Molecular Size, Complexity, and Potential Targets for Phloem Small RNA Species.
(A) and (B) Size distribution and complexity, respectively, of the small RNA species contained within a phloem database (10,000 clones) generated from summer-grown pumpkin (sap collected from mature petioles). nt, nucleotides.
(C) Representative putative target genes of phloem small RNA with identified homology to cucurbit ESTs and/or Arabidopsis genes. Distribution of sense (above target gene; black, 0; green, 1; red, 2; and blue, 3 mismatches, respectively) and antisense (below target gene; colors as described for sense) clones directed against the indicated genes. Targets: cucurbit TnL1 and TnL2; cucurbit small RNA identical to Arabidopsis miR159 proposed to target a MYB transcription factor (GenBank accession number At2g32460); putative MT (homologous to a spinach gene [GenBank accession number AF237633]); bifunctional End (homologous to a Zinnia elegans gene [GenBank accession number O80326]) and RNA Hel (homologous to a Vigna radiata gene [GenBank accession number AF156667]). The size classes directed against the TnL and Myb genes were centered on 21 nucleotides, whereas those associated with MT, End, and Hel were in the 23- to 24-nucleotide range.

Identification of RNA Species in the Phloem of Spontaneously Silencing CP Transgenic Squash Lines.
(A) RNA gel blot analysis of RNA extracted from spontaneously silencing [3(S) and 127(S)] and nonsilencing [22(NS)] squash lines expressing the CP of Squash mosaic virus ([SqMV]; Pang et al., 2000). Top panels: hybridization analysis performed with CP and 18S probes. Bottom panels: small RNA detected using antisense (as) CP riboprobe; loading control provided by ethidium bromide staining (EtBr).
(B) RNA gel blot analyses performed on phloem sap collected from squash lines in (A), using antisense and sense (s) riboprobes. LC, loading control. Bottom panel: phloem sap integrity confirmed by RT-PCR using rbcS primers.
(C) Comparison of small RNA populations present in the phloem sap of wild-type and CP transgenic spontaneously silencing (line 127) squash plants.
(D) ssRNA-specific RNase assay.
(E) RNA gel blot analysis performed on phloem sap collected from heterografted plants, using antisense riboprobe. Positive signals were detected in phloem sap collected from both the stock (St, squash) and cucumber scion (Sc) samples taken from heterografted 127(S) plants, a spontaneously silencing line. Signal was not detected in the phloem from heterografted nonsilencing CP transgenic line 22(NS) nor from homografted wild-type squash or cucumber (C) plants. Equivalent results were obtained using sense riboprobe.
(F) RNA gel blot analysis of SqMV CP RNA (top panel) and siRNA (bottom panel) extracted from the scion apex of control [homografted 3(S) and 22(NS)] and heterografted [3(S) stock:22(S) scion] plants. Loading controls: 18S and 5S riboprobes.
(G) RT-PCR analysis detected sense and antisense CP transcripts in phloem sap collected from summer-grown squash. CP primers were used to amplify full-length transcripts (600 bp) from phloem sap and leaf RNA samples collected from wild-type and CP expressing squash lines. RT-PCR performed with internal CP primers gave similar results. Controls for these experiments used primers for CmPP16 (400 bp) and rbcS (500 bp). Lanes are as follows: 1, CmPP16; 2, rbcS; 3, CP sRNA; 4, CP antisense RNA.

Identification of a Small RNA Binding Protein Present in Pumpkin Phloem Sap.
(A) Pumpkin phloem sap fast protein liquid chromatography (FPLC)-fractionated proteins.
(B) RNA overlay-protein blot assay performed on FPLC-fractionated proteins from (A) using a CmRINGP-specific riboprobe (Ruiz-Medrano et al., 1999). Note the complement of phloem RNA binding proteins (PRB) capable of recognizing this phloem-mobile mRNA.
(C) Detection of a 27-kD PRB (arrowhead) by a monoclonal anti-His₆ antibody.
(D) to (I) Northwestern assays performed on FPLC-fractionated phloem proteins from (A) using the indicated forms of small RNA riboprobes. Note that all probes bound to a 27-kD PRB.

Purification, Cloning, and Expression of PSRP1.
(A) Purification of the 27-kD PSRP1 from pumpkin phloem sap using a combination of Q-Sepharose and metal chelation chromatography. Protein profiles contained within the anion-exchange fractions (L, loading; FT, flow-through; E, elution) were resolved by 10% SDS-PAGE (an equal volume [20 μL] was loaded for each fraction). Protein profiles for metal chelation resolved as above (FT, flow-through; W1/ 2, washes).
(B) Conceptual translation of CmPSRP1 (GenBank accession number AY326308) yielded a 20,454-D protein. The predicted molecular mass was consistent with the value of 21,004 D as determined by mass spectroscopy with the 27-kD PSRP1 in (A).
(C) Purification of recombinant (R)-PSRP1 using metal chelation chromatography. Proteins from each step were resolved by 10% SDS-PAGE and visualized using GelCode Blue.

In Situ RT-PCR–Based Detection of CmPSRP1 Transcripts in the Vascular System.
(A) Schematic transverse section of a portion of the pumpkin petiole. Vascular bundles are comprised of internal and external phloem (IP and EP, respectively) and xylem (X), with an intervening cambium (yellow).
(B) Confocal laser scanning microscopy image of a pumpkin petiole (transverse section) demonstrating the presence of CmPSRP1 RNA (green signal represents incorporation of Alexa Fluor–labeled nucleotides) in phloem cells.
(C) Equivalent cellular pattern detected for CmPP16 (Xoconostle-Cázares et al., 1999).
(D) Negative control in which primers were omitted from the RT-PCR reaction mixture. Red fluorescence represents tissue autofluorescence, and the green signal associated with the xylem reflects residual nonspecific binding of unincorporated Alexa Fluor–labeled nucleotides. Bar in (D) = 100 μm, common to (B) and (C).

Small RNA Population Detected in the Pumpkin Phloem Translocation Stream.
(A) Small RNA species present within the phloem sap and vegetative tissues of pumpkin were extracted, end-labeled with ³²P-phosphate, separated using PAGE, and then visualized by autoradiography. Left top and bottom panels: samples from summer- and winter-grown plants, respectively. Loading control (LC): a constant high molecular weight band present in the unfractionated phloem sap RNA was used for between sample calibration. Right top and bottom panels: apical and mature leaf tissues from summer-grown plants and ethidium bromide–stained 5S rRNA as loading control, respectively (0.3 μg per lane). nt, nucleotides.
(B) Small RNA species detected in the phloem sap of cucumber, white lupin, caster bean, and yucca.
(C) ssRNA-specific RNase assay performed on control (synthetic 25-nucleotide ssRNA and 2-nucleotide 3′ 25-nucleotide dsRNA) and phloem small RNA preparations. Note the absence of signal associated with the synthetic 25-nucleotide ssRNA and low residual level in the phloem RNA population after treatment.

RNA Gel Blot Analysis of miRNA in Various Pumpkin Tissues and Phloem Sap.
Total RNA was extracted from shoot apices (SA), stem tissue (S), mature leaves (L), and phloem sap (P) and the small RNA species extracted by size fractionation. Duplicate RNA samples were separated on a denaturing polyacrylamide gel, transferred to Hybond-N⁺ nylon membrane, and hybridized with either radiolabeled DNA sense (s) or antisense (as) probes complementary to four identified plant miRNAs (miR156, miR159, miR167, and miR171; Reinhart et al., 2002). Position of RNA oligonucleotide standards is indicated on the right. RNA loading was normalized by spectrometry, and 2 μg of small RNA was used per lane. nt, nucleotides.

Identification of RNA Species in the Phloem of Virus-Infected Pumpkin Plants.
(A) Distribution of sense (green) and antisense (red) clones directed against both RNA 1 and RNA 2 of the CuYV genome. Viral open reading frames are as described (Hartono et al., 2003), and the presence of viral transcripts in the phloem sap was confirmed using standard protocols.
(B) Size distribution of clones reflected in phloem small RNA databases prepared from healthy and CuYV-infected pumpkin plants. Inset, small RNA populations present in healthy (H) and infected (I) phloem sap. Bioinformatic analysis revealed that 57% of the small RNA clones from CuYV-infected plants displayed 100% identity to this viral genome, a majority 20- to 21-nucleotide size class. Database for healthy plants lacked any clones having sequence homology to CuYV. nt, nucleotides.

Identification of a Small RNA Binding Protein Present in Cucumber and Lupin Phloem Sap.
(A) Cucumber phloem sap FPLC-fractionated proteins.
(B) Northwestern assay performed on FPLC-fractionated proteins from (A) using a CmRINGP-specific riboprobe. Note the set of cucumber PRBs capable of recognizing this pumpkin phloem-mobile mRNA.
(C) and (D) RNA overlay-protein blot assays performed on FPLC-fractionated phloem proteins from (A) using the indicated forms of small RNA riboprobes. Note that both probes bound to a single PSRP1 in the 27-kD size range.
(E) Detection of a 27-kD PSRP1 by a monoclonal anti-His₆ antibody.
(F) Lupin phloem sap FPLC-fractionated proteins.
(G) RNA overlay-protein blot assay performed on FPLC-fractionated proteins from (A) using a CmRINGP-specific riboprobe. Note the set of lupin PRB capable of recognizing this pumpkin phloem-mobile mRNA.
(H) and (I) RNA overlay-protein blot assays performed on FPLC-fractionated phloem proteins from (F) using the indicated forms of small RNA riboprobes. Note that the ssRNA probe bound to a 27-kD protein, whereas the dsRNA probe bound to a 55-kD protein.
(J) Detection of a 27-kD PSRP1 by a monoclonal anti-His₆ antibody.

Recombinant PSRP1 Displayed Form- and Size-Specific RNA Binding Properties.
(A) Gel mobility-shift assays performed using R-PSRP1 and ssRNA and dsRNA probes (10 fmol). nt, nucleotides.
(B) and (C) Competition experiments performed by preincubating R-PSRP1 (0.25 μg) with different concentrations of unlabeled ssRNA or dsRNA, respectively, followed by competition with ³²P-labeled 25-nucleotide ssRNA (10 fmol). R-PSRP1 dissociation constants (K_d) for 25-nucleotide ssRNA and 2-nucleotide 3′ 25-nucleotide dsRNA were 3.13 × 10⁻⁸ M and 3.06 × 10⁻⁵ M, respectively.
(D) Competition experiments performed with ssRNA of various lengths. Purified R-PSRP1 (0.2 μg) was mixed with different amounts (molar excess indicated) of unlabeled 25-nucleotide (lanes 3 to 5), 45-nucleotide (lanes 6 to 8), 100-nucleotide (lanes 9 to 11), 400-nucleotide (lanes 12 to 14), or 1000-nucleotide (lanes 15 to 17) ssRNA molecules, followed by addition of radioactively labeled 25-nucleotide ssRNA (10 fmol) probe. Complexes were analyzed by 5% PAGE. Lane 1, free probe only; lane 2, probe with R-PSRP1 only. Different length for each competitor RNA was taken into account in calculating the molar excess concentration. Note that R-PSRP1 bound preferentially to ssRNA molecules in the following order: 25 nucleotides > 45 nucleotides > 100 nucleotides = 400 nucleotides = 1000 nucleotides.

PSRP1 Can Mediate Form-Specific Cell-to-Cell Movement of Small RNA.
(A) and (B) Retention in the target cell of microinjected fluorescently labeled synthetic 25-nucleotide ssRNA (A) or dsRNA (B). Inset shows low-magnification image of entire cell.
(C) and (D) Cell-to-cell trafficking of KN1 through PD potentiated extensive movement of FITC-labeled 20-kD dextran ([D], green signal), but the coinjected 25-nucleotide ssRNA (red signal) was unable to diffuse out of the target cell (C).
(E) Phloem-purified CmPSRP1 mediated cell-to-cell movement of coinjected fluorescently labeled (green) 25-nucleotide ssRNA.
(F) Equivalent experiment to that presented in (E), demonstrating movement of both 20-kD dextran (green) and 25-nucleotide ssRNA (red). Note the confinement of the fluorescent signals to neighboring cells ([E] and [F]).
(G) and (H) Neither 25-nucleotide dsRNA (G) nor 25-nucleotide ssDNA (H) moved from the target cell when coinjected with phloem-purified CmPSRP1. All images were collected by confocal microscopy 20 min after injection into mesophyll cells in mature leaves of Nicotiana benthamiana. Bars = 100 μm; (C) common to (D) to (H).