Supporting Experimental Procedures
Plasmid Construction
To engineer CmPSRP1 mutants, CmPSRP1 cDNA was used as template and amplified by polymerase
chain reaction (PCR), using the primer sets for mutagenesis provided in Table S1. Amplified cDNAs
were subcloned into pCR-Blunt II TOPO vector (Invitrogen), and confirmed by sequencing. For co-IP
assays and in-planta recombinant protein expression, CmPSRP1, CmPSRP1Qm and CmPSRP1ΔC were
subcloned into a Zucchini yellow mosaic virus (ZYMV)-based viral vector, as described previously (Ma
et al., 2010; Yoo et al., 2013). For E. coli-based c-Myc4-His8-tagged recombinant protein expression,
pET30b (Novagen) was employed, except for GFP, which carried a His6-tag. CmPSRP1, CmPSRP1Qm,
CmPSRP1ΔC, CmPSRPK1 and GFP subcloned in the ZYMV vector were used as templates, and
amplified using the primer pairs listed in Table S1. PCR products were subcloned into pCR-Blunt II
TOPO vector. NdeI-XhoI fragments from the pCR-Blunt II TOPO vector with CmPSRP1, CmPSRP1Qm,
CmPSRP1ΔC, CmPSRPK1, or GFP were recloned into the NdeI-XhoI site of pET30b.
Cloning CmPSRPK1
The ion-exchange fractions with CmPSRP1-specific phosphorylation activity were separated on a 13%
SDS-PAGE gel, and then the 40 kDa protein band with phosphorylation activity was excised for in-gel
trypsin digestion. The proteolytic peptides were processed for mass spectrometry, as described previously
(Ham et al., 2009). These mass spectral data were interrogated to identify sequences associated with a
protein kinase of the appropriate mass. To identify the CmPSRPK1 cDNA, total RNA extracted from
pumpkin vascular tissue was reverse transcribed by using SuperScript II (Invitrogen), according to
manufacturer’s instructions. The open reading frame region of the CmPSRPK1 cDNA was amplified by
PCR using the primer set provided in Table S1 online. Amplified CmPSRPK1 cDNA was subcloned into
pCR-Blunt II TOPO vector and confirmed by sequencing.
Immunoblot Analysis
Immunoblot analyses were performed, as described previously (Ham et al., 2009). Briefly, the relevant
primary antibody preparations were used for immunoblotting on nitrocellulose membrane; anti-c-Myc
monoclonal antibody (mAb) (Sigma) at 1:5,000 dilution, anti-ZYMV CP Ab at 1:1,000 dilution, anti-PP2
Ab at 1:2,000 dilution, anti-Rubisco Ab at 1:1,000 dilution, and anti-phospho-serine mAb, anti-phosphothreonine mAb and anti-phospho-tyrosine mAb (each from Calbiochem) at 1:1,000 dilution in the
blocking buffer (5% nonfat milk in 1 X tris-buffered saline). Conditioned membranes were incubated
with horseradish peroxidase-conjugated anti-rabbit or -mouse (1:20,000 or 1:50,000 dilution, respectively;
Sigma) in the blocking buffer and then developed with chemiluminescence reagent (Perkin-Elmer Life
Science).
In-gel Kinase Assays
In-gel kinase assays were performed as previously described (Lee et al., 2005). Briefly, pumpkin phloem
exudate proteins were fractionated by fast protein liquid chromatography (FPLC), as described previously
(Yoo et al., 2004). E. coli-expressed CmPSRP1, CmPSRP1ΔC, CmPSRP1Qm and GFP were purified
using a nickel affinity column, as described previously (Yoo et al., 2004) and employed as substrate.
Fractionated pumpkin phloem exudate proteins were separated on a substrate-containing SDS-PAGE (100
µg/mL of each substrate). The SDS-PAGE gels were washed (4x15 min) in SDS removal buffer (20%
[V/V] 2-propanol, 50 mM Tris-HCl, pH8.0), followed by incubation with the denaturation buffer (50 mM
Tris-HCl, pH8.0, 20 mM DTT, and 6M guanidine hydrochloride). Fractionated phloem exudate proteins
were renatured by incubation in renaturation buffer (50 mM Tris-HCl, pH8.0, 5 mM DTT, 0.04% Tween20, 100 mM NaCl, and 5 mM MgCl2) and then gels were equilibrated in kinase assay buffer (25 mM
HEPES, pH7.4, 20 mM MgCl2, 1 mM MnCl2, 10 mM NaF, 1 mM DTT, and 10 mM β-glycerophosphate).
Conditioned gels were incubated with kinase assay buffer containing 50 µM ATP and [γ 32P]-ATP (20
Ci/mL) for in-gel phosphorylation and the reaction was terminated by incubating the gels with gel wash
solution (5% TCA [V/V] and 1% [W/V] sodium pyrophosphate).
In-situ RT-PCR Analysis
In situ RT-PCR analyses were conducted as described previously (Ham et al., 2009). Briefly, pumpkin
stem sections were pretreated with DNaseI for 15 min to eliminate genomic DNA and then followed by in
situ RT-PCR. The primer pairs were used for amplifying CmPSRPK1 and CmPP16, listed in Table S1. A
confocal laser scanning microscope (model DM RXE 6 TCS-SP2 AOBS; Leica Microsystems) using an
Ar/ArKr and a GHeNe laser was applied for fluorescence analysis. All images were processed with
Adobe Photoshop version CS2.
Supporting References
Lee, J.-Y., Taoka, K., Yoo, B.-C., Ben-Nissan, G., Kim, D.-J., and Lucas, W.J. (2005) Plasmodesmalassociated protein kinase in tobacco and Arabidopsis recognizes a subset of non-cell-autonomous proteins.
Plant Cell 17, 2817-2831.