Conserved Threonine Residues within the A-Loop of the Receptor NIK Differentially Regulate the Kinase Function Required for Antiviral Signaling

Abstract

NSP-interacting kinase (NIK1) is a receptor-like kinase identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues. Mutations at Thr-474 or Thr-468 impaired autophosphorylation and were defective for kinase activation. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. To dissect the functional significance of these results, we used NSP-expressing virus infection as a mechanism to interfere with wild type and mutant NIK1 action in plants. The NIK1 knockout mutant shows enhanced susceptibility to virus infections, a phenotype that could be complemented with ectopic expression of a 35S-NIK1 or 35S-T469A NIK1 transgenes. However, ectopic expression of an inactive kinase or the 35S-T474A NIK1 mutant did not reverse the enhanced susceptibility phenotype of knockout lines, demonstrating that Thr-474 autophosphorylation was needed to transduce a defense response to geminiviruses. Furthermore, mutations at Thr-474 and Thr-469 residues antagonistically affected NIK-mediated nuclear relocation of the downstream effector rpL10. These results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation.

Plant immunity against viruses: antiviral immune receptors in focus

“…These interactions suppress the NIK kinase activity and prevent the activation of the antiviral signal transduction pathway, creating a suitable environment for begomovirus infection (Santos et al, 2009(Santos et al, , 2010. Consistent with a role for NIK in antiviral defence, loss-of-function nik1, nik2 and nik3 mutants showed enhanced susceptibility to CaLCuV infection (Fontes et al, 2004;Rocha et al, 2008;Santos et al, 2009). In addition, overexpression of NIK1 delays viral infection and attenuates symptom development in tomato (Carvalho et al, 2008).…”

“…NIK homologues from arabidopsis, tomato and soybean interact with NSPs from Cabbage leaf curl virus (CaLCuV) and from tomato-infecting begomoviruses, such as Tomato golden mosaic virus (TGMV), Tomato crinkle leaf yellow virus (TCrLYV) and Tomato yellow spot virus (ToYSV) (Fontes et al, 2004;Mariano et al, 2004;Sakamoto et al, 2012). These interactions suppress the NIK kinase activity and prevent the activation of the antiviral signal transduction pathway, creating a suitable environment for begomovirus infection (Santos et al, 2009(Santos et al, , 2010. Consistent with a role for NIK in antiviral defence, loss-of-function nik1, nik2 and nik3 mutants showed enhanced susceptibility to CaLCuV infection (Fontes et al, 2004;Rocha et al, 2008;Santos et al, 2009).…”

“…In addition, overexpression of NIK1 delays viral infection and attenuates symptom development in tomato (Carvalho et al, 2008). Finally, mutations in the activation loop (A-loop) of NIK1 that prevent its autophosphorylation also compromise the capacity of NIK1 to elicit a response against begomoviruses (Santos et al, 2009). …”

“…This region is highly conserved among members of the LRR-RLK II subfamily and other members of the extended LRR-RLK family. The phosphorylation status of the activation segment has been shown to dictate NIK1 kinase activity (Carvalho et al, 2008;Fontes et al, 2004;Santos et al, 2009). NIK1 is phosphorylated in vitro at the conserved positions Thr474 and Thr469, and mutations in the A-loop compromise its autophosphorylation capacity (Santos et al, 2009).…”

“…The phosphorylation status of the activation segment has been shown to dictate NIK1 kinase activity (Carvalho et al, 2008;Fontes et al, 2004;Santos et al, 2009). NIK1 is phosphorylated in vitro at the conserved positions Thr474 and Thr469, and mutations in the A-loop compromise its autophosphorylation capacity (Santos et al, 2009). Replacement of Thr474 with alanine strongly inhibits the autophosphorylation activity and the capacity of NIK1 to elicit a defence response, whereas replacement of Thr474 with a phosphomimetic aspartate residue increases autophosphorylation activity and results in constitutive activation of a NIK1 mutant receptor that it is no longer inhibited by begomovirus NSP (Santos et al, 2009).…”