Fontes* Abstract Conserved Threonine Residues within the A-Loop of the Receptor NIK Differentially Regulate the Kinase Function Required for Antiviral Signaling Ane´ sia A. Santos, Claudine M. Carvalho¤, Lilian H. Florentino, Humberto J. O. Ramos, Elizabeth P. B. Departamento de Bioqu´ımica e Biologia Molecular/BIOAGRO, Universidade Federal de Vic¸osa, Vic¸osa, Minas Gerais, Brazil 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. Citation: Santos AA, Carvalho CM, Florentino LH, Ramos HJO, Fontes EPB (2009) Conserved Threonine Residues within the A-Loop of the Receptor NIK Differentially Regulate the Kinase Function Required for Antiviral Signaling. PLoS ONE 4(6): e5781. doi:10.1371/journal.pone.0005781 Editor: Juergen Kroymann, CNRS UMR 8079/Universite´ Paris-Sud, France Received February 2, 2009; Accepted May 12, 2009; Published June 3, 2009 Copyright: 2009 Santos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Brazilian Government Agencies CNPq grants 573600/2008-2 and 470249/2008-0, FAPEMIG grant EDT 523/07, and FINEP grant 01.07.0610.00 (to EPBF). AAS and HJOR are CNPq (150275/2007-2) and CAPES (Prodoc) postdoctoral fellows, respectively. LHF was supported by a graduate fellowship from CAPES. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bbfontes@ufv.br € Current address: Departamento de Fitopatologia, Universidade Federal de Vic¸osa, Vic¸osa, Minas Gerais, Brazil Introduction The perception of external stimuli through cell surface receptors is a common mechanism of multicellular organisms that allows communication among cells and between cells and the external environment. In plants, an extensive battery of Ser/Thr receptor- like kinases (RLK) may transduce external signals into cells through the reversible phosphorylations that allow the cells to sense and respond to external signals in a precise, regulated and adaptive way [1]. In the Arabidopsis genome, the RLK family is represented by 417 sequences that are organized into a typical receptor configuration harboring an N-terminal extracellular domain followed by a transmembrane segment and a kinase domain at the C-terminus [1]. These receptors have been shown to be predominantly involved in developmental events and defense strategies. Characterized members of this family include BRI1 and BAK1 involved in brassinosteroid signaling [2,3,4], HAESA associated with floral abscission regulation [5], SERK1 associated with early embryogenesis [6], BONZAI responsible for growth capacity under different temperature conditions [7], ERECTA and CLAVATA1 that control size and shape of flowers [8,9], RLK2 that controls anther development [10] and typical defense proteins, such as FLS2 [11], BAK1 [12,13] and the rice Xa-21 protein [14]. The representatives of the Arabidopsis RLK superfamily have been phylogenetically organized into families based on the structural identity of extracellular domains and conservation of C-terminal kinase domains [1,15,16]. The major family comprises the leucine-rich repeats (LRR)-RLKs which contain three to 26 LRR motifs in their extracellular domains [1]. Further subdivision into 13 sub-families (LRRI-LRRXIII) is based on sequence identity and organization of the LRR domain. One of these, the LRRII-RLK subfamily, is constituted by 14 proteins harboring four complete LRRs (with 24 residues) and a fifth incomplete LRR (with 16 residues) arranged in a single continuous block within the extracellular domain [17]. Phylogenetic analysis based on sequence conservation and evolutionary structural features of LRRII-RLKs has demonstrated that the members of this subfamily are clustered into three distinct branches of functional relatedness: (i) antiviral defense proteins, (ii) developmental proteins and (iii) functionally unassigned proteins. The NSP- PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5781