Supplemental Methods
Plasmid Constructions and Generation of Transgenic Arabidopsis Plants
The PAP1-ENTRY vector and the PAP2-pENTR vector were described previously
(Zimmermann et al., 2004). For generation of transgenic 35S:HA-PAP1 and 35S:HA-PAP2
lines, the ORF of PAP1 or PAP2, respectively, was cloned into pEarleyGate201 (Earley et
al., 2006) using Gateway® LR technology. For generation of 35S:PAP1 plants, the ORF of
PAP1 was cloned into pEarleyGate100 (Earley et al., 2006). All constructs were transformed
into Col-0 wild-type plants.
To screen for COP1/SPA-interacting proteins using the yeast two-hybrid system, the
ORFs of SPA1, SPA4 and COP1 were recombined into pDEST32 (Invitrogen) and
subsequently used as bait vectors, in addition to COP1-GBKT7 (Hoecker and Quail, 2001).
For yeast two-hybrid and three-hybrid analyses, the vectors AD-PAP1, AD-PAP2
(Zimmermann et al., 2004) and SPA4-GBKT7 (BD-SPA4) (Laubinger and Hoecker, 2003)
were described previously. For SPA1-GBKT7 (BD-SPA1), SPA2-GBKT7 (BD-SPA2), the fulllength ORFs of SPA1 or SPA2, respectively, were ligated into pGBKT7 (Clontech). The BDSPA3 construct was created by recombining the ORF of SPA3 into the pAS2-1-attR
destination vector (Clontech, modified; (Uhrig et al., 2004)) using Gateway® technology. To
compare the interaction of PAP2 with COP1 and COP1K550E, the ORFs of COP1, GFP and
HY5 were first cloned into pDONR207 using Gateway technology. To generate pDONR207COP1K550E and pDONR207-GFP-CID, inverse PCR was performed on pDONR207-COP1
and pDONR207-GFP, respectively, with subsequent re-ligation. Sequences of the primers
used are provided in Table S1. These pDONR207 entry vectors were subsequently
recombined into the destination vectors pAS2-1-attR, pACT-attR or pBridge (Clontech,
modified; Uhrig et al., 2004) to obtain BD-GFP, BD-COP1 (Figure 2a), BD-COP1K550E, BDHY5, AD-COP1, AD-GFP and pBridge (BD/ProMet25:GFP-CID or GFP, respectively).
Subsequently, BD was replaced in these pBridge vectors by BD-HY5 (HpaI/BamHI) or BDCOP1 (HpaI/SalI), respectively, which were excised from pAS2-1-HY5 and pAS2-1-COP1,
respectively. These cloning steps generated BD-HY5/ProMet25:GFP-CID or GFP, respectively,
and BD-COP1/ProMet25:GFP-CID or GFP, respectively.
The SPA1-pENSG-CFP (CFP-SPA1) CFP-talin constructs used for co-localization
experiments in onion epidermal cells were described previously (Saedler et al., 2004; Zhu et
al., 2008). To generate CFP-COP1, the ORF of COP1 was cloned by BP reaction into
pDONR201 (Life technologiesTM) and subsequently into pENSG-CFP (Laubinger et al., 2006)
as destination vector. YFP-PAP1 and YFP-PAP2 were generated by LR reaction using
pEarleyGate104 (Earley et al., 2006) and PAP1-pENTRY or PAP2-pENTR as entry clones.
For co-localization experiments in Arabidopsis suspension culture and pull down assays from
Nicotiana benthamiana leaves, the ORF of COP1 was cloned into pNmR (A. Schrader and
J.F. Uhrig, unpublished) using Gateway® technology to obtain 35S:RFP-HA-COP1.
The
ORF of PAP2 was recombined from PAP2-pENTR into pEarleyGate104 (Earley et al., 2006)
using Gateway® technology to obtain 35S:YFP-PAP2. As negative controls, 35S:RFP-HA
(pNmR) and 35S:YFP (pEarleyGate104) were used.
Yeast Two-Hybrid and Three-Hybrid Interaction Assays
To generate the SPA1-pDEST32, SPA4-pDEST32, COP1-pDEST32 and COP1-GBKT7
vectors used as baits, the ORFs were cloned into pDEST32 (Invitrogen).
The spotted
libraries were screened as described previously (Soellick and Uhrig, 2001; Castrillo et al.,
2011) on dropout media containing 0.5 - 3 mM 3-AT. To confirm interactions, constructs
were transformed into the yeast strain AH109. Selection was carried out on synthetic dropout
medium lacking leucine (-Leu), tryptophan (-Trp) and histidine (-His) according to the
manufacturer’s instructions (Clontech). To analyze the strength of interactions media were
supplemented with 3-AT at the indicated concentrations. For serial yeast drop test single
colonies from transformed yeast cells were grown over-night in liquid dropout medium
lacking leucine and tryptophan until OD600 of 0.6. Ten μl cell suspension was then
resuspended in 100 μl sterile water of which 10 μl was subsequently dropped onto drop out
selection media as indicated.
In yeast three-hybrid experiments, pBridge vectors coding for the binding domain (BD),
BD-COP1 or BD-HY5 were used in co-transformation with AD-COP1, AD-PAP2 or AD-GFP.
pBridge contains a methionine (Met) suppressible promoter positioned upstream of a
Gateway cassette. GFP or GFP-CID expression was gradually suppressed using increasing
methionine concentrations in -Leu -Trp -His -Met dropout plates supplemented with 3 mM
(HY5) or 5 mM (COP1) 3-AT. For each combination, 10 colonies selected on dropout
medium -Leu -Trp were resuspended in water, the OD600nm was adjusted to 0.7 and 20 µl
was streaked out on the respective plates. Co-transformation of BD-COP1 and AD-COP1
served as a control for yeast growth affected by lack or addition of methionine.
The -galactosidase assay was performed according to the Clontech manual (Yeast
Protocols Handbook) for MTP scale.
In Vivo Pulldown Assays
For in vivo protein-protein interaction assays, Arabidopsis cell suspension cultures were
transfected with Agrobacterium tumefaciens strain LBA4404.pBBR1MCS.virGN54D (van der
Fits et al., 2000) carrying PAP1-pEarleyGate201, PAP2-pEarleyGate201 or empty vector
pEarleyGate201, respectively, (Earley et al., 2006) were grown for 24 h in liquid YEB
medium at 28 °C while shaking at 180 rpm. The bacterial cultures were spun down and
resuspended in 1 ml of Arabidopsis cell culture medium. Ten ml of the Arabidopsis cell
culture was diluted with 40 ml of cell culture medium, and 250 µl of Agrobacterium
suspension was added. Additionally, anti-silencing strain Agrobacterium 19K (Voinnet et al.,
1999) was added and cell cultures were grown for 6 days. For in vivo protein pull-down
assays, 1.25 g of the cell suspension culture was ground to a fine powder under liquid
nitrogen. Two ml of lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 10%
glycerol, 5 mM DTT, 1% protease inhibitor (complete Mini, Roche), 10 µM MG132, 0.4%
Triton X-100) was added. After thawing, the samples were spun down 12 min, 20.000 x g at
4 °C. Fifty µl of the supernatant served as input sample. The lysates were incubated with 50
µl anti-HA affinity matrix (Roche) for 3 h at 4 °C on a rotary shaker. After incubation, the resin
was washed three times with lysis buffer and resuspended in 2 x Laemmli buffer.
Leaves of N. benthamiana were transiently co-infiltrated with supervirulent A.
tumefaciens strain LBA4404.pBBR1MCS.virGN54D (pEGATE104-PAP1 and pEGATE104PAP2) or LBA4404pBBR1MCS-5.virGN54D (for all other plasmids) (van der Fits et al., 2000)
harboring the different plasmids and the antisilencing Agrobacteria strain 19K (Voinnet et al.,
1999) as described earlier (Gigolashvili et al., 2007). Plants were kept at 24°C under long
day conditions for 4 days after infiltration and prior to protein analysis. Protein extracts were
prepared from co-infiltrated leaves of N. benthamiana expressing YFP-PAP2 and RFP-HACOP1 fusions under control of the Ca MV35S promoter. Proper expression of both fusion
proteins was tested by CLSM. Approx. 530 – 540 mg of the successfully infiltrated leaf areas
(as determined with a Leica MZ FL III fluorescence binocular) were lysed as described
previously (Kirik et al., 2007) and subsequently used for co-immunoprecipitation with antiGFP MicroBeads according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch
Gladbach, Germany). RFP-HA and YFP fusion proteins were visualized by protein gel
blotting (primary antibody: rat anti-HA or mouse anti-GFP (Roche, Mannheim, Germany);
secondary antibody: horseradish peroxidase–conjugated goat anti-rat or goat anti-mouse
(Jackson, Suffolk, UK)). Chemiluminescence signals were visualized with a LAS-4000 mini
luminescent image analyzer (Fujifilm Europe, Düsseldorf, Germany). RFP-HA or YFP served
as negative controls.
Expression of GST-PAP2 in E. coli and in vitro ubiquitination assays
GST-PAP2 protein was expressed and purified as described in Zimmermann et al., (2004).
In vitro ubiquitination assays were performed as previously described (Datta et al., 2008) with
minor modifications. Ubiquitination reactio
Biochem), 50ng rice His-Rad6 E2 (Yamamoto et al. 2004)
ng yeast E1 (Boston
µg unlabelled ubiquitin
(Boston Biochem), 25ng GST-PAP2 (or 200 ng HA-STH3 in the case of the positive control)
and 2µg maltose-binding-protein-COP1 (MBP-COP1) (previously incubated with 20 µM
ZnCl2) in reaction buffer containing 50mM Tris at pH 7.5, 5mM MgCl2, 2mM ATP and 0.5mM
DTT. MBP-COP1 that was not incubated with ZnCl2 (-Zn) was used as a negative control.
After 2h incubation at 30oC, reaction mixtures were stopped by adding sample buffer, and an
half of the mixtures (30µl) were separated onto 7.5% SDS-PAGE gels. GST-PAP2 and HASTH3 were detected using anti-GST (Sigma) and anti-STH3 (Datta et al., 2008) antibodies,
respectively.
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