Conserved threonine residues within the A-loop of the receptor NIK differentially regulate the kinase function required for antiviral signaling.
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.
Santos
Anésia A
AA
Departamento de Bioquímica e Biologia Molecular/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
Carvalho
Claudine M
CM
Florentino
Lilian H
LH
Ramos
Humberto J O
HJ
Fontes
Elizabeth P B
EP
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins; D002467:Cell Nucleus / Q000378:metabolism; D017791:Geminiviridae / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000737:chemistry*; D014026:Nicotiana / Q000378:metabolism; D014780:Viruses / Q000378:metabolism
D000998:Antiviral Agents; D029681:Arabidopsis Proteins; D012269:Ribosomal Proteins; C000630691:ribosomal protein L10, Arabidopsis; D013912:Threonine; D000409:Alanine