agaaagctgggtctctaggaccagagagctccat-39), re-introduced by recombi- nation into the entry vector pDONR201 and then transferred to the binary vector pK7FWG2 [46] to yield pK7F-NIK1G473VT474A, pK7F-NIK1T469A, pK7F-NIK1T474A and pK7F-NIK1T474D. These resulting clones harbor a GFP gene fused in-frame after the last codon of the respective mutant cDNAs under the control of the CaMV 35S promoter A ribosomal L10 cDNA was isolated from an Arabidopsis cDNA two-hybrid library by its capacity of interacting with the kinase domain of NIK1 [22]. The L10 cDNA was amplified by PCR with appropriate extensions provided by the primers rpL10Fwd (59-aaaaagcaggcttcacaatgggaagaagacctgt-39) and rpL10Rvs (59-agaaagctgggtctcagtagtgggctggcaaaaa-39), introduced by recombination into the entry vector pDONR201 and then transferred to pDEST15 to generate GST-fused L10 (pGST-L10). To obtain the YFP gene fused before the first codon of L10, the respective cDNA was transferred from pDONR207 to 35S-YFP- casseteA-Nos-pCAMBIA1300, yielding pYFP-L10 [21]. The plasmid pGST-NSP, harboring the CaLCuV NSP sequence fused to GST, has been described previously [18]. Expression in E. coli and purification of GST-fused proteins E. coli, strain BL21::DE3 pLysS, was transformed with plasmids containing different fusions and the synthesis of the recombinant proteins was induced with 0.4 mM isopropyl-dithiogalactopyrano- side (IPTG) for 16 h at 20uC and 200 rpm. The accumulation of recombinant proteins was monitored by SDS–PAGE in whole cell extracts, as well as in soluble and insoluble fractions. Cells were pelleted by centrifugation, resuspended in lysis buffer [140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 0.4% (v/v) Triton X-100 supplemented with 0.15 mg/ mL of lysozyme, 0.8 mM PMSF, 1 mM benzamidine, 1 mM thiourea], incubated at 4uC for 30 min, disrupted by sonication, and centrifuged at 14,000 g for 20 min at 4uC. The GST fusions were affinity-purified using GST-Sepharose beads (GE health- care), according to manufacturer’s instructions. The efficiency of protein purification was monitored by SDS-PAGE. Protein kinase assay The purified GST-fused proteins (KDNIK1T474A, KDNIK1T474D, KDNIK1T474E, KDNIK1G473V/T474A, KDNIK1T469A, KDNIK1T468A/T469A, KDNIK1T474A/ T468A, KDNIK1T474A/T469A, KDNIK1T474A/S465A) were incubated alone or with the substrate GST-L10 at 25uC for 45 min in 20 mL of kinase buffer containing 18 mM HEPES pH 7.4, 10 mM MgCl2, 10 mM MnSO4, 1 mM DTT, 10 mM ATP and 5–10 mCi [c-32P]ATP (3000 Ci/mmol). We also performed in vitro kinase assay under the same conditions as described for SERK1 mutants [33]. Phosphoproteins were resolved by SDS-PAGE. The gel was stained with Coomassie brilliant blue to verify protein loading, dried, and subjected to autoradiography. Incorporated radioactivity in protein bands was quantified by phosphoimaging and protein loading by densitom- etry using the Multi Gauge V3.0 software (Fujifilm). Alternatively, the protein bands were excised from the dried gel, dissolved in 30% (v/v) H2O2 and the radioactivity incorporated was measured by scintillation counting. MALDI-TOF/TOF analyses For MS/MS analyses, GST-fused proteins (KDNIK1, KDT474A) were phosphorylated by incubating 10–25 mg of the purified recombinant protein at 25uC for 75 min in 20 mL of Regulation of NIK1 Activation kinase buffer containing 18 mM HEPES pH 7.4, 10 mM MgCl2, 10 mM MnSO4, 1 mM DTT, 200 mM ATP. Phosphoproteins were resolved by SDS-PAGE. GST-KDNIK1 or GST-KDT474A spots were excised from SDS-PAGE gel, rinsed with distilled water and destained twice with 30-min washes in 50 mM NH4HCO3/ 50% acetonitrile. After dehydration in 100% acetonitrile, the gel pieces were incubated with 30 ml of 12.5 ng/ml sequencing grade trypsin (Promega, Madison, WI) for 12 h at 37uC. Tryptic peptides were extracted from the gel pieces twice with 15 ml of 1% formic acid/50% acetonitrile for 20 min. The pooled extracts were dried in speed-vac and tryptic digests were resuspended in 50% acetonitrile/1% phosphoric acid/1% TFA. MALDI-TOF/TOF experiments were conducted on an Ultra- flex MALDI-TOF/TOF Analyzer (Bruker Daltonics). Peptides from each spot were mixed 1:1 with matrix solution [2,5- dihydroxybenzoic acid (2,5-DHB) in 50% acetonitrile/0.1% trifluoroacetic acid/1% phosphoric acid] and 1 ml were applied to wells of a sample target plate. Peptide mass fingerprints were obtained using the reflector and positive ion mode. Mass spectra were collected from the sum of 200–800 laser shots, and monoisotopic peaks were obtained. LIFT mass spectra were acquired and metastable fragmentation was induced without the further use of collision gas. Peptides mass spectra were matched in silico against the GST- KDNIK1 theoretical tryptic mass using the PIUMS software with mass tolerance of 100 ppm, one missed trypsin cleavage, fixed modification of carbamidomethyl cysteine, and variable modifi- cations of methionine oxidation and threonine, tyrosine and serine phosphorylation. Peptides sequencing was made by visual inspection of mass spectrum using FlexAnalysis software. Phos- phorylated peptides were confirmed by an increase in mass of 80 Da and a decrease of 98.0 Da corresponding to the addition of PO3 and neutral loss of H3PO4, respectively. RT-PCR and qRT-PCR analyses Total RNA was extracted from Arabidopsis seedlings using TRIzol (Invitrogen). Reverse transcription (RT)-PCR assays were performed with 2 mg of total RNA, 0.5 mM of poly-dT and 1U of M-MLV reverse transcriptase (Invitrogen Life Technologies, Inc.), as previously described [47,48]. PCR was carried out with NIK1- specific primers (AtNIK1-Fwd, 59–accgcgatgtcaaagcagcg–39 and AtNIK1-Rvs, 59- atgtgacccaccgtgcctct –39) and with actin-specific primers (AtACTIN-Fwd, 59–atgtcgtgagccatcctgtc–39 and AtAC- TIN-Rvs, 59–acaccggattcgtgcggcat–39) to assess the quantity and quality of the cDNA. The PCR comprised 30 cycles of 45 s at 94uC, 30 s at 55uC, and 2 min at 72uC. Real-time RT-PCR reactions were performed on an ABI7500 instrument (Applied Biosystems, Foster City, CA), using SYBRH Green PCR Master Mix (Applied Biosystems), as described [49,50]. The amplification reactions were performed as follows: 2 min at 50uC, 10 min at 95uC, and 40 cycles of 94uC for 15 sec and 60uC for 1 min. To confirm quality and primer specificity, we verified the size of amplification products after electrophoresis through a 1.5% agarose gel, and analyzed the Tm (melting temperature) of amplification products in a dissociation curve, performed by the ABI7500 instrument. The RNA actin was used as endogenous control to normalize all values in the real-time RT-PCR assays [49]. Gene expression was quantified using the 2DCT method. Plant material, growth conditions, and plant transformation The Columbia (Col-0) ecotype of Arabidopsis thaliana was used as the wild type control and nik1 knockout line [18] was used for complementation experiments. Seeds were surface sterilized and PLoS ONE | www.plosone.org 13 June 2009 | Volume 4 | Issue 6 | e5781