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PLANTS INTERACTING WITH OTHER ORGANISMS

Extracellular Proteins in Pea Root Tip and Border Cell Exudates^1,[OA]

Department of Plant Sciences, Division of Plant Pathology and Microbiology (F.W., H.D.V., M.C.H.) and Center for Toxicology, Pharmacy 205 (G.T.), University of Arizona, Tucson, Arizona 85721

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Newly generated plant tissue is inherently sensitive to infection. Yet, when pea (Pisum sativum) roots are inoculated with the pea pathogen, Nectria haematococca, most newly generated root tips remain uninfected even though most roots develop lesions just behind the tip in the region of elongation. The resistance mechanism is unknown but is correlated spatially with the presence of border cells on the cap periphery. Previously, an array of >100 extracellular proteins was found to be released while border cell separation proceeds. Here we report that protein secretion from pea root caps is induced in correlation with border cell separation. When this root cap secretome was proteolytically degraded during inoculation of pea roots with N. haematococca, the percentage of infected root tips increased from 4% ± 3% to 100%. In control experiments, protease treatment of conidia or roots had no effect on growth and development of the fungus or the plant. A complex of >100 extracellular proteins was confirmed, by multidimensional protein identification technology, to comprise the root cap secretome. In addition to defense-related and signaling enzymes known to be present in the plant apoplast were ribosomal proteins, 14-3-3 proteins, and others typically associated with intracellular localization but recently shown to be extracellular components of microbial biofilms. We conclude that the root cap, long known to release a high molecular weight polysaccharide mucilage and thousands of living cells into the incipient rhizosphere, also secretes a complex mixture of proteins that appear to function in protection of the root tip from infection.

The infection of pea (Pisum sativum) roots by the soilborne pea pathogen Nectria haematococca was used to test predictions of this hypothesis (Gunawardena and Hawes, 2002). Even under conditions highly conducive to infection, including direct inoculation of roots with a suspension of fungal spores, followed by incubation in warm, moist conditions, most root tips escaped infection. Surprisingly, considering the absence of infection within root tips, was the discovery that when viewed with a microscope, a conspicuous mantle of fungal hyphae was present on the cap periphery of most inoculated roots (Hawes et al., 1998; Gunawardena and Hawes, 2002). Such hyphae, whose identity and pathogenicity were established as the inoculating strain of N. haematococca by expression of specific marker genes, spontaneously detached from the tip along with border cells when the root was immersed in water. After detachment of the mantle, the root tip was confirmed to be free of infection by plating onto culture medium. Border cells remained in the rhizosphere in association with detached mantles, left behind as the root tip moved downward. When root tips occasionally developed visible lesions, root cap turnover was induced and the effect was to jettison the invading fungus (Hawes et al., 1998; Gunawardena et al., 2005). A similar mechanism of parasite expulsion has been reported to occur in mammalian intestinal cells induced to undergo turnover in response to microbial infection (Cliffe et al., 2005).

In the pea-N. haematocca interaction, removal of border cells from roots prior to inoculation with N. haematococca spores resulted in a significant increase in frequency of root tip infection (Gunawardena and Hawes, 2002). These observations supported the hypothesis that border cells play a role in protecting the root tip. Moreover, when border cells were cocultivated with N. haematococca independently of the root, a complete suppression of fungal growth occurred within 48 h (Gunawardena et al., 2005). Detailed examination of hundreds of border cells growing together with N. haematococca, however, did not reveal hyphal penetration of any border cells. This result suggested that a product or products released into the extracellular environment might be responsible for growth inhibition and, perhaps, for preventing invasion of root tips surrounded by mantles of actively growing fungal hyphae.

In a previous study, two-dimensional gel electrophoresis was used to demonstrate that profiles of proteins synthesized by border cells are distinct from those of progenitor cells in the root cap of pea (Brigham et al., 1995). To our surprise, an array of proteins was found to be synthesized and secreted into the extracellular environment from the root cap and/or border cells as border cells undergo the process of cell wall solubilization and separate from the cap periphery. These proteins can be collected without tissue injury or cell death, because they disperse spontaneously into solution when root tips of intact seedlings are immersed in water. In this study, the amount of extracellular protein released from pea root tips, termed the root cap secretome, was quantified and found to be correlated with the number of border cells present on the cap periphery. The possible impact of the secretome in protecting the root tip from infection was tested directly by measuring the ability of N. haematoccca to infect root tips, with and without treatment with a broad spectrum protease to destroy the secretome at the time of inoculation. To follow up on previous observations using two-dimensional gel electrophoresis to document the presence of extracellular proteins released from the root tip (Brigham et al., 1995), we used multidimensional protein identification technology (MudPIT) to profile the nature of the secreted proteins. MudPIT allows analysis of complex mixtures of proteins by combining two-dimensional liquid chromatography (LC) directly with tandem mass spectrometry (MS/MS) and therefore is ideally suited for profiling the root cap secretome directly as it is released into the rhizosphere (Washburn et al., 2001).

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Dynamics of Extracellular Protein Delivery in Root Tip Exudates

In previous studies, pea root tips with a full set of approximately 4,000 border cells present on the root cap periphery were exposed for 1 h to ³⁵S Met on filter paper (Brigham et al., 1995). The root tips (2–3 mm from the apex) of intact seedlings then were immersed into water to disperse border cells into suspension, which was centrifuged to pellet the border cells. Labeled proteins from excised root caps (1 mm from the apex) and the border cell pellet then were extracted and analyzed by two-dimensional electrophoresis (Brigham et al., 1995). The supernatant from the washed border cells, included as a control, unexpectedly was found to contain an array of >100 proteins whose functions were unknown. Cell viability was measured based on uptake of the vital stain flourescein diacetate and the observation of cytoplasmic streaming, as described previously (Hawes and Wheeler, 1982). Viability of the population of washed, detached border cells remained at 90% ± 5% from the beginning to the end of the test period, so the presence of extracellular proteins could not be attributed to leakage from dead cells.

In this study, the extracellular proteins of the root cap secretome, defined here as all the proteins released into the medium by border cells and progenitor root cap cells during the process of border cell separation, were quantified using Bradford's assay (Bradford, 1976). Exudates from each root tip of pea, collected when roots were >25 mm in length, yielded 1.25 ± 0.15 µg of soluble protein. Removing border cells induces renewed production of border cells by the root cap (Brigham et al., 1998; Hawes and Lin, 1990; Wen et al., 1999). Within 5 to 10 min after existing cells are removed, new cells can be collected from the tip and after approximately 20 h a full set of approximately 4,000 new cells is present on the cap periphery, at which time cap turnover and border cell separation cease. Inducing root cap turnover by this method was found to induce renewed production of extracellular protein in correlation with the number of border cells present (Table I ). Once a full set of border cells was present, the level of protein remained stable at approximately 1.3 µg of soluble protein, per root tip.

Dynamics of Extracellular Protein Delivery in Border Cell Exudates

To determine whether populations of detached border cells continue to secrete protein after separation from the cap, protein in the supernatant of washed border cells was measured. Residual protein in the supernatant was reduced to 0.0001 µg/4,000 border cells by washing, at time 0 (Table I). When the border cell pellet was immersed in fresh water, significant levels of renewed extracellular protein were detected within 15 min, and levels increased for 60 min. At 15, 30, and 60 min, respectively, 0.08 ± 0.04, 0.13 ± 0.06, and 0.26 ± 0.09 µg/4,000 cells was detected. The level of extracellular protein at 2, 4, and 6 h was 0.28 ± 0.04, 0.27 ± 0.03, and 0.27 ± 0.06 and was not significantly higher than at 1 h, suggesting that a plateau in protein release occurs after border cells are detached from the root cap. This plateau was not correlated with an increase in number of dead cells, whose viability remained at 90% ± 4% for >6 h. Indeed, deliberately killing border cells (by freezing or high speed centrifugation) did not result in significant release of extracellular protein. Border cell walls presumably provide a significant barrier to passive diffusion of proteins even after death; cell walls of actively growing suspension cultured cells have a pore size that would prevent diffusion of most proteins (Fleischer et al., 1999). Border cells have walls that are >1 µm in diameter and undergo secondary wall development (Hawes and Wheeler, 1982). These results suggested that viable cells capable of active secretion are required for release of proteins into the extracellular environment. When roots or border cells were treated with the secretion inhibitor Brefeldin A (Nebenfuhr et al., 2002) after removing border cells, no additional protein was detected in the extracellular milieu over a 24 h period (<0.001 µg/root).

Functional Impact of the Root Cap Secretome on Root Tip Infection

Most root tips escape infection when whole pea roots are inoculated with spores of the root rot pathogen, N. haematocca (Gunawardena and Hawes, 2002). Because meristems are uninvaded, growth is indistinguishable from that of uninoculated controls (Fig. 1A ). Yet most root tips are enshrouded by fungal hyphae, which grow among border cell populations at the tip to develop into a mantle (Fig. 1B). In response to immersion in water, mantles detach physically from the root tip, leaving the root tip uninvaded. The fact that invasion is somehow prevented while mantles of pathogenic hyphae are appressed to the cap periphery suggested the presence of a physical or chemical barrier that somehow blocks penetration into the cap tissue. The hypothesis that the proteins of the root cap secretome play a role in providing such a barrier was explored by using global destruction of the root cap secretome during inoculation with N. haematococca.

View larger version (101K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Proteolytic destruction of the root cap secretome resulted in increased root tip infection (A and C) and increased fungal growth on root tips (B and D). A, Most pea roots (94% ± 4%) inoculated with 105 spores of N. haematococca show no obvious signs of infection 3 d later and growth proceeded at approximately 2.5 cm/d (black arrow denotes the site of the root tip at the time of inoculation). B, At 10x magnification, the presence of a mantle of fungal hyphae (white arrows) growing among border cells was evident. No necrosis at the root tip was visible. C, When roots were treated with proteinase K at the time of inoculation with N. haematococca spores, root tip infection was apparent within 2 to 3 d; a brown-black necrosis encompassed the root apex (arrowhead) of every inoculated root, and growth ceased within 24 to 48 h after inoculation (black arrow denotes the site of the root apex at the time of inoculation). D, Root tip necrosis (arrowhead) and cessation of root growth occurred in correlation with a marked increase in hyphal growth (white arrows). Each experiment was repeated at least seven times, with 10 replicate seedlings for each experiment, and photos (C and D) show results representative of all replicate roots; there were no escapes.

Identification of Proteins of the Root Cap Secretome

Border cells from pea were collected from the root tip and centrifuged, as described above, and proteins in the supernatant were analyzed using MudPIT. Like previous studies using two-dimensional gel electrophoresis (Brigham et al., 1995), this analysis revealed that a discrete set of >100 plant proteins is released into solution when roots with border cells are immersed into water (Table II ). Some proteins represented carbohydrate metabolizing and cell wall proteins known to be components of cell walls (e.g. Albersheim et al., 1969). Such proteins, which in this study included extensin, Hyp-rich glycoprotein, endoxyloglucan transferase, cell wall invertase, -galactosidase, cellulase, -mannosidase, and oligosaccharyl transferase, have been confirmed to be present in cell walls of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) recently (Bayer et al., 2006; Zhu et al., 2006b). The presence of a rhizosphere -galactosidase was previously identified (Price, 2002; Celoy, 2004). Calmodulin, proteases, and other signal pathway-associated proteins including ATPase also were found. The largest class of proteins was comprised of stress and defense-related enzymes and proteins including histone H4 (Patat et al., 2004), chitinase, heat shock and dehydration responsive proteins, and reactive oxygen-related enzymes such as lipoxygenase, peroxidase, and superoxide dismutase (Kawano, 2003). Also present were such cytoplasmic markers as 14-3-3 proteins (Stankovic et al., 1995), ribosomal proteins, and a cytochrome P450. Only 20 proteins, including the cytochrome P450, matched proteins with predicted signal peptides (Table II).

View larger version (105K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Localization of extracellular 14-3-3 proteins. Immunolocalization with GF14-omega 14-3-3 antibody (Ferl, 2004) highlighted a sheath of material surrounding border cells from maize (arrows) and pea (inset). No intracellular penetration of the antibodies was detected.

The presence of 14-3-3 proteins has been reported in the extracellular matrix of Chlamydomonas and these proteins also are thought to be secreted from human fibroblasts (Voigt and Frank, 2003; Ghahary et al., 2005). The expression of 14-3-3 proteins in plant tissues is induced in response to pathogens (Brandt et al., 1992). Because 14-3-3 proteins coordinate activity of diverse proteins through direct protein-protein interactions, their presence in the secretome could yield clues to how so many proteins can function outside the plant cell (Roberts, 2000; Berg et al., 2003; Ferl, 2004). Thus, 14-3-3 proteins could function in transport, localization, stability, and/or activity of the diverse proteins and enzymes secreted into the rhizosphere environment. If so, then inhibiting the function of the 14-3-3 proteins would be predicted to inhibit the ability of the secretome to function in protecting the root tip from infection. This prediction was tested using R18, a high-affinity peptide antagonist of 14-3-3 proteins (Wang et al., 1999). When roots were inoculated with R18 together with fungal spores, frequency of root tip infection increased significantly (P < 0.05) from 6% ± 4% to 40% ± 5% of inoculated roots. These results are consistent with the hypothesis that 14-3-3 proteins influence root tip resistance to N. haematococca. However, the possibility that intracellular effects of R18 could be involved in altered root tip infection cannot be ruled out.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The balance of microbial populations in the rhizosphere, stimulated by exudates from plant roots, has long been known to play a critical role in plant health and crop productivity (Atkinson et al., 1975; Hirsch et al., 2003). The components of root exudates, including those from the root cap, have been analyzed and found to contain diverse products including sugars, amino acids, carbohydrates, and secondary metabolites (Teplitski et al., 2000; Knee et al., 2001; Bais et al., 2006). Several investigators noted enzyme activities in the rhizosphere (e.g. Knudson and Smith, 1919; Knudson, 1920; Rogers et al., 1942), but confirming the presence of proteins was difficult because most experimental systems include contributing microbial contaminants that remain difficult to control (Curl and Truelove, 1986). Antibiotics used to inhibit bacterial colonization are toxic to roots and create additional artifacts of interpretation. Indeed, pea remains our primary experimental model for border cell function because it is the only species among many tested (e.g. Hawes and Pueppke, 1986) in which we can reliably obtain roots whose root caps are free of culturable microbial species. The release of an array of newly synthesized proteins from living cells, free of microbial contamination, confirmed the secretion of proteins from the pea root cap during border cell separation (Brigham et al., 1995). In recent years the secretion of several specific endogenous proteins from the root cap, including a Gly-rich glycoprotein and ribosome inactivating proteins, has been documented (Matsuyama et al., 1999; Park et al., 2002). Basu et al. (2006) used MudPIT and LC-MS/MS to document that 52 extracytosolic proteins are released from roots of Arabidopsis grown in liquid shake culture for 11 d. There was little overlap among the proteins identified in that study and those in the pea root cap secretome (Table II). This may reflect the divergence in species, collection methods, and seedling age, the possible presence of microbial activities, or the fact that root caps of Brassicacae species do not produce populations of living border cells (Hawes et al., 1997, 1998).

Sharp and coworkers (Zhu et al., 2006b) have summarized limitations of current protein identification technology, given existing limitations in understanding plant proteome function. Large gene families, of which most pea root cap secretome proteins are members (Table II), are especially problematic with methods like MudPIT, which may not distinguish among closely related protein sequences. Additional problems are inherent in the use of databases for plant species like pea whose genome sequences are not complete, and therefore extensive comparative analysis of probable function based on predicted function is not warranted. In general, however, the identification of the root cap secretome proteins has revealed that the profile of the secreted proteins is, in some respects, similar to apoplast proteins extracted from other cells of the root (Alvarez et al., 2006). Thus, the cell wall proteome in the maize primary root elongation zone and root tip also includes energy-related, carbohydrate processing, and defense-related proteins (Zhu et al., 2006a, 2006b). Many of the secreted proteins found within the root cap secretome, including the pathogenesis-related chitinase and glucanase, have been known to be present within the plant apoplast for many years (Bol et al., 1990). Carbohydrate processing enzymes as well as peroxidases and other components of reactive oxygen pathways have been known to be an integral component of cell wall-associated defense responses (Albersheim et al., 1969; Brisson et al., 1994; Cassab, 1998). Proteases also have been implicated in peptide-based extracellular signaling responses, and the presence of ATP in the rhizosphere has recently been reported (Bergey et al., 1996; Segarra et al., 2003; G. Stacey, personal communication). That the root cap secretome is not strictly synonymous with the cell wall proteome was indicated by the observation that two cell wall enzymes known to be present in peripheral cells of the pea root cap were not represented (Hawes and Lin, 1990; Wen et al., 1999). Thus, a polygalacturonase and a pectinmethylesterase can be extracted from cell walls by treatment with NaCl but neither activity was detected in the water-soluble materials collected from pea root tips (Table II). Conversely, a -galactosidase that is secreted from pea border cells is not detectable within root cap tissue (Price, 2002; Celoy, 2004).

Actin, 14-3-3 proteins, glyceralde-3-P dehydrogenase, heat shock proteins, and histones, generally viewed as cytoplasmic markers, increasingly have been implicated in extracellular functions (Chivasa et al., 2005). Actin was reported to be associated with plasmodesmata within the cell wall by White et al. (1994) who speculated that it might play a role in cell-cell communication. Secreted 14-3-3 proteins appear to play a role in cross linking of Hyp-rich glycoproteins in the Chlamydomonas cell wall (Voigt and Frank, 2003) and also are associated with human fibroblasts (Ghahary et al., 2005). Secretion of proteins such as ribosomal proteins and cytochrome P450s has not been reported from plant cells and their presence might represent leakage that occurs during the cell separation process. However, the periwinkle cytochrome P450 related to a pea root cap secretome protein is among the minority predicted to have a signal peptide (Table II). Moreover, recent studies have revealed that such intracellular components are found outside the cells of gram-positive bacteria (Bendtsen et al., 2005). Thus, ribosomal proteins, enolase, elongation factor, and chaperonins are components of extracellular matrices produced by on-filter growth and analyzed by LC-MS/MS analysis (Gallaher et al., 2006). Histones are a component of the extracellular defense system in some vertebrates, and an extracellular complex of proteins including ribosomal proteins and histones is a key component in the innate immunity of Atlantic cod (Gadus morhua; Patat et al., 2004; Bergsson et al., 2005).

Current understanding of how proteins are secreted in diverse organisms is evolving rapidly, and even in bacteria, whose secretion machinery has been studied extensively, there continue to be surprises emerging from secretome studies. For example, analysis of the complete Bacillus subtilis genome revealed that 300 proteins have structural motifs that predict they would be exported from the cytoplasm (Tjalsma et al., 2004). However, direct analysis under a wide range of conditions yielded only 90 that were exported and among those, nearly 50% would not have been predicted to be exported based on sequence. The export of proteins from the plant cytoplasm past the plasma membrane into the cell wall and apoplast is inherent in plant function, but little is known about the detailed mechanisms for secretion into the membrane or the cell wall, let alone into the extracellular spaces beyond the wall (for review, see Bais et al., 2006). Secretion from plant cells can occur by a default pathway when proteins are targeted to the endoplasmic reticulum by signal peptide-mediated translocation, but may also occur by nonspecific migration through exocytosis (Denecke et al., 1990). The root cap has long been known to be a secretory organ whose active secretion of a high M_r polysaccharide-based mucilage made it a favored model for studies of Golgi function (e.g. Pickett-Heaps, 1967; Feldman, 1984). Our data suggest that its role in the secretion of soluble proteins into the rhizosphere may have been underestimated by those of us working in the field of root-rhizosphere interactions.

Understanding the relative contribution of the root cap versus border cells to the secretome will require additional research. Newly synthesized proteins from these two cell types have very different protein profiles in two-dimensional gel electrophoresis, but spots common to both can be found in extracellular proteins (Brigham et al., 1995). In this study, border cells were found to continue to secrete at least a subset of the secretome, after they are dispersed into suspension. It will be of interest to define the ways this process may be influenced by challenges such as pathogenic bacteria and aluminum, which induce a marked increase in the dimensions of the extracellular mucilage layer around individual border cells (Hawes et al., 1998; Miyasaka and Hawes, 2001). By virtue of the fact that border cells can be isolated into suspension without osmotica, hormones, or other artifactual conditions, these cell populations may provide a convenient system to explore the ways diverse proteins are delivered to the extracellular environment of plant cells. Alternatively, this process may constitute a unique system that is not representative of normal secretion mechanisms. In any case, the proteins we identified can be collected simply by transient immersion of the root tip into water, an action that closely duplicates conditions normally encountered by roots in diverse growing conditions. Therefore, it is reasonable to conclude that the proteins identified here are an integral component of the complex of materials delivered into the rhizosphere of plants under natural conditions. Our results support the hypothesis that proteins released along with the root cap mucilage during the process of border cell separation play a vital role in the plant's system of innate immunity, by protecting the vulnerable root tip as it moves through the soil environment.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material

Seeds of pea (Pisum sativum L. cv Little Marvel; Meyer Seed Company) and maize (Zea mays L. cv Golden Bantam; Vegetable Seed Warehouse) were germinated as described (Brigham et al., 1995). This protocol includes incubation for 15 min in 95% ethanol followed by 1 h in full-strength commercial bleach (12.5% NaOH), and imbibition with repeated rinses in sterile water for 6 h during which seeds that float are discarded leaving only those with an intact seed coat. In pea, this yields roots from which no microbial colonies emerge when root tips are immersed into water that is then plated onto rich media such as Luria broth or potato (Solanum tuberosum) dextrose agar (e.g. Gunawardena et al., 2005). In maize, Arabidopsis (Arabidopsis thaliana), Medicago truncatula, and numerous other species examined (e.g. Hawes and Pueppke, 1986), significant residual microbial contamination remains on root tips of aseptically grown seedlings, and antibiotic treatment sufficient to eliminate the contamination results in browning and root growth inhibition. This confounds interpretation of the components of extracellular material from the root tip. Therefore, the proteomic analysis presented here is limited to pea, and immunolocalization using a specific antibody was used to confirm the presence of 14-3-3 proteins in the maize border cell extracellular matrix (below).

Root tips (approximately 2–3 mm of the root apex including the root cap and apical meristem) of intact 2-d-old seedlings with a radicle length of 2.5 cm were immersed in 1 mL of water and washed by gently pipetting with water, as described (Zhu et al., 1997; Brigham et al., 1998). Border cells were removed from the collected exudates by centrifugation at 3,500g for 1 min, and washed twice to remove all extracellular material. Root tip exudate refers to the supernatant component obtained after this centrifugation step, and root cap secretome refers to proteins within root tip exudate that includes material from root cap and from border cells accumulated during the process of border cell separation. Border cell exudate refers to material collected exclusively from border cells over time, after washing to remove all residual extracellular material at the time of border cell collection.

Global Destruction of Root Cap Secretome: Impact on Root Tip Resistance to Infection

Nectria haematococca mating population VI strains were maintained, collected, and quantified as described previously (Wassmann and Van Etten, 1996; Gunawardena and Hawes, 2002). Conidia of isolate 77-13-4 (10⁵ or 10⁶/mL) were added, with and without proteinase K (0.5 mg/mL) to 200 µL pipette tips. Roots were placed horizontally into cellophane growth pouches (Mega International) with root tips inserted into the pipette tip containing conidia in water for 24 h, to retain contact among the root tip, spores, and proteinase K (Sigma Aldrich). Then, the pipette tips were removed and growth pouches containing the seedlings were placed upright. Root tip infection was evaluated by direct observation of lesion development and measurement of root growth, as described (Gunawardena and Hawes, 2002). Results are based on at least seven independent experiments with at least 10 replicate seedings for each treatment.

Protein Sample Isolation and Preparation

Protein was isolated as described (Brigham et al., 1995), and quantified by the method of Bradford (1976). Absorption of the supernatant was measured at 595 nm wavelength (A₅₉₅), which was plotted on the standard curve to find the protein concentration. To collect proteins in border cell exudate, the border cell pellet was washed once and fresh water was added to the cells that were incubated at room temperature for 1 to 2 h. Protein from the extracellular medium was harvested by centrifuging the cells and collecting the supernatant. For each root tip exudate sample, protein (approximately 50 µg) was collected in 2 mL water from tips of 50 pea roots. For border cell samples, protein (approximately 20 µg) was collected from washed border cells from 300 roots. Samples that yielded microbial colonies when plated onto nutrient media were discarded. Protein samples were collected four times from independently grown batches of pea roots. Protein analysis was carried out essentially as described (Eng et al., 1994; Yates et al., 1995; Washburn et al., 2001; Andon et al., 2003; Wilkins et al., 2006). Ammonium bicarbonate was added to a concentration of 0.1 M, and 40 µL of 10 mM dithiothreitol was added before reducing at 56°C for 45 min (Washburn et al., 2001). Reduced Cyss were alkylated by addition of 40 µL of 55 mM iodoacetamide (10 mM final concentration) and incubation for 30 min at room temperature. Proteolysis was initiated with a 1:50 ratio (by weight) of sequencing grade modified trypsin (Promega) and allowed to proceed for 2 h at 37°C. A second aliquot of 1:50 trypsin was added and the digestion continued overnight at 37°C. The digest was stored at –20°C prior to analysis.

Protein Separation and Identification by MudPIT

Mass spectra of the four independent samples each were acquired in the Arizona Cancer Center/Southwest Environmental Health Sciences Center (AZCC/SWEHSC) Proteomics Core, University of Arizona, Tucson. For the nano-LC-MS/MS used to fractionate peptides from the digest, a microbore HPLC system (Paradigm MS4, Michrom) was used with two separate SCX and RP columns: a 100 µm i.d. capillary packed with 10 cm of 5 µm Vydac C18 reversed-phase resin and a separate 250 µm i.d. capillary packed with 8 cm of 5 µm Partisphere strong cation exchanger resin (Whatman). The sample (23 µg) was acidified using trifluoroacetic acid (TFA) and manually injected onto the SCX column, the effluent from the column being fed through RP column.

A representative 12-step MudPIT analysis would be as follows. Solutions used are 10% methanol/0.1% formic acid, 0.01% TFA (buffer A), 95% methanol/0.1% formic acid, 0.01% TFA (buffer B), 10% methanol/0.1% formic acid, 0.01% TFA (buffer C), and 500 mM ammonium acetate/10% methanol/0.1% formic acid, 0.01% TFA (buffer D). Step 1 consists of a 5 min equilibration step at 100% buffer A, followed by another equilibration step for 5 min at 25% buffer B (75% buffer A), followed by a 40 min gradient from 25% buffer B to 65% buffer B, followed by a 10 min 65% buffer B and 10 min of 100% buffer A. Chromatography steps 2 to 12 follow the same pattern: 15 min of the appropriate percentage of buffers C and D followed by a 2 min 100% buffer C wash, a 5 min wash with 100% A, equilibration with 25% buffer B for 5 min, followed by a gradient from 25% buffer B to 65% buffer B in 40 min, followed by a 10 min 65% buffer B and 10 min of 100% buffer A. The buffer C/D percentages used were 95%/5%, 90%/10%, 85%/15%, 80%/20%, 70%/30%, 60%/40%, 40%/60%, 20%/80%, 0%/100%, 0%/100%, and 0%/100%, respectively, for the 11 salt steps. The flow rate is approximately 350 nL/min, with elution directly into the electrospray ionization source of a ThermoFinnigan LCQ-Deca XP Plus ion trap mass spectrometer (ThermoFinnigan). Eluting peptides were electrosprayed into the mass spectrometer with a distally applied liquid junction spray voltage of 1.6 kV. Spectra are scanned over the range 380 to 2,000 mass units. Automated peak recognition, dynamic exclusion, and daughter ion scanning of the most intense ion was performed using the Xcalibur software as described previously (Andon et al., 2002, 2003).

MS/MS data were analyzed using SEQUEST (Eng et al., 1994; Yates et al., 1995). The criteria for a positive peptide identification for a doubly charged peptide are a correlation factor (Xcorr) greater than 2.0, a cross correlation factor (Xcorr) greater than 0.08, a minimum of one tryptic peptide terminus, and a high preliminary scoring. For triply and singly charged peptides the correlation factor threshold is set at 3.5 and 1.5, respectively. All spectra were searched against the nonredundant database from the National Center for Biotechnology Information. Of proteins listed, five were detected in all four samples, 73 were detected in three samples, 27 in two samples, and 16 were present in a single sample. A survey for the presence of signal peptides was predicted using Signal-P V3 program (Bendtsen et al., 2004; www.cbs.dtu.dk/services/TargetP).

In Situ Immunolocalization of Extracellular 14-3-3 Protein

Washed border cells were immersed in phosphate-buffered saline (PBS) for 5 min, then treated with 3% blocking reagent (Roche Molecular Biochemicals) in PBS for 1 h, and washed twice in PBS, then incubated 2 h with a 1:1,000 dilution of Arabidopsis 14-3-3 monoclonal antibody GF14-omega raised in mouse (a gift from Robert J. Ferl, University of Florida, Gainesville; www.hos.ufl.edu/ferllab/14-3-3's/index.htm), followed by two 10-min rinses in PBS. The second antibody, Hilyte Fluor488-labeled goat anti-mouse IgG (1:1,000 in PBS; AnaSpec) was incubated with border cells for 1 h, followed by 30 s rinses in PBS and water, respectively. P88C, the virus peptide induced antiserum from mouse (a gift from Dr. Z. Xiong, University of Arizona) was used as negative control antibody. Specimens were examined under an Olympus fluorescence microscope under UV radiation (480–500 nm filter). The images were captured with an Olympus digital camera using Micfire software.

Effects of R18, an Oligopeptide Conserved in the 14-3-3 Protein Binding Sites, on Root Tip Resistance to Infection

The effect of R18 was tested by adding to roots at a concentration of 4 µg/mL (Wang et al., 1999), as described above. N. haematococca spores were added, and infection was measured after 24, 48, and 72 h (Gunawardena and Hawes, 2002, 2005). Three independent experiments, with 10 replicate seedlings in each experiment, were carried out to test the R18 effect.

	ACKNOWLEDGMENTS

 
We thank Robert J. Ferl, University of Florida, for GF14-omega antibody for these experiments. Mass spectra were acquired in the AZCC/SWEHSC Proteomics Core.

Received October 19, 2006; accepted November 17, 2006; published December 1, 2006.

	FOOTNOTES

 
¹ This study was supported by the Department of Energy, Division of Energy Biosciences, and the University of Arizona College of Agriculture and Life Sciences (grant no. DE–FG02–04ER15551). The Arizona Cancer Center/Southwest Environmental Health Sciences Center Proteomics Core was supported by National Institute of Environmental Health Sciences (grant no. ES06694) and National Institutes of Health/National Cancer Institute (grant no. CA023074–26).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Martha C. Hawes (mhawes{at}u.arizona.edu).

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.106.091637

^* Corresponding author; e-mail mhawes{at}u.arizona.edu; fax 520–621–9290.

	LITERATURE CITED

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