Download Systemic Delivery of siRNA by a Plant PHLOEM SMALL RNA

Systemic Delivery of siRNA by a Plant PHLOEM SMALL
RNA-BINDING PROTEIN1-Ribonucleoprotein Complex
Byung-Kook Ham, Gang Li, Weitao Jia, Julie A. Leary, and William J. Lucas
FULL LEGENDS FOR SUPPORTING INFORMATION
Supporting Figure Legends
Figure S1. Screen for protein kinase activity in fractionated pumpkin phloem exudates with a capacity to
phosphorylate mutated forms of CmPSRP1.
In-gel kinase assays performed on ion-exchange fractionated pumpkin phloem exudate proteins that were
separated on 12% SDS-PAGE gels containing purified recombinant CmPSRP1∆C or CmPSRP1Qm (100
µg/mL) as substrate. Recombinant CmPSRP1∆C and CmPSRP1Qm were expressed in and purified from
E. coli. - CmPSRP1, no embedded substrate in the SDS-PAGE gel.
Figure S2. CmPSRPK1 transcripts are confined to phloem companion cells.
(a) to (b) Transverse pumpkin vascular sections analyzed by in situ RT-PCR using CmPSRPK1 and
CmPP16 gene-specific primers. All images were collected with a confocal laser scanning microscope;
Green and yellowish signals indicate incorporation of Alexa Fluor-labeled nucleotides. Red signal
represents tissue autofluorescence. Bars = 300 µm in (B), (C) and (E) and 70 µm in (D) and (F).
(a) Negative control in which primer sets were omitted from the in situ RT-PCR mixture. IP and EP,
internal and external phloem, respectively; X, xylem; CA, cambium.
(b) Presence of CmPSRPK1 and (D) CmPP16 in phloem tissues. Note that yellowish (positive) signal is
produced by merging the green signal from Alexa Fluor-labeled nucleotides with the red tissue
autofluorescence. Note that CmPP16 transcripts are detected in both companion cells and sieve element
(Xoconostle-Cázares et al., 1999).
(c) and (e) High magnification of the boxed are in (B) and (D), respectively. CC, companion cell; SE,
sieve element.
Figure S3. CmPSRP1 phosphorylation is not required for its entry into the sieve tube system.
(a) and (b) Total proteins were extracted from infected tissues (A) and phloem exudate (B) of pumpkin
plants in which c-Myc4-His8 tagged rbcS, GFP, CmPSRP1 WT, Qm and ∆C were expressed using a
ZYMV vector. Total proteins extracted from infected tissues (10 µg) and phloem exudate (40 µg) were
separated on 13% SDS-PAGE gels and analyzed by protein gel blot analysis using anti-c-Myc mAb.
(b) CmPSRP1, Qm and ∆C were detected in phloem exudates, thereby indicating that phosphorylation of
CmPSRP1 is not required for its entry into the sieve tube system.
Figure S4. Dissociation constant (Kd) determined for the CmPSRP1∆C mutant.
In-planta expressed and purified CmPSRP1∆C was incubated with increasing concentrations of unlabeled
24-nt single-stranded sRNA, followed by addition of radiolabeled 24-nt single-stranded sRNA probe (10
fmol). CmPSRP1∆C sRNA mixtures were separated on a 5% polyacrylamide gel. Dissociation constant
(Kd) for CmPSRP1∆C was 34.8 nM.
Figure S5. Phosphatases in sink phloem exudate as well as in source and sink vascular tissues can
dephosphorylate a specific CmPSRP1 fraction.
Phloem exudate and vascular tissues were obtained from source and sink (shoot apex) regions of pumpkin
plants. In vitro CmPSRPK1-phosphorylated CmPSRP1 was mixed with phloem exudate, or co-IP
reaction buffer and then co-immunoprecipitated with anti-c-Myc monoclonal antibody. Co-IP products
were incubated with 400 µg of the indicated phloem exudate, or extracted proteins from vascular tissue
(VT) for 0 and 3 h, followed by separation on 12% SDS-PAGE gels, and then subjected to
autoradiography. Western blot analysis with anti-c-Myc monoclonal antibody served as the loading
control for these experiments.
(a) Phloem exudate collected from sink (SiPE), but not source (SoPE) regions of pumpkin plants could
partially dephosphorylate both the purified CmPSRP1 and CmPSRP1-sRNPC.
(b) Total protein extracts of VT harvested from source (SoVT) and sink (SiVT) regions of pumpkin plants
could partially dephosphorylate both the purified CmPSRP1 and CmPSRP1-sRNPC.
(c) CmPSRP1 co-IP products incubated in phosphatase reaction buffer, alone, retained their phosphorpeptide signal.
(d) Time course experiment in which CmPSRP1 co-IP products were mixed with SoVT or SiVT to assess
the ability of phosphatases to dephosphorylate the purified CmPSRP1 and CmPSRP1-sRNPC.
(e) Relative intensity of co-IP product bands present in (d). Each band intensity in (d) was normalized to
phosphatase reaction buffer treated co-IP products at each corresponding time point. Data represent mean
+ SD (n = four independent experiments) (*, p < 0.01, ANOVA).
Figure S6. Assembly of the CmPSRP1-sRNP complex prevents full dephosphorylation of CmPSRP1 in
the presence of CIP.
(a) In vitro CmPSRPK1-phosphorylated CmPSRP1 was mixed with co-IP reaction buffer, and subjected
to co-immunoprecipitation with anti-c-Myc monoclonal antibody. Co-IP products were then incubated
with sink phloem exudate (SiPE), calf intestinal alkaline phosphatase (CIP), SiPE + CIP, or phosphatase
reaction buffer (Buffer) for 0 and 60 min, separated on a 12% SDS-PAGE gel, and subjected to
autoradiography. Western blot assays were employed as loading controls for these experiments.
(b) Relative intensity of co-IP product bands present in (A). Data represent mean + SD (n = four
independent experiments) (*, p < 0.01, ANOVA).
Supporting Table 1. PCR primer sets employed in the current study.
Supporting Table 2. Sequence information for identified CmPSRP1 interacting proteins.
Supporting Experimental Procedures