascade that impairs virus replication and/or movement. Regulation of kinase Regulation of NIK1 Activation activity may be dictated by two components: a conserved Ser/Thr kinase activation component that results from autophosphoryla- tion at Thr-474 and a novel inhibitory component at a distinct residue (Thr-469) within the A-loop that down regulates substrate phosphorylation. Counteracting the pathway proactivation mech- anism, binding of NSP to the kinase domain promotes steric constraints that impair intermolecular phosphorylation at Thr-474 within the NIK A-loop. The argument that NSP acts upstream of Thr-474 autophosphorylation is supported by the finding that the hyperactive T474D mutant, in which the introduced carboxylate may mimic the phosphorylated state of Thr-474, keeps the capacity to form a stable complex with NSP in vitro but it is not inhibited by the viral protein. This molecular mechanism for NSP inhibition of NIK1 prevents activation of the NIK-mediated signaling pathway creating an intracellular environment that is more favorable to virus proliferation and spread. In summary, the relevance of the present study is two-fold. Firstly, it uncovers a novel scheme for receptor serine/threonine kinase activation with different roles for distinct conserved threonine residues within the A-loop of the kinase domain. The positive regulatory role of the Thr-474 phosphorylation site promotes kinase activation triggering the antiviral response, whereas the inhibitory role of Thr-469 on substrate phosphory- lation would allow the kinase to control more efficiently the extent of the response in a sustained signaling pathway. Secondly, it provides further evidence that regulated nucleocytoplasmic trafficking of rpL10 couples activation of the receptor NIK with an antiviral response. In fact, mutations at conserved threonine residues that antagonistically regulate kinase activation affect similarly the NIK capacity to elicit an antiviral response and to mediate a phosphorylation-dependent nuclear relocalization of the rpL10 downstream component. Materials and Methods DNA constructs and generation of point mutants The NIK1 activation loop mutations were obtained through the Gene TailorTM Site-directed Mutagenesis system (Invitrogen Life Technologies, Inc.) using the recombinant plasmid pDON-AtNIK1 [18] as template and partially overlapped primers (Table S1) that generated the nucleotide changes: Thr474 to Ala474 (pDON- NIK1T474A), or Glu474 (pDON-NIK1T474E) or Asp474 (pDON- NIK1T474D), Thr469 to Ala469 (pDON-NIK1T469A) as well as Gly473/Thr474 to Val473/Ala474 (pDON-NIK1G473VT474A). The resulting clones harboring single mutations were used to generate the double mutants: pDON-NIK1T468A/T469A, pDON-NIK1T474A/T468A, pDON-NIK1T474A/T469A, and pDON-NIK1T474A/S465A with the primers shown in Table S1. To create plasmids for E. coli expression, the mutant NIK1 C- terminal kinase domains (KD, encoding amino acids 297–638) were amplified from the appropriate mutated clone with the primers NBSAtNIK1F-GA (59-aaaaagcaggcttcacaatgaggagatttggtttc-39) and KDAtNIK1RG (59-agaaagctgggtctcatctaggaccagagagctc-39), intro- duced by recombination into the entry vector pDONR201 and then transferred to the bacterial expression vector pDEST15, resulting in GST fused to mutant kinase domains, such as pGST- KDNIK1T474A, pGST-KDNIK1T474D, pGST-KDNIK1T474E, pGST-KDNIK1G473V/T474A, pGST-KDNIK1T469A, pGST- KDNIK1T468A/T469A, pGST-KDNIK1T474A/T468A, pGST- KDNIK1T474A/T469A and pGST- KDNIK1T474A/S465A. The double mutant NIK1G473V/T474A cDNA, as well as T469A, T474A and T474D mutant cDNAs were also amplified from the respective mutated clones with the primers AtNIK1FG (59- aaaaagcaggcttcacaatggagagtactattgtt-39) and AtNIK1RGNS (59- PLoS ONE | www.plosone.org 12 June 2009 | Volume 4 | Issue 6 | e5781