| dc.description | 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. | |
