The Plant Cell Wall Matrix Harbors a Precursor of Defense Signaling Peptides

The plant cell wall matrix harbors a precursor of defense signaling peptides

Javier Narva´ ez-Va´ squez*, Gregory Pearce, and Clarence A. Ryan†

Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340

Contributed by Clarence A. Ryan, July 22, 2005 Proteins of plant cell walls serve as structural macromolecules and powder, and stored at Ϫ80°C until used. Genomic DNA was play important roles in morphogenesis and development but have extracted from Ϸ5 g of fresh weight leaf tissue by using DNAzol not been reported to be the origins of peptide signals that activate (Invitrogen) and following the manufacturer’s protocol. Five genes for plant defense. We report here that the mRNA coding the micrograms of genomic DNA was separately digested with tomato leaf polyprotein precursor of three hydroxyproline-rich restriction enzymes, fractionated on agarose gels, transferred to glycopeptide defense signals (called LeHypSys I, II, and III) is nylon membranes, and probed with a random-primed 32P- synthesized in phloem parenchyma cells in response to wounding, labeled LepreproHypSys cDNA, and the hybridized membranes systemin, and methyl jasmonate, and the nascent protein is se- were washed as described in ref. 10. questered in the cell wall matrix. These ﬁndings indicate that the plant cell wall can play an active role in defense as a source of In Situ Hybridization. In situ hybridization analyses to visualize peptide signals for systemic wound signaling. LepreproHypSys mRNA were carried out by using 10-␮m par- affin sections of leaf and petioles of tomato plants with methods phloem parenchyma ͉ tomato leaves ͉ wound signals ͉ plant defense described in ref. 9. Sections were hybridized with in vitro transcribed digoxigenin-labeled LepreproHypSys sense or anti- eptide hormone signaling in plants is a growing area of sense RNA probes, synthesized from linearized pBluescript Presearch in which nearly 20 peptide signals (hormones) have plasmids containing a full-length LepreproHypSys cDNA (7). been identified to date that regulate genes for cell division, Hybridized probes were colorimetrically detected with anti- development, reproduction, nodulation, and defense (1–8). digoxigenin antibodies conjugated with alkaline phosphatase Plant peptide signals have several characteristics that are found (Roche Applied Science, Indianapolis). Sections were mounted in animals and yeast peptide hormones (1) that include being in DPX embedding medium (Electron Microscopy Science, derived from larger precursor proteins, being receptor mediated, Hatfield, PA), examined, and photographed with an Olympus and being active at low nanomolar concentrations. Three novel BH2 light microscope. hydroxyproline-rich glycopeptide signals (LeHypSys I, II, and III) were recently isolated from tomato (Lycopersicon esculen- Expression of a LepreproHypSys-GFP Fusion Gene. A LepreproHyp- tum) leaves that are powerful activators of the same intracellular Sys-GFP fused gene was prepared first by amplifying the LepreproHypSys gene by PCR from the full-length cDNA clone defense-related genes that are activated by wounding and sys- Ј temin, mediated by the octadecanoid pathway (7). The three (7) with both a sense primer (5 -CGGAATTCATGATCAGCT- TCTTCAGAGCTTTCT-3Ј) and an antisense primer (5Ј- peptides are composed of 18, 20, and 15 amino acids, respec- Ј tively, and are derived from a single polyprotein precursor (7), CGGGATCCATAGGAAGCTTGAAGAGGCAAAGTA-3 ). here called LepreproHypSys. Whereas the tomato prosystemin The amplified PCR product contained the LepreproHypSys ORF without a stop codon and with an introduced EcoRI and precursor is synthesized and compartmentalized in the cytosol Ј Ј and nucleus of phloem parenchyma cells (9), the cell types and BamHI recognition sites at the 5 and 3 ends, respectively. The subcellular localization of the precursor of the HypSys peptides PCR product was digested with EcoRI and BamHI and was has not been known. Here, we report that the LeproHypSys ligated to the N terminus of GFP within the pEZR-LN vector precursor protein is synthesized in the phloem parenchyma cells (11). The LepreproHypSys-GFP chimeric gene constructs was of the vascular bundles of tomato leaves and is localized in the introduced into Agrobacterium tumefaciens and stably trans- cell wall matrix. The results reveal a role for the cell wall matrix formed into tomato plants (cv. Microtome) as described in ref. in harboring the precursor of peptides that are known to be 12. At least 15 independent primary transformants were regen- powerful signals for the activation of defense genes of tomato erated and assayed for the expression of the different gene constructs by Northern blotting and confocal laser scanning leaves. microscopy. As a negative control for the localization of the Materials and Methods LeproHypSys-GFP fusion protein, tomato plants were also in- dependently transformed with the pEZR-LN vector alone. The Plant Material and Treatments. Wild-type tomato plants (Lycop- GFP protein was visualized with a Bio-Rad MRC-1024 confocal ersicon esculentum Mill. cv. Castlemart) and transgenic tomato laser scanning microscope by using blue laser excitation light plants were grown in growth chambers under 18 h of light (300 Ϫ Ϫ (488 nm). Optical sections were digitally processed by using ␮mol photons m 2 s 1) at 28°C and 6-h nights at 18°C. To assay PHOTOSHOP 8.0 (Adobe Systems, San Jose, CA). wound inducibility, terminal leaflets of the lower leaves of 2-week-old plants were wounded twice across the mid-vein with Preparation of LeproHypSys Peptide Antibodies. Rabbit antibodies a hemostat. Plants were then incubated for 24 h under contin- were prepared against synthetic peptide sequences of N- and uous light at 28°C, and undamaged tissue from wounded and C-terminal regions of the LepreproHypSys protein that were unwounded leaves was collected and processed for LeproHypSys distal from putative glycosylation sites and did not include amino mRNA and protein localization analyses. Two-week-old plants were also treated with methyl jasmonate vapors as described in ref. 10. *Present address: Department of Botany and Plant Sciences, University of California, Riverside, CA 92521-0001. DNA Blot Analyses. Tissues from wild-type and transgenic tomato †To whom correspondence should be addressed. E-mail: [email protected]. plants were immersed in liquid nitrogen, ground to a fine © 2005 by The National Academy of Sciences of the USA

12974–12977 ͉ PNAS ͉ September 6, 2005 ͉ vol. 102 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505248102 Downloaded by guest on September 29, 2021 tube and stored at Ϫ20°C until use. Proteins in the supernatant were quantified with the BCA kit (Pierce) with BSA as a protein standard. Fifty micrograms of total soluble proteins were mixed with an equal volume of 2ϫ Laemmli’s sample buffer, boiled for 5 min, and loaded into 12% polyacrylamide gels. After electrophoresis in duplicate gels, proteins in one gel were stained with Coomassie blue, and the others were transferred to nitrocellulose membranes, probed with LeproHypSys peptide antibodies, and exposed to anti-IgG secondary antibodies conjugated with alkaline phosphatase, followed by colorimetric development.

Immunocytochemical Staining. Immunocytochemistry was per- formed as described in ref. 9. Briefly, leaf samples were fixed overnight at 4°C in 50 mM Pipes buffer (pH 7.2), containing 2% Fig. 1. DNA blot analysis of the LeproHypSys precursor gene. Five micro- (vol͞vol) formaldehyde and 0.5% (vol͞vol) glutaraldehyde, de- grams of genomic DNA isolated from tomato leaves was digested with the hydrated, and embedded in L.R. White resin (Ted Pella, Inc., restriction enzymes EcoRI (E), NdeI (N), SpeI (S), and XhoI (X). Digested and Redding, CA). Leaf cross-sections (0.1 ␮m) were incubated 2 h undigested (Und) DNA were separated on an agarose gel, transferred to nylon at 25°C in blocking solution [100 mM Tris, pH 7.2͞500 mM membranes, and probed with the LeHypSys precursor cDNA. Nucleic acid NaCl͞0.3% (vol/vol) Tween-20͞0.5% (wt/vol) PVP (Mr 10,000)͞ lengths (kb) are indicated on the left. 0.5% (vol/vol) donkey serum͞1% (wt/vol) BSA͞0.02% (wt/vol) NaN3], followed by 3–18 h in blocking solution containing acids from the bioactive peptide sequences (7). The N-terminal protein-G affinity purified anti-LeproHypSys peptide antibodies peptide consisted of 17 amino acids (from Asn-30 to Asn-46), (1:50–1:200 dilution). A similar dilution of the corresponding and the C-terminal peptide included 14 amino acids (from preimmune serum IgGs was always used as a negative control. Gln-90 to Thr-103). Synthetic peptides were conjugated to Thereafter, sections were washed four times with blocking keyhole limpet hemocyanin (KLH) by using the Imject mcKLH solution alone, incubated2hwithblocking solution containing kit (Pierce) and following the manufacturer’s recommendations. LeproHypSys-KLH conjugated peptides were injected into rab- bits (Pocono Rabbit Farm and Laboratory, Canadensis, PA), and the presence of cross-reacting antibodies in rabbit serum were monitored by ELISA and protein immunoblot analyses. For immunocytochemistry, primary antibodies were affinity purified by using protein-G coated magnetic beads (Dynal Biotech, Oslo), as described in ref. 9.

Protein Immunoblots. Leaves from 2-week-old tomato plants were frozen in liquid nitrogen, ground to a fine powder with a mortar and pestle, extracted with an equal volume of 0.1 M Na- phosphate buffer (pH 7.0) in a 1.7-ml Eppendorf tube, and incubated on ice for at least 1 h. Cell debris was pelleted at 15,000 ϫ g for 10 min at 4°C. The supernatant was transferred to a new PLANT BIOLOGY

Fig. 3. Localization of LepreproHypSys precursor mRNA in tissue sections from leaves and petioles of tomato by in situ hybridization. Sections were hybridized with digoxigenin-labeled LepreproHypSys sense or antisense RNA probes. Hybridized probes were detected with digoxigenin antibodies conju- Fig. 2. Protein immunoblot analysis of LeproHypSys in leaves of wild-type gated to alkaline phosphatase, as described in ref. 9. (A) Cross-section through (WT) tomato plants and in plants overexpressing the prosystemin gene in its the mid-vein of a leaf from a control untreated plant hybridized with the sense sense [S(ϩ)] and antisense [AS(Ϫ)] orientations. Fifty micrograms of total probe (negative control). (B) Similar section as in A, hybridized with the soluble proteins extracted from leaves in 0.1 M phosphate buffer (pH 7) were antisense probe, showing very low constitutive levels of LepreproHypSys mixed with equal volume of Laemmli’s sample buffer, boiled for 5 min, and mRNA accumulation. (C) Section from a wounded leaf, hybridized with the loaded into 12% polyacrylamide gels (SDS͞PAGE). After electrophoresis, pro- antisense RNA probe (6 h after wounding). (D) Methyl jasmonate-induced teins were electrotransferred to nitrocellulose membranes and probed with LepreproHypSys mRNA accumulation in phloem bundles of a mid-vein, 6 h LeproHypSys peptide antibodies, followed by exposure to anti-IgG secondary after treatment. (E) Cross-section of a petiole from a wounded leaf hybridized antibodies conjugated with alkaline phosphatase and color development. The with the sense probe. (F) Wound-induced accumulation of LepreproHypSys arrow on the right indicates a cross-reacting band (Ϸ16 kDa) corresponding to mRNA in phloem bundles of a petiole. P, phloem bundles; X, xylem. (Scale bars: the LeproHypSys precursor protein. 50 ␮m.) Arrows show concentrations of label.

Narva´ez-Va´squez et al. PNAS ͉ September 6, 2005 ͉ vol. 102 ͉ no. 36 ͉ 12975 Downloaded by guest on September 29, 2021 Fig. 4. Visualization of a LeproHypSys-GFP fusion protein in leaves of transgenic tomato plants visualized by confocal laser scanning microscopy. (A and B) Leaf epidermal cells of a control plant transformed with the GFP gene construct alone. (C and D) Confocal image showing GFP ﬂuorescence localized to the cell walls of epidermal cells of transgenic plants expressing the Lepro- HypSys-GFP fusion protein.

18-nm gold-labeled donkey anti-rabbit polyclonal antibodies (1:20 dilution, Jackson ImmunoResearch), and washed four times with blocking solution and three times with distilled water. Sections were poststained 5 min with a 1:3 mixture of 1% (vol͞vol) potassium permanganate and 2% (vol͞vol) uranyl acetate in water and examined with a transmission electron microscope, Model JEM 1200 EX (JEOL) at 100 kV.

Results and Discussion The protein precursor of HypSys I, II, and III peptide defense signals, LepreproHypSys, is composed of 146 amino acids with a leader sequence that directs its synthesis through the secretory Fig. 5. Electron micrographs showing immunogold labeling of the Lepro- pathway (7), where the protein is postranslationally decorated HypSys protein in leaf cross-sections from tomato plants. Leaf tissue was with hydroxyl groups and pentose residues. LepreproHypSys, obtained from wild-type plants that had been unwounded (A and B), like prosystemin (12), is encoded by a single copy gene (Fig. 1) wounded (C), and methyl-jasmonate-treated (D), and from transgenic tomato that is up-regulated in tomato leaves by wounding, systemin, and plants overexpressing the prosystemin gene (E and F). Tissue was processed for immunocytochemical analysis under the transmission electron microscope as methyl jasmonate treatments (7). When each of the three described in ref. 9. (A) A section through a vascular bundle of a leaf from peptides were supplied to young, excised tomato plants through unwounded wild-type tomato plant treated with preimmune serum, followed their cut petioles, they activated the synthesis and accumulation by treatment with 18-mm gold-labeled secondary donkey anti-rabbit poly- of proteinase inhibitor protein in leaves (7), demonstrating that clonal antibody. (B) Similar section as in A but treated with afﬁnity puriﬁed the peptides were behaving as signals for defense. The Lepro- anti-LeproHypSys protein serum (1:100 dilution). (C) Leaf section from HypSys precursor protein was shown to accumulate in the leaves wounded wild-type plant treated as in B.(D) Leaf section from a methyl of plants constitutively overexpressing the prosystemin gene (13) jasmonate-treated wild-type tomato plant treated as in B.(E and F) Transgenic tomato leaf sections treated as in B. CW, cell wall; Ch, chloroplast, Mi, (Fig. 2), indicating that the peptide precursor gene is regulated mitochondria; V, vacuole; Cyt, cytoplasm; PP, phloem parenchyma cell; CC, by systemin. The rabbit antibodies used to identify the protein companion cell; SE, sieve element. (Scale bars: 0.5 ␮m.) was prepared against synthetic peptide sequences of the N- and C-terminal regions of LeproHypSys protein that were distal from putative glycosylation sites and did not include any sequences were analyzed by using in situ hybridization techniques. Lepre- from the regions containing the bioactive peptides. The protein proHypSys mRNA was found to be synthesized exclusively did not accumulate in leaves of plants overexpressing the pro- within the vascular bundles of mid-veins of leaves and petioles, systemin gene in its antisense orientation (Fig. 2), and the associated with parenchyma cells of phloem bundles (Fig. 3). LeproHypSys mRNA accumulation was suppressed in wounded Phloem parenchyma cells have been shown to be the sites of leaves of antisense transgenic plants (data not shown). In synthesis of the prosystemin precursor mRNA (9), where the release of the peptides in cells near the companion cells of wild-type plants, the LeproHypSys mRNA has been shown to be the phloem facilitate the amplification of systemic signaling systemically induced to accumulate in response to wounding (7). (9, 14, 15). The experiments demonstrated that the gene is among the The specific localization of newly synthesized LeproHypSys ͞ wound systemin responsive genes in tomato plants, suggesting protein was visualized in leaves of tomato plants transformed that the role of HypSys peptides, like systemin, may be to amplify with a constitutively expressed LepreproHypSys-GFP fusion the wound response in tissues throughout the plants (13). gene. The visual detection of the gene, monitored by using To identify the specific cell types in which the LepreproHypSys confocal microscopy, was found to be associated primarily with gene is expressed, leaves and petioles of young tomato plants cell walls (Fig. 4).

12976 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505248102 Narva´ez-Va´squez et al. Downloaded by guest on September 29, 2021 Transmission electron microscopy analyses of the subcellular changes in extracellular pH may have a role in the activation of localization of the LeproHypSys protein in leaves of wounded processing peptidases. It is also possible that specific wound- tomato plants, methyl jasmonate-treated plants, and plants inducible processing proteases are synthesized through the Golgi overexpressing the prosystemin gene, demonstrated that under and secretory pathway in response to wounding and are trans- all of these conditions, the accumulation of the precursor protein ported to the cell walls. Proteinases released by invading patho- was found to be in the cell wall matrix of vascular parenchyma gens may also produce peptides from the precursor, signaling cells (Fig. 5). intracellular defense responses. In any event, the production of Hundreds of hydroxyproline-rich proteins (HPRGs) have multiple signals produced from HypSys peptide precursors in the been reported to be components of cell walls of plants, including cell walls may be among the plant’s earliest events in response to green algae (16). All HPRGs contain motifs with hydroxyproline pest and pathogen attacks. The generation of extracellular residues that are unique to specific classes of proteins. All of peptide signals by proteolysis is a strategy that occurs in animals these proteins have been suggested to be members of a super- during growth and differentiation (19). family, related to HPRG proteins found in early life forms (17). The identification of a hydroxyproline-rich glycopeptide pre- The hydroxyproline-rich glycoprotein precursors of HypSys de- cursor protein that produces multiple peptide defense signals fense-signaling peptides present in leaves of tobacco (6) and within the matrix of cell walls provides a previously uncharac- tomato (7) plants do not fit into this structural scenario, because terized paradigm for cell wall matrix proteins. More than 100 they are very small (Ϸ150 amino acids) and they lack repeating species of plants from diverse families exhibit systemic wound motifs as found in HPRGs. However, the functional HypSys signaling for defense (20). In addition to being present in tomato peptides do contain regions rich in hydroxyproline and proline plants (7), multiple hydroxyproline-rich glycopeptide signals residues interspersed with serine and threonine residues and derived from single protein precursors have been purified from flanked by charged amino acids (6, 7), and they appear to tobacco (6), petunia, nightshade, and potato (G.P., W. Siems, represent a previously uncharacterized subfamily of cell wall and C.A.R., unpublished data). The next step should be to find HPRGs. small, related HypSys peptide signals in species throughout the plant kingdom where they may have a general role in amplifying The localization of the precursor of the three HypSys defense ͞ signaling peptides in the cell wall indicates that the peptides may defense gene activation in response to herbivores and or result from regulated processing events that occur in the cell wall pathogens. matrix in response to trauma. Several scenarios for the generation of processing activity are possible. Wounding may cause This work is dedicated to the memory of Prof. Vincent Franceschi. We thank the Washington State University Electron Microscopy Center staff proteases present in the extracellular matrix to mix with the for their technical advice. This research was supported by National precursor as a consequence of cell damage, resulting in process- Science Foundation Grant IBN 0090766, the Charlotte Y. Martin ing. The alkalinization of the cell wall apoplast is one of the Foundation, and the College of Agricultural, Human, and Natural earliest events induced after wounding of tomato leaves (18), and Resources Sciences.

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Narva´ez-Va´squez et al. PNAS ͉ September 6, 2005 ͉ vol. 102 ͉ no. 36 ͉ 12977 Downloaded by guest on September 29, 2021