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Experimental procedures
Introduction of Arabidopsis BACs into the recombineering E.coli host SW102
Arabidopsis BAC clones pBELOBAC11_T4P13 (CmR), carrying the AKIN10 (At3g01090)
gene, and pBELOBAC-KAN_ F7G19 (KmR) harbouring SNF4 (At1g09020) were obtained
from the Arabidopsis Biological Resource Center (ABRC, Ohio, USA). BAC DNAs were
isolated and introduced by electroporation (0.2 cm cuvettes, 2.5 kV, 25 μF, 200 Ω) into the
recombineering host E. coli SW102 strain as described (Warming et al., 2005). To exclude
possible rearrangements and deletions, BACs were isolated both from original ABRC stocks
and E. coli SW102 by CsCl-ethidium bromide equilibrium density gradient centrifugation
(Sambrook et al., 1989) and subjected to fingerprinting with at least three different restriction
endonucleases yielding 10 to 15 fragments, the size of which was predicted based on known
BAC sequences available at the TAIR database (http://www.arabidopsis.org). SW102 BAC
strains were propagated at 30°C by maintaining continuous selection for their
chloramphenicol (Cm, 25μg/ml) and kanamycin (Km, 25μg/ml) markers in LB (LuriaBertani) medium (Sambrook et al., 1989). Induction of Redαβγ was performed by incubation
of cultures grown to OD600 of 0.5 for 15 min at 42°C before preparation of electro-competent
cells for recombineering according to (Warming et al., 2005 and Experimental procedures).
Construction of pGAP-Km and pGAP-Hyg Agrobacterium binary gap-repair vectors
To construct the gap-repair vector pGAP-Km, a pBR322 segment of pPCV002
Agrobacterium binary vector (Koncz et al., 1994), which shares short sequence homology
upstream of the promoter of ampicillin resistance (β-lactamase, bla/AmpR) gene with vector
backbones of all sequenced Arabidopsis BACs, was modified. A ClaI-EcoRI pBR322
segment of pPCV002 was removed and replaced by a region of pBlueScript SK- (pBSKStratagene), which lacks the lacZ gene and flanking polylinker region between the promoter
of bla/AmpR gene and replication origin (positions 280 and 1001). This pBSK- segment was
PCR amplified using the primers Ecorev (5’-ggggtgttgGAATTCgggcgaaaaaccgtctatcaggg-3’)
and Clafor (5’-gggaagaggATCGAT gggagaggcggtttgcgtattg-3’), and following EcoRI and
ClaI digestion was ligated to a 5.6 Kb fragment of pPCV002. The resulting vector pGAP-Km
(7890bp) carries a polylinker with multiple cloning sites, a Tn5 aph(3’)II amynoglycoside
phosphotransferase plant selectable marker gene between nopaline synthase (Nos) promoter
and Ocs (octopine synthase) polyadenylation sequences, left and right T-DNA borders (LB
and RB), AmpR bacterial selectable marker, and conditional replication and transfer origins
(oriV and oriT) of plasmid RK2, which are activated by the trfA and Tra (i.e., TraJ,K) gene
products encoded by the helper Ti plasmid of Agrobacterium strain GV3101 (pMP90RK)
(Koncz et al., 1994; Figure 2).
To provide an alternative selectable marker for potential combination of gene
constructs obtained by BAC-recombineering in plants, a second gap-repair vector pGAP-Hyg
was constructed with a hygromycin-B phosphotransferase (hph) gene as plant selectable
marker using segments of pPCV002, pPCV812-δGUS and pBSK- vectors. pBSK- sequences
carrying the AmpR gene and replication origin were PCR amplified using the H3 rev (5’ggggtgttgAAGCTTgggcgaaaaaccgtctatcaggg-3’) and above described Clafor primers, and
digested with HindIII and ClaI. A ClaI-KpnI fragment of 0.58Kb, carrying the T-DNA left
border was isolated from pPCV002. These fragments were ligated to a HindIII-KpnI fragment
of pPCV812-δGUS, which carried the hph gene between Nos promoter and terminator, the
right T-DNA border sequence, and conditional replication and conjugational transfer origins
of plasmid RK2 (oriV and oriT). To introduce an EcoRI site into the polylinker, the HindIIIKpnI pBSK- region of resulting intermediate plasmid pGAP-H1 was replaced with that of
pGAP-Km to construct pGAP-Hyg-EcoRI. This second plasmid intermediate carried a
duplicated segment of Nos polyadenylation region between the polylinker and Nos promoter.
Because even a short sequence duplication would promote internal recombination in the
recombineering host, this region was removed. A BamHI-NcoI PCR fragment extending from
the 5’-end of the Nos-promoter to the NcoI site of hph gene was PCR amplified from pGAPHyg-EcoRI, using the primers NOSp5’ (5’-ggggtgttgGGATCCcgctgccgcaagcac tcagg-3’) and
NOS-Nco (5’-cgaacccgctcgtctggctaag-3’), and inserted between the BamHI site of polylinker
and unique NcoI site of hph gene of the same plasmid to obtain pGAP-Hyg (7942 bp, Figure
1). Complete nucleotide sequence and annotation of pGAP-Km and pGAP-Hyg vectors were
deposited in the GenBank database under the accession numbers EU933992 and EU933993,
respectively.
Similarly to all other pPCV vectors (Koncz et al., 1994), both pGAP-Km and pGAPHyg are transferrable into Agrobacterium GV301 (pMP90RK) by conjugation from E. coli
stains (e.g., S17-1) that provide chromosomally integrated plasmid RK2 helper functions
(Simon et al., 1983; Koncz et al., 1994). As the helper Ti-plasmid pMP90RK also carries RK2
genes for plasmid conjugation (Koncz et al., 1994), the binary gap-repair vectors can be reconjugated by the help of their RK2 oriT sequences from GV3101(pMP90RK) into E. coli for
testing the stability of large plant DNA inserts in Agrobacterium. As conjugation frequencies
range between 0.1 and 1.0% of recipient cell number, it is also sufficient to streak out bacteria
from the conjugation spots on selective medium to select for plasmid transfer between E. coli
and GV301(pMP90RK) in both directions. Alternatively, the gap-repair vectors can be
introduced into GV3101(pMP90RK) by electroporation using conditions described for E. coli
(Warming et al., 2005) and recovered after plasmid preparation from Agrobacterium by E.
coli transformation (Sambrook et al., 1989).
Co-immunopecipitation assays
Plant material was grown in solid MS medium with 0.5% sucrose at short-day conditions (8h
light/16h dark). Seedlings transformed with the pXCSG-YFP-HA construct expressing a
control YFP protein with a C-terminal hemagglutinin (HA) tag were used as control in the
immunoprecipitation and protein kinase assays (García et al., 2010). Total protein extracts
were prepared from 9 days old seedlings carrying the AKIN10-GFP, SNF4-YFP and control
35S-YFP constructs using extraction buffer (50 mM Tris-HCl (pH 7.8), 1 mM EGTA, 1 mM
EDTA, 2 mM DTT, 5 mM NaF, 1 mM Na3VO4, 0.05 Nonidet P-40, 10 % glycerol, 0.5 mM
PMSF and 1 x protease inhibitor cocktail [Sigma]). 500 μg protein extract was mixed with 25
μL GFP-Trap®-M magnetic beads (Chromotek), and incubated for 2h at 4°C with rotation.
After collecting the beads on a magnetic platform, the supernatant was removed and beads
were washed extensively three times with 10 volumes of wash buffer (10 mM Tris-HCl [pH
7.5], 150 mM NaCl, 0.5 mM EDTA, 1mM PMSF and 1x protease inhibitor cocktail). Beads
were resuspended in SDS-PAGE protein loading buffer. An aliquot of eluted proteins was
separated on 10% SDS-PAGE and subjected to western blot by incubating with either antiAKIN10 antibody (1:200 dilution) or anti-SNF4 antibody (1:500 dilution). The western blot
were then treated with goat anti-rabbit antibody (1: 10,000 dilution BioRad) and subjected to
Enhanced Chemoluminesce Detection (ECL) detection by autoradiography. The anti-AKIN10
antibody was raised in rabbit by the Eurogentec Co. (Belgium) against an AKIN10-specific
peptide (TMEGTPRMHPAESV) and affinity purified on the same peptide carrying a Cterminal cysteine residue for immobilization, as described by Németh et al. (1998) for an antiPRL1 peptide antibody. After stripping, the western blot membranes were incubated with a
mouse anti-actin antibody (clone C4, Millipore, 1:5000 dilution) and developed by further
incubation with a goat anti-mouse secondary antibody (1: 10,000 dilution, Thermo Scientific)
followed by ECL detection.
Protein kinase assays
To perform the SnRK1 protein kinase assays, total protein extracts from 9 days old AKIN10GFP, SNF4-YFP and control 35S-YFP seedlings were prepared as described above. 100 μg of
total protein was incubated for 2 h with 10 μg of anti-GFP antibody (Roche). Subsequently,
20 μL of Protein G Sepharose (Amersham) was added and incubated for 3 hrs at 4°C with
rotation, washed three times with 10 volumes of extraction buffer (see above) and
resuspended in 20μl kinase buffer (50mM Tris-HCl [pH 7.8], 15mM MgCl2, 5mM EGTA,
1mM DTT). Subsequently, 10 μg of each substrate and 5 μCi of [γ-32P]ATP were added to the
matrix-bound immunoprecipitates in a final volume of 50 μl to perform kinase assays for 30
minutes at 30°C. Equal aliquots (15 μl) from the kinase assays were loaded on 10% SDSPAGE and, following electrophoresis the gels were exposed to X-ray film for 15 min to detect
the phosphorylated kinase substrates.
Expression and purification of recombinant SnRK1 substrate protein TRX-KD,
carrying a SnRK1 substrate peptide from spinach sucrose-phosphate synthase fused to Nterminal thioredoxin and C-terminal His6 tags was performed as described by (Bhalerao et al,
1999). Coding regions ABI5 (At2G36270) and EEL/DPBF4 (At2G41070) cDNAs lacking the
stop codons were PCR amplified with primers DPBF4fwd-DPBFrev and ABI5fwr-ABI5rev
(Supplementary Table S1). The PCR amplified EEL/DPBF4 coding region was cloned as
NdeI-SalI fragment, whereas the ABI5 coding region was inserted as XhoI fragment in SalIXhoI sites of vector pET201 (Bhalerao et al., 1999). To express ABI5 and EEL/DBPF4 fused
to N-terminal thioredoxin (TRX) and C-terminal His6 tags, the pET201 constructs were
transformed into E. coli strain BL21DE3 pLysS (Promega). Protein expression was induced
by addition of 1mM IPTG to bacterial cultures grown to optical density at OD600: 0.6 in 500
ml LB medium (Sambrook et al., 1989), and the cultures were incubated for 5 hrs at 37°C.
Subsequently, cells were harvested by centrifugation and homogenized by sonication in
lysis/binding buffer (40mM Na-phosphate [pH 8.0], 300mM NaCl, 10% glycerol, 1%
TritonX-100, 5mM 2-mercaptoethanol, protease inhibitors [1 mM PMSF, 5 µg/ml leupeptine,
1 mM benzamidine hydrochloride and 1 µg/ml of each pepstatin and aprotinin]). A cleared
lysate was prepared by centrifugation (25,000g for 30min) at 4°C, and the supernatant was
applied onto a gravity flow Ni-NTA column (Qiagen) of 2 ml bed volume (binding capacity
5-10 mg protein/ml bed volume), which was pre-equilibrated with lysis/binding buffer at 4°C.
The column was washed with 100 ml wash buffer (lysis/binding buffer supplemented with
40mM imidazole), and then the TRX-His6-tagged ABI5 and EEL/DPBF4 proteins were
eluted by 8 x 1 ml elution buffer (40mM Na-phosphate [pH 8.0], 300mM NaCl, 5mM 2mercaptoethanol, 250mM imidazole). The eluted fractions were analyzed by 10% SDS-PAGE
followed by Coomassie brilliant blue staining. The peak fractions were pooled and dialysed
against storage buffer 20mM Tris-HCl (pH 8.0), 50mM NaCl, 5mM 2-mercaptoethanol, 10%
glycerol.
Supplementary References
García, A.V., Blanvillain-Baufumé, S., Huibers, R.P., Wiermer, M., Li, G., Gobbato, E.,
Rietz, S. and Parker, J.E. (2010) Balanced nuclear and cytoplasmic activities of EDS1 are
required for a complete plant innate immune response. PLoS Pathog. 6, e1000970.
Koncz, C., Martini, N., Szabados, L., Hrouda, M., Bachmair, A. and Schell, J. (1994)
Specialized vectors for gene tagging and expression studies. In Gelvin S and Schilperoort B
(eds.), Plant Molecular Biology Manual, Kluwer Academic, Dordrecht, B2, pp. 1-22.
Németh, K., Salchert, K., Putnoky, P., Bhalerao, R., Koncz-Kálmán, Z., StankovicStangeland, B., Bakó, L., Mathur, J., Ökrész, L., Stabel, S., Geigenberger, P., Stitt, M., Rédei,
G.P., Schell, J. and Konczm C, (1998) Control of glucose and hormone responses by PRL1, a
nuclear WD-protein, in Arabidopsis. Genes Dev. 12, 3059-3073.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Simon, R., Priefer, U.B., and Pühler, A (1983) A broad host range mobilization system for in
vivo genetic engineering; transposon mutagenesis in Gram negative bacteria. Bio/Technology
1, 784-891.
Warming, S., Costantino, N., Court, D.L., Jenkins, N.A. and Copeland, N.G. (2005) Simple
and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33, e36.