Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Plant Soil (2012) 355:1–16 DOI 10.1007/s11104-012-1218-3 MARSCHNER REVIEW Roles of root border cells in plant defense and regulation of rhizosphere microbial populations by extracellular DNA ‘trapping’ Martha C. Hawes & Gilberto Curlango-Rivera & Zhongguo Xiong & John O. Kessler Received: 2 November 2011 / Accepted: 11 March 2012 / Published online: 27 March 2012 # Springer Science+Business Media B.V. 2012 Abstract Background As roots penetrate soil, specialized cells called ‘border cells’ separate from root caps and contribute a large proportion of exudates forming the rhizosphere. Their function has been unclear. Recent findings suggest that border cells act in a manner similar to that of white blood cells functioning in defense. Histone-linked extracellular DNA (exDNA) and proteins operate as ‘neutrophil extracellular traps’ to attract and immobilize animal pathogens. DNase treatment reverses trapping and impairs defense, and mutation of pathogen DNase results in loss of virulence. Scope Histones are among a group of proteins secreted from living border cells. This observation led to the discovery that exDNA also functions in defense of root caps. Experiments revealed that exDNA is synthesized and exported into the surrounding mucilage which attracts, traps and immobilizes pathogens in a host-microbe specific manner. When this plant exDNA is degraded, the normal resistance of the root cap to infection is abolished. Conclusions Research to define how exDNA may operate in plant immunity is needed. In the meantime, the specificity and stability of exDNA and its association with distinct microbial species may provide an important new tool to monitor when, where, and how soil microbial populations become established as rhizosphere communities. Responsible Editor: Philippe Hinsinger. M. C. Hawes (*) : G. Curlango-Rivera Department of Soil, Water and Environmental Sciences, University of Arizona, 429 Shantz Building, #38, 1177 E. Fourth St., POB 210038, Tucson, AZ 85721-0038, USA e-mail: [email protected] Z. Xiong Division of Plant Pathology and Microbiology, School of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA J. O. Kessler Physics Department, University of Arizona, Building 81, Tucson, AZ 85721, USA Keywords Root border cells . Mucilage . Root cap . Extracellular DNA (exDNA) . Root exudates . Rhizosphere colonization Abbreviations exDNA Extracellular DNA DAPI 4′,6-diamidino-2-phenylindole One of the take-home messages is that spatial and temporal variability act to confound root research (Zobel and Wright 2005). There is an urgent need to develop new approaches and methods for probing rhizodeposition (Jones et al. 2009). 2 Background Critical needs for sustainable practices in agriculture have been considered in many excellent articles (e.g. Brady and Weil 2010; Compant et al. 2005; Donato et al. 2010; Pinton et al. 2007; Sylvia et al. 1998; Zobel and Wright 2005). The root-soil interface is a target where positive changes can yield stable improvement in fertility, water use, and disease control leading to increased crop productivity with reduced damage to the environment (Bruehl 1987; Gilbert et al. 1996; Marschner et al. 2011; Rovira 1991; Schroth and Snyder 1961; Uren 2001). Efforts to apply biological control to root systems have been a focus of interest for decades with promising results and progress in understanding mechanisms (Handelsman and Stabb 1996; Hirsch 2004; Loh et al. 2002; Morris and Monier 2003; Pierson and Pierson 2007; Weller 1988; Zentmyer 1963). Of special interest are carbon allocation to the root and its delivery to the soil environment (Curl and Truelove 1986; Kuzyakov 2001; Lynch and Whipps 1990). If exudates control microbial growth, then controlling the composition, timing, and localization of root exudation would seem to be a reasonable approach to stimulate the growth of beneficial microorganisms at the expense of pathogens (Bednarek et al. 2010; Broeckling et al. 2008; Liu et al. 2005). Unfortunately, despite ever-increasing precision in measuring carbon deposition and microbial colonization in the rhizosphere, the goal of developing predictive models, let alone controlling the process for crop improvement, has eluded researchers (Bowen and Rovira 1976; Cooper and Rao 2006; Darrah and Roose 2001; Handelsman 2004; Hinsinger 2001; Hinsinger et al. 2011; Luster et al. 2009). Apart from the extremes of environment and composition encountered in soils, the process of root exudation per se, as detailed below, is an intrinsically dynamic process that can be difficult to predict even under controlled conditions (Brady and Weil 2010; Lynch and Whipps 1990; Watt et al. 2006). Here we describe challenges and opportunities presented by the recent discovery that extracellular DNA (exDNA) is a component of exudates whose delivery into the rhizosphere is controlled by metabolically active cells at the root apex. Plant Soil (2012) 355:1–16 Lots of exudates at the root tip, not much microbial colonization: why? Microbial growth in the rhizosphere, by definition, is increased relative to that in bulk soil (Rovira 1969). This phenomenon is attributed to the plant’s release of nutrient-rich exudates that can support the growth of diverse microbiota. Therefore, regions of the root that release more exudates might be predicted to support a corresponding increase in microbial growth relative to that in other regions. The root cap has been reported to be a primary source of exudate in experiments using diverse species and conditions (Dennis et al. 2010; Jones et al. 2009; Lundegardh and Stenlid 1944; Lynch and Whipps 1990; McDougall and Rovira 1970; Odell et al. 2008; VanEgeraat 1975; Wood 1967). In direct measurements from whole roots of young legume seedlings grown in hydroponic or plate culture under aseptic conditions, for example, more than 90 % of the total fresh or dry weight derives from the root cap (Griffin et al. 1975; Gunawardena et al. 2005). Therefore it would seem reasonable to predict that root exudate-stimulated microbial populations would predominate at the root cap under more complex conditions. Instead, root caps of cereals, legumes, and other agronomically important species repeatedly have been found to be free of infection and colonization. In fieldgrown wheat Foster et al. (1983) reported that, ‘Unlike the rest of the root surface, the root cap as seen in scanning electron micrographs is generally quite devoid of microbial colonies.’ On tomato roots inoculated with Fusarium, ‘the root cap is not an important site of colonization’ (Lagopodi et al. 2002). On tomato inoculated with Pseudomonas fluorescens, ‘the root cap was always devoid of bacteria’ (Gamalero et al. 2005). Similar results occurred on maize root caps inoculated with P. fluorescens, but upon removal of root caps colonization of the apex developed (Humphris et al. 2005). On pea roots inoculated with spores of pathogenic fungi, then incubated in warm, moist conditions, the root cap remains sterile despite being ensheathed within a mantle of fungal hyphae (Gunawardena and Hawes 2002). Newly synthesized plant cells like those in the region of elongation are more susceptible to infection than older tissue with lignified cell walls (Hawes et al. 2000). Because root caps Plant Soil (2012) 355:1–16 also are comprised of newly synthesized cells generated by meristems in the root apex, this was an especially surprising observation (Curlango-Rivera and Hawes 2011). New insight into the nature and function of root cap defense systems may yield an answer to this long-standing mystery: Sometimes, the carbon-based ‘exudates’ may act to trap, immobilize and inhibit microbial growth rather than serving as a passive nutrient base. Extracellular DNA (exDNA) and protein in root tip defense The recognition that exDNA is a key component of root exudates involved in border cell ‘extracellular trapping’ (Hawes et al. 2011) followed a long history of clues whose significance was overlooked until Brinkmann et al. (2004) documented the importance of exDNA in mammalian defense. VanEgeraat (1975) documented that the primary source of root exudates from young healthy seedlings under laboratory conditions is the root apex. Seedlings were placed onto damp filter paper for 24 h, then removed and the paper was dried and sprayed with ninhydrin (2,2-dihydroxyindane-1,3-dione) which reacts with lysine present in peptides and proteins. Positive reactions were limited to sites where root caps had been in contact with the filter paper. In older seedlings, an additional source is the site of lateral root emergence from the pericycle. However, chromatographic profiles of the material released from these natural wound sites are similar to those of root extracts, while profiles of material released from the root cap are distinct. As VanEgeraat (1975) recognized, ‘The process by which compounds are exuded from the root tip region is completely different from the release following damage of the root....Exudation by the root tip might be more selective so that certain specific compounds would be liberated.’ This prediction proved correct, despite the longstanding presumption that apart from a high molecular weight ‘slime’ or mucilage secreted from root caps, exudates from root tips primarily are the product of cytoplasmic contents leaking from dead ‘sloughed’ cells (Esau 1967; Levy-Booth et al. 2007; Voeller et al. 1964). Synonyms for ‘sloughed’ are ‘putrid’ and 3 ‘gangrenous.’ Border cells, once termed ‘sloughed root cap cells,’ instead are metabolically active cells which exhibit host specific susceptibility and resistance to infection (Goldberg et al. 1989; Sherwood 1987). The border cell gene expression profile is distinct from that of progenitor cells in the root cap but parallel across diverse species (Brigham et al. 1998; Wen et al. 2008). Two-dimensional gel electrophoresis of proteins synthesized by the root cap during a 1h test period (Fig. 1a) also yielded a profile markedly distinct from that of border cells (Fig. 1b) (Brigham et al. 1995). Most surprising was that the profile of proteins extracted from intact border cells (Fig. 1b) was markedly similar to that of a secretome with >100 proteins synthesized and exported during the same experiment (Fig. 1c). Extracellular proteins were found to play a key role in defense of the root tip: when treated with protease at the time of inoculation with spores of a pathogenic fungus, the normal resistance to root tip infection was abolished (Wen et al. 2007b). Among the proteins were antimicrobial enzymes long known to be associated with plant and mammalian defense (De-la-Pena and Vivanco 2010; Kwon et al. 2008). Therefore, it was perhaps not surprising that their destruction altered the normal root defense processes. Treatment with protease also resulted in disintegration of a surrounding mucilage layer and release of bacteria within the layer (Wen et al. 2007a). These data support the suggestion by Matsuyama et al. (1999) that proteins may play a role in the structural integrity of the matrix, even though protein comprises only a small fraction of the matrix composition (Bacic et al. 1986; Chaboud and Rougier 1990; Moody et al. 1988). The discovery that histone H4 was among the proteins synthesized and exported into the extracellular matrix was a surprise, given the long-established role of histones in assembly of genetic material inside the cell (Wen et al. 2007a). However, emerging research provided insights into alternative functions of histones, including potent antimicrobial activity in the extracellular environment (Bergsson et al. 2005; Kawasaki and Iwamuro 2008; Patat et al. 2004; Wang et al. 2009; Xu et al. 2009). Of special interest were reports of a role for histones in extracellular chemotaxis and ‘trapping’ of pathogens by neutrophils in the 4 Plant Soil (2012) 355:1–16 Root cap Border cells Supernatant b c a Fig. 1 The root cap secretome. After a 1-h period of labelling, large differences in protein profiles from (a) root caps and (b) border cells of Pisum sativum L. are evident using twodimensional gel electrophoresis. c More than 100 proteins are synthesized and exported from living cells during the test period (from Brigham et al. 1995). Examples of proteins common to root caps and border cells (black arrows), specific to root caps (open triangles), or specific to border cells (closed triangles) are denoted. Plant Physiol 143:773–783 (www.plantphysiol.org) “Copyright American Society of Plant Biologists” mammalian immune response, because a very similar process occurs in border cells in response to plant pathogens (Gochnauer et al. 1990; Goldberg et al. 1989; Gunawardena et al. 2005; Hawes and Pueppke 1987; Hawes et al. 1988; Zhu et al. 1997). ‘Neutrophil extracellular traps’ (NETs) were first described by Zychlinsky and coworkers (Brinkmann et al. 2004), and now have been implicated in defense against diverse pathogens and other aspects of immune responses in mammals (Abdallah et al. 2012; Amulic and Hayes 2011; Brinkmann and Zychlinsky 2007; Harding and Kubes 2012; Medina 2009; Mitroulis et al. 2011; Park et al. 2012; Urban et al. 2006; Wardini et al. 2010; Wen et al. 2012; Yost et al. 2009; Young et al. 2011). As with the border cell slime layer (Fig. 2), NET formation can occur rapidly in response to specific signals, in the absence of cell death (Pilszik et al. 2010). Experiments therefore were carried out to determine (1) whether the presence of extracellular histone surrounding border cells, like neutrophils, is associated with exDNA; and if so, (2) to determine if enzymatic degradation of border cell exDNA, like NET exDNA, interferes with resistance to infection (Gunawardena and Hawes 2002). The results of these experiments revealed that, like the plant proteins exported from the root cap and border cells (Brigham et al. 1995), plant DNA is synthesized and exported into the root cap extracellular matrix during a 1-h period when no cell death occurs (Wen et al. 2009). Initial sequence analysis revealed that the exDNA structure is related to nuclear DNA, but is enriched in repetitive sequences. When this exDNA was degraded by addition of DNase I concomitant with the inoculation by a pathogenic fungus, the frequency of root cap infection increased from a mild local necrosis in <5 % of inoculated roots to 100 % infection, with rotting of each root tip and proliferation of fungal hyphae (Wen et al. 2009). As in exDNAbased extracellular trapping in mammals, the root tip resistance to fungal infection is associated with aggregation of the fungus and inhibition of its growth (Gunawardena et al. 2005; Medina 2009). The extracellular trapping phenomenon is hostmicrobe specific, with no aggregation or growth inhibition of nonpathogenic fungi (Gunawardena and Hawes 2002; Jaroszuk-Scisel et al. 2009). Host specific chemotaxis and extracellular trapping of pathogens by border cells was described previously, but was presumed to involve aspects of pathogenesis, not defense (Goldberg et al. 1989; Hawes and Pueppke 1987; Hawes and Smith 1989). Agrobacterium tumefaciens chemotaxis toward border cells of a host species was measured using swarm agar assays (Fig. 2a) (Hawes et al. 1988) or direct microscopic observation (Fig. 2b,c). Within hours, strings and strands of immobilized bacteria develop (Fig. 2b). Bacteria adhere to the surface in a layer that is impervious to removal by washing in water (Fig. 2c). Plant Soil (2012) 355:1–16 5 a b c d e f Fig. 2 Host specific binding of bacteria within an inducible extracellular ‘trap’ produced by border cells. a Within seconds of adding the pathogen Agrobacterium tumefaciens to border cells of a host species (P. sativum L.), chemotaxis toward the cells is evident. The large arrow denotes the leading edge of the bacterial swarm; the small arrow denotes border cell sample. b Within 1–2 h, interconnecting strands of bacteria (white arrow) develop between border cells (black arrow). c Trapping of bacteria is evident within the surrounding mucilage of individual border cells of the host species, pea (arrow); d No chemotaxis occurs in response to cells from a nonhost species (Avena sativa L.), and bacteria are excluded from rather than trapped within the surrounding mucilage layer (visualized using India ink, which does not penetrate the mucilage) (arrow). No mucilage layer is induced when a nonpathogenic strain of E. coli is added to border cells of pea (e) or oats (f), and no trapping occurs at the cell surface. Scale bar: 15 μm Adding the plant pathogen to border cells of a nonhost species triggers no chemotaxis or attachment within the surrounding mucilage (Fig. 2d). The human pathogen E. coli added to border cells (Fig. 2e, f) was not associated with chemotaxis, attachment, or production of a mucilage layer in either plant species. Minimal growth can be measured in remaining unattached bacteria or in bacteria growing on mucilage as a sole carbon source, but whether the trapped pathogenic bacteria are viable is unclear (Knee et al. 2001; Zhu et al. 1997). Similar patterns of specificity were reported in association between maize border cells and bacterial species including Rhizobium, E. coli, Pseudomonas, Bacillus, Streptomyces and Cytophaga (Gochnauer et al. 1990). It will be of interest to examine the role of exDNA in this phenomenon, and to explore the possibility that clusters and strings of viable but not culturable (VBNC) colonies found in the rhizosphere might be related to exDNA based trapping (Gamalero et al. 2004). The high molecular weight polysaccharide-based mucilage exported from root caps has been studied 6 Plant Soil (2012) 355:1–16 in various species but DNA has not been included in analyses to date (Bacic et al. 1986; Chaboud and Rougier 1990; Foster 1981a, b, 1982; Jones and Morre 1973; Knee et al. 2001; Lynch and Staehelin 1995; Miki et al. 1980; Newcomb 1967; Oades 1978; Read et al. 1999; Sealey et al. 1995; Watt et al. 1993). Therefore details of how exDNA is synthesized, exported, and integrated into the extracellular matrix remain to be established (Hawes et al. 2011). However, the presence of nucleic acids among exudates of healthy roots was reported (Curl and Truelove 1986; Fries and Forsman 1951; Lundegarth and Stenlid 1944; Stenlid 1944), and its active synthesis and export into the extracellular matrix of the root cap periphery also were documented (Phillips and Torrey 1971). Using the fluorescent stain DAPI, which binds to A-T rich strands of DNA and can pass through intact cell membranes to reveal DNA within cells or outside the cell boundaries (Kubista et al. 1987), exDNA is readily detected within border cell mucilage (Wen et al. 2009). In the presence of stimulating bacteria, DAPI staining occurs within border cells, throughout the surrounding expanded mucilage layer (Fig. 3a) and within trapped bacteria (Fig. 3a, arrow). Fig. 3 exDNA from the root tip of pea. a DAPI staining of a pea border cell with bacteria trapped within the surrounding mucilage layer. Scale bar: 15 μm. b SYTOX green staining of border cell exDNA strands and c other structures. Scale bar: 10 μm. 3A, 3 C, photos by Fushi Wen. 3B, photo by Sarah O’Connor Staining border cell populations and associated mucilage with SYTOX green, a high-affinity nucleic acid stain which is not taken into living cells, reveals extracellular material ranging from strands (Fig. 3b) to distinctive structures (Fig. 3c). These structures are similar in appearance to those produced by neutrophils (Patel et al. 2010; Pilszik et al. 2010). Questions of particular interest are the nature of the exDNA structure(s) involved in trapping and how they might interface with other polymers within the root cap mucilage (Bacic et al. 1986; Knee et al. 2001). One possibility is that the ‘stickiness’ of DNA alone might be sufficient to trap added microorganisms. If so, then addition of DNA alone would be predicted to result in trapping. No such result occurred upon addition of salmon sperm DNA or pea genomic DNA to microbes (Wen et al. 2009). An alternative hypothesis is that distinct sequences organized in specific structures are required. In support of this model are observations by Van’t Hof and colleagues (Van’t Hopf and Bjerknes 1982; Kraszewska et al. 1985), who described a distinct class of DNA produced by P. sativum root caps during the G2-M transition, the point in the root cap meristem a b c Plant Soil (2012) 355:1–16 7 The presence of DNA from plants and other organisms in the soil is well established (Izano et al. 2008; Vlassov et al. 2007; Whitchurch et al. 2002). Plant exDNA has been presumed to be derived by leakage from dead cells (Levy-Booth et al. 2007). The discovery that secretion of exDNA from root caps instead is a component of a complex, inducible, and carbonexpensive defense mechanism may be useful in tracking as well as modelling rhizosphere community structure. The programmed separation of cells from the root cap was long presumed to be a product of continuous cell cycle activity within the root cap meristem in parallel with such activity in the apical meristem (e.g. Clowes 1971; Whipps and Lynch 1983). If correct, then a continuous detection of exudates at the tip would be a predicted result. Direct observations of rhizosphere structure even under controlled conditions do not support this paradigm (Iijima et al. 2003). The viability and number of border cells that a root cap can release daily are conserved within families and can range from 0 to 10,000 cells a day (Hawes et al. 2003; Hawes and Pueppke 1986). For a given root, the process of root cap turnover is not continuous but instead is induced or repressed in a species- and genotype- specific manner in response to endogenous Fig. 4 Variation in border cell delivery from root caps under controlled conditions. The length of the root at time zero is denoted with white arrows. As roots elongate side by side in the same plate of water agar, the presence of border cells detectable by direct microscopic observation ranges from none (inset photo, left) to intermittent clumps (inset photo, center) to a continuous robust sheath (center). (Plant Physiol 119:417–428, (www.plantphysiol.org) Copyright American Society of Plant Biologists). Inset, center: Border cells released from tips of elongating root of Lithospermum erythrorhizon. The border cells from this species express a red pigment, shikonin, which facilitates detection of their presence at intervals along the root surface (arrows). (Plant Physiol 119:417–428, (www.plantphysiol.org) Copyright American Society of Plant Biologists) cell cycle when border cell separation occurs (Brigham et al. 1998). Like root cap exDNA (Wen et al. 2009), this ‘extrachromosomal DNA’ is related to nuclear DNA but is distinguishable based on the prevalence of repetitive sequences (Kraszewska et al. 1985). The programmed delivery of characteristic exDNA patterns as an integral component of the matrix could provide a tool to examine underlying patterns of rhizosphere carbon deposition and microbial colonization and allow progress toward exploiting the system for crop improvement. Factors known to influence border cell delivery are summarized below. Factors controlling delivery of exDNA-based traps from root caps Border cell populations 8 Plant Soil (2012) 355:1–16 and environmental signals (Brigham et al. 1998; Ponce et al. 2005). Therefore, when seedlings are grown under identical conditions side by side in petri dishes, the delivery of mucilage and border cells can vary from nothing to intermittent clumps to a continuous sheath surrounding the root from base to tip (Fig. 4). The variation is illustrated schematically because even with direct microscopic observation on sterile plates the differences can be difficult to detect (Fig. 4, inset photos). Some species exhibit border cell specific expression of pigmented metabolites which provide a convenient marker for cell dispersal (Brigham et al. 1999). Thus, Saccharum officinarum, Sorghum vulgare and Lithospermum erythrorhizon have pink, purple, and red border cells, respectively. This pigmentation facilitates recognition of rhizosphere distribution patterns that otherwise would be obscure (Fig. 4, inset center). Variation in root exudation and rhizosphere colonization has been proposed to be a major obstacle to agronomic application of promising discoveries like biological control (Cooper and Rao 2006; Sylvia et al. 1998). Understanding factors controlling carbon delivery via border cells may be key to monitoring and controlling rhizosphere community structure (Lee and Hirsch 2006; Smucker and Erickson 1987). a b Fig. 5 Instantaneous swelling and dispersal of P. sativum border cells in response to immersion in free water. a At 99+% humidity, the root tip is smooth, and the presence of border cells is undetectable except with scanning electron microscopy (inset). Scale bar: 0.5 mm. b Addition of a droplet of water results in swelling of border cells away from the tip within 10–15 s. For roots of any given plant, one major factor controlling border cell release is the availability of free water (Fig. 5) (Odell et al. 2008). Roots of legumes, cereals, cucurbits and most other crop species are programmed to produce a species-specific number of cells (Hawes and Pueppke 1986). When that number has accumulated on the cap periphery the same set may remain on the root for an extended period without any new cells being produced. This appears to result from the accumulation of an extracellular signal within the surrounding mucilage to a level that suppresses cap turnover except when diluted in water (Brigham et al. 1998). The properties of the mucilage are such that it can hold 1000X its weight in water (Guinel and McCully 1986). Yet even at 99 % humidity, in the absence of free water, the mucilage remains ‘dry’ like a sponge without moisture (Fig. 5a) and highresolution microscopy is necessary to detect the ensheathed border cells (Fig. 5a, inset). Upon addition of water–including, for example, a drop resulting from condensation on the inside of a petri plate falling onto the root–the mucilage immediately expands (Fig. 5b) and border cells are dispersed into suspension (Fig. 5c). It is important to note that the drop of water not only causes the dissociation of the existing group of ca 4,000 cells from the cap periphery, but also c Scale bar: 0.5 mm. c Gentle agitation of the plate by tapping one side results in immediate dispersal of border cells into the suspension, in the direction of the applied force. Scale bar: 0.5 mm. Staining with the vital stain fluorescein diacetate, which only accumulates inside living cells, reveals border cell viability of 95–100 % (inset). Scale bar: 10 μm Plant Soil (2012) 355:1–16 9 triggers renewed cell cycle instantaneously (Brigham et al. 1998). Within 5 min, mitosis increases within the root cap meristem, and dozens of new cells emerge from the periphery. Activation of the quiescent center also occurs, and cell production proceeds until a new set has accumulated within 24 h (Ponce et al. 2005). It seems obvious that within the soil environment, where a continuous film of free water at the root tip would be intermittent for most crops in most conditions, this factor alone could account for much of the variability in root tip carbon deposition that occurs. Other factors that can vary the number of border cells and associated products released into the rhizosphere, include soil type, physical abrasion, day length, root age and growth rate (Iijima et al. 2000, 2003; Odell et al. 2008; Somasundaram et al. 2008; Wuyts et al. 2006). Sodium fluoride added to wheat roots can stimulate changes in number of border cells and in level of protein secretion (Bozhkov et al. 2007). Carbon dioxide, aluminum, boron, and plant pathogens stimulate changes in border cell production in a plant species- and genotype-specific manner with distinct responses at different developmental stages (Cannesan et al. 2011; Chen et al. 2008; Liu et al. 2007; Miyasaka and Hawes 2001; Pan et al. 2004; Tamas et al. 2005; Zhao et al. 2000; Zhu et al. 2003). For example, increased carbon dioxide inhibits border cell production in P. sativum during germination, but results in increased cell production in seedlings (Fig. 6). Border cell production in Medicago sativa seedlings, in contrast, is impervious to similar changes in carbon dioxide (Zhao et al. 2000). Continuous culture of roots in high concentrations of certain sugars and secondary metabolites results in marked increases in mucilage production by maize roots (Jones and Morre 1973; Knudson 1917). Transient exposure of roots to metabolites including rhamnose, caffeine, and flavonoids for several minutes, a condition more likely to occur under natural conditions, can specifically induce or repress border cell production without affecting rate of root growth (Curlango-Rivera et al. 2010). Altered expression of genes controlling cell cycle or cell wall solubilization at the cap periphery results in altered border cell production, and transient changes in their expression due to diverse environmental signals could influence the process as well (Wen et al. 1999; Woo et al. 2004). A new study reporting an ‘extraordinary sheath’ of material triggered on roots of Acacia magnum grown in hydroponic culture, highlights the importance of understanding factors controlling this avenue of carbon deposition and their impact on rhizosphere structure (Endo et al. 2011). Fig. 6 Effects of carbon dioxide on border cell separation from root tips (arrows) of P. sativum seedlings. a Cells from a single root tip observed with a dissecting microscope after 3 days in (A) ambient (0.03 % CO2 vs 21 % O2); or (b) 6 % CO2 vs 15 % O2). Increased O2 alone had no effect on cell production. From Plant Physiology 122:181–188, used with permission. (www. plantphysiol.org) Copyright American Society of Plant Biologists Single cells Morphology of border cell detachment from the cap periphery can range from a population of single cells in suspension to finger-like strands of cells to an entire root cap (Endo et al. 2011; Hamamoto et al. 2006; Vicre et al. 2005; Wen et al. 2008). The significance of these variations with respect to exDNA-based trapping is unknown, but the variation in amount and composition of carbon-based material can be substantial even on a single-cell basis. For many years, border cells were called ‘sloughed root cap cells’ to reflect the presumption that delivery of the cell populations must 10 Plant Soil (2012) 355:1–16 reflect a process of falling away from the root as a consequence of cell death (e.g. Uren 2001). This notion prevailed, despite repeated documentation that the cells from most species are metabolically active as they detach from the root cap and can survive for extended periods in liquid culture (Caporali 1983; Gautheret 1933; Hawes and Wheeler 1982; Stubbs et al. 2004). Knudson (1919) reported that border cells released from Zea mays or P. sativum grown in hydroponic culture, with or without glucose, remained 100 % viable for more than one month. Even more surprising was the observation that the cells export enzymes and other proteins (Rogers et al. 1942) and can remain metabolically active after detachment into the soil environment (Vermeer and McCully 1982). Continued secretion of mucilage from border cells can occur for days after detachment from roots grown in soil (Hawes and Brigham 1992; Hawes et al. 1998). Like white blood cell ‘granules’, border cells contain abundant storage particles which may provide energy for survival and response to signals in the extracellular environment (Feldman 1985; Newcomb 1967). The mucilage produced by individual border cells after separation from the root cap also is a dynamic process. An increase in the diameter of the mucilage layer is induced almost instantaneously in a speciesand genotype-specific manner in response to exposure to bacteria (Figs. 2, 3), fungi (Wen et al. 2009), and aluminum (Miyasaka et al. 2000). Border cells from pea, for example, can form aggregates containing hundreds of cells and associated mucilage (Fig. 7a), or exist as isolated cells with variable layers of surrounding mucilage (Fig. 7b) (Wen et al. 2007a). Given that such variation can occur in controlled environments and that each cell can trap thousands of bacterial cells, the potential for creating variable islands that confound efforts to measure carbon deposition and its impact on rhizosphere colonization in the soil, is obvious. With recognition of the ‘trapping’ function of border cells, on the other hand, these seemingly inexplicable phenomena may be easier to understand. The observation by Guinel and McCully (1987) that border cells can continue to expand after detachment from the root as single cells, also is less surprising in the context of their proposed functions in ‘border patrol.’ If border cells trap heavy metals and pathogens and control the growth of deleterious microorganisms in the vicinity of plant roots, then a capacity to achieve an increased surface area would be a predictable benefit to the plant rather than an egregious waste of fixed carbon (Fig. 8). In addition to proteins, DNA and polysaccharides, the root cap and border cell exudates include primary and secondary metabolites that function in signalling and recognition of beneficial as well as pathogenic microbes (e.g. Baluska et al. 1996; Graham 1991; Maxwell and Phillips 1990; Peters and Long 1988). The mixture also contains feedback signals that may influence rate and direction of root growth and development (Baluska et al. 1996; Caffaro et al. 2011; Moore and Fondren 1986). The potential for creating changes in the composition of border cell products has been demonstrated by studies of cotton engineered to resist insect damage by expression of crystal (CRY) proteins from Bacillus thuriengensis. BT toxin is delivered through exudates of engineered plants into the Fig. 7 Variation in aggregation of detached border cell populations ranges from (a) a cohesive mass containing hundreds of border cells to (b) individual cells. Mucilage layers, detected by staining with India ink which is excluded, are present on viable cells but disintegrate rapidly after cell death (block arrow). Scale bar: 20 μm a b Plant Soil (2012) 355:1–16 11 Fig. 8 Border cell expansion of the volume of a single cell (black arrows denote each end of the cell) by >10-fold 7 days after detachment from the root cap. The nucleus (white arrow) and cytoplasmic strands are evident within the living cell. Inset: The original size of border cells within this sample is illustrated by showing for comparison a cell within the same population which died before any growth occurred. Scale bar: 30 μm soil where it can exhibit a half-life of up to 234 days (Saxena and Stotzky 2001; Tapp and Stotzky 1997). Direct measurements of Cry proteins revealed that roots of all genetically modified lines tested synthesize and export BT toxin, and that root caps, border cells and root mucilage are sources of this material (Knox and Vadakattu 2005; Knox et al. 2007). The environmental impact is not clear at this time, but the results suggest that reproducible changes in the soil environment already have been accomplished via changes in root cap delivery systems of genetically modified crops. dynamics of the rhizosphere and its components in the interest of fostering sustainable methods for agriculture (Ceccherini et al. 2009; Levy-Booth et al. 2007; Pietramellara et al. 2009). If used in conjunction with holistic tracking methods that combine laboratory and field assessment (e.g. Knox et al. 2009), a goal of harnessing the plant’s ability to control root exudation and rhizosphere community structure may not be unrealistic (Atkinson et al. 1975; Knox et al. 2009; Liu et al. 2005). Conclusions The discovery that exDNA plays a role in plant defense raises more questions than it answers, and additional research is needed before conclusions can be drawn regarding a general role in plant immunity. The new data do reinforce the premise that a simple model of nutrient rich material leaking from roots and feeding microbial growth in general is inadequate (De-La-Pena et al. 2010). The important role of metabolites secreted into the ‘apoplast’ has long been recognized (Brisson et al. 1994; Kwon et al. 2008). Understanding the nature and function of the ‘exudates’ delivered by the root cap may offer insights into how the natural immunity of the root cap might be extended to more vulnerable sites including the region of elongation, where most soilborne pathogens initiate infections (Hawes et al. 2000). The controlled delivery of exDNA may complement new tools available to define the structural and functional Acknowledgements We gratefully acknowledge support for our research in this area from the National Science Foundation (NSF# 1032339 to MCH and ZX) and the Department of Energy (DOE DEAC02-06CH11357 to JOK). We thank Dr. Virginia Rich for critical reading of the manuscript. We dedicate this review to the memory of W. D. ‘Dietz’ Bauer. References Abdallah DS, Lin C, Ball CJ et al (2012) Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps. Infection Immun 80:768–777 Amulic B, Hayes G (2011) Neutrophil extracellular traps. Curr Biol 21:R297–R298 Atkinson TG, Neal JL, Larson RI (1975) Genetical control of the rhizosphere of wheat. In: Bruehl GW (ed) Biology and control of soil-borne plant pathogens. American Phytopathological Society, St. Paul Bacic A, Moody SF, Clarke AE (1986) Structural analysis of secreted root slime from maize. Plant Physiol 80:771–777 Baluska F, Volkmann D, Hauskrecht M, Barlow PW (1996) Root cap mucilage and extracellular calcium as modulators of cellular growth in postmitotic growth zones of the maize root apex. Bot Acta 109:25–34 12 Bednarek P, Kwon C, Schulze-Lefert P (2010) Not a peripheral issue: secretion in plant-microbe interactions. Curr Opin Plant Biol 13:378–385 Bergsson G, Agerberth B, Jornvall H, Gudmundsson GH (2005) Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua). FEBS J 272:4960–4969 Bowen GD, Rovira AD (1976) Microbial colonization of plant roots. Ann Rev Phytopathol 114:121–144 Bozhkov AI, Kuznetsova YA, Menzyanova NG (2007) Interrelationship between the growth rate of wheat roots, their excretory activity and the number of border cells. Russian J Plant Physiol 54:97–103 Brady NC, Weil RR (2010) Elements of the nature and properties of soils. Prentice Hall Brigham LA, Woo HH, Nicoll SM, Hawes MC (1995) Differential expression of proteins and messenger-RNAs from border cells and root tips of pea. Plant Physiol 109:457– 463 Brigham LA, Woo HH, Wen F, Hawes MC (1998) Meristemspecific suppression of mitosis and a global switch in gene expression in the root cap of pea by endogenous signals. Plant Physiol 118:1223–1231 Brigham LA, Michaels PJ, Flores HE (1999) Cell-specific production and antimicrobial activity of naphthoquinones in roots of Lithospermum erythrorhizon. Plant Physiol 119:417–428 Brinkmann V, Zychlinsky A (2007) Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol 5:577– 582 Brinkmann V, Brichard U, Goosmann C et al (2004) Neutrophil extracellular traps kill bacteria. Science 303:1532–1535 Brisson RF, Tenhaken R, Lamb C (1994) Function of oxidative cross linking of cell wall structural proteins in plant disease resistance. Plant Cell 6:1703–1712 Broeckling CD, Broz AK, Bergelson J, Manter DK, Vivanco J (2008) Root exudates regulate soil fungal community composition and diversity. Appl Environ Microbiol 74:738– 744 Bruehl GW (1987) Soilborne plant pathogens. Macmillan Publishing Company, NY, USA Caffaro MM, Vivanco JM, Gutierrez BFH et al (2011) The effect of root exudates on root architecture in Arabidopsis thaliana. Plant Growth Regul 64:241–249 Cannesan MA, Gangneux C, Lanoue A et al (2011) Association between border cell responses and localized root infection by pathogenic Aphanomyces euteiches. Ann Bot 108:459– 469 Caporali L (1983) Cytological study of cultured cells from maize root cap. Plant Sci Lett 31:231–236 Ceccherini MT, Ascher J, Agnelli A et al (2009) Experimental discrimination and molecular characterization of the extracellular soil DNA fraction. Antonie Van Leeuwenhoek 96:653–657 Chaboud A, Rougier M (1990) Comparison of maize root mucilages isolated from root exudates and root surface extracts by complementary cytological and biochemical investigations. Protoplasma 156:163–173 Chen W, Liu P, Xu G et al (2008) Effects of aluminum on the biological characteristics of cowpea root border cells. Acta Physiol Plant 30:303–308 Plant Soil (2012) 355:1–16 Clowes FAL (1971) The proportion of cells that divide in root meristems of Zea mays L. Ann Bot 35:249–261 Compant S, Duffy B, Nowak J et al (2005) Use of plant growth promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action and future prospects. Appl Environ Microbiol 71:4951–4959 Cooper JE, Rao JR eds (2006) Molecular approaches to soil, rhizosphere and plant microorganism analysis. CABI, Oxfordshire, UK, Cambridge MA, USA Curl EA, Truelove B (1986) The rhizosphere. Advanced series in agricultural sciences, vol. 15, Springer-Verlag, BerlinHeidelberg-New York-Tokyo Curlango-Rivera G, Hawes MC (2011) Root tips moving through soil: an intrinsic vulnerability. Plant Signal Behavior 6:1–2 Curlango-Rivera G, Duclos DV, Ebolo JJ, Hawes MC (2010) Transient exposure of root tips to primary and secondary metabolites: impact on root growth and production of border cells. Plant Soil 332:267–275 Darrah PR, Roose T (2001) Modeling the rhizosphere. In: Pinton R, Varanini Z, Nannipieri P (eds) The rhizosphere: biochemistry and organic substances at the soil-plant interface. Marcel Dekker, Inc., New York, pp 327–372 De-la-Pena C, Vivanco JM (2010) Root-microbe interactions: the importance of protein secretion. Curr Proteonomics 7:265–274 De-la-Pena C, Badri DV, Lei Z et al (2010) Root secretion of defense-related proteins is development-dependent and correlated with flowering time. J Biol Chem 285:30654– 30666 Dennis PG, Miller AJ, Hirsch PR (2010) Are root exudates more important than other sources of rhizodeposits in structuring rhizosphere bacterial communities? FEMS Microbiol Ecol 72:313–327 Donato JJ, Moe LA, Converse BJ et al (2010) Metagenomic analysis of apple orchard soil reveals antibiotic resistance genes encoding predicted bifunctional proteins. Appl Environ Microbiol 76:4396–4401 Endo I, Tange T, Osawa H (2011) A cell-type-specific defect in border cell formation in the Acacia mangium root cap developing an extraordinary sheath of sloughed-off cells. Ann Bot 108:279–290 Esau K (1967) Plant anatomy. Wiley, New York Feldman LJ (1985) Root gravitropism. Physiol Plant 65:341–344 Foster RC (1981a) Polysacharrides in soil fabrics. Science 214:665–667 Foster RC (1981b) The ultrastructure and histochemistry of the rhizosphere. New Phytol 89:263–273 Foster RC (1982) The fine structure of epidermal cell mucilages of roots. New Phytol 91:727–740 Foster RC, Rovira AD, Cock TW (1983) Ultrastructure of the root-soil interface. American Phytopathological Society, St. Paul Fries N, Forsman B (1951) Quantitative determination of certain nucleic acid derivatives in pea root exudate. Physiol Plant 4:410–420 Gamalero E, Lingua G, Capri FG et al (2004) Colonization pattern of primary tomato roots by Pseudomonas fluorescens A6RI characterized by dilution plating, flow cytometry, fluorescence, confocal and scanning electron microscopy. FEMS Microbiol Ecol 48:79–87 Plant Soil (2012) 355:1–16 Gamalero E, Lingua G, Tombolini R et al (2005) Colonization of tomato root seedling by Pseudomonas fluorescens 92rkG5: spatio-temporal dynamics, localization, organization, viability and culturability. Microbial Ecol 50:289–297 Gautheret MR (1933) Cultures of cells isolated from the root cap. CR Acad Sci 186:638–640 Gilbert GS, Clayton MK, Handelsman J, Parke JL (1996) Use of cluster and discriminant analysis to compare rhizosphere bacterial populations following biological perturbation. Microbial Ecol 32:123–147 Gochnauer MB, Sealey LJ, McCully ME (1990) Do detached root cap cells influence bacteria associated with maize roots? Plant Cell Environ 13:793–801 Goldberg NP, Hawes MC, Stanghellini ME (1989) Specific attraction to and infection of cotton root cap cells by zoospores of Pythium dissotocum. Can J Bot 67:1760–1767 Graham TC (1991) Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates. Plant Physiol 95:594–603 Griffin GJ, Hale MG, Shay FJ (1975) Nature and quantity of sloughed organic matter produced by roots of axenic peanut plants. Soil Biol Biochem 8:29–32 Guinel FC, McCully ME (1986) Some water-related physical properties of maize root cap mucilage. Plant Cell Environ 9:657–666 Guinel FC, McCully ME (1987) The cells shed by the root cap of Zea: their origin and some structural and physiological properties. Plant Cell Environ 10:565–578 Gunawardena U, Hawes MC (2002) Tissue specific localization of root infection by fungal pathogens: role of root border cells. Mol Plant Microbe Int 15:1128–1136 Gunawardena U, Rodriguez M, Straney D et al (2005) Tissue specific localization of root infection by Nectria haematococca: mechanisms and consequences. Plant Physiol 137:1363–1374 Hamamoto L, Hawes MC, Rost TL (2006) The production and release of living root cap border cells is a function of root apical meristem type in dicotyledonous angiosperm plants. Ann Bot 97:917–923 Handelsman J (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68:669–685 Handelsman J, Stabb EV (1996) Biocontrol of soilborne plant pathogens. Plant Cell 8:1855–1869 Harding M, Kubes P (2012) Innate immunity in the vasculature: interactions with pathogenic bacteria. Curr Opin Microbiol 15:85–91 Hawes MC, Wheeler H (1982) Factors affecting victorininduced cell death: temperature and plasmolysis. Physiol Plant Pathol 20:137–144 Hawes MC, Pueppke SG (1986) Sloughed peripheral root cap cells: yield from different species and callus formation from single cells. Am J Bot 73:1466–1473 Hawes MC, Pueppke SG (1987) Correlation between binding of Agrobacterium tumefaciens by root cap cells and susceptibility of plants to crown gall. Plant Cell Rep 6:287–290 Hawes MC, Smith LY (1989) Requirement for chemotaxis in pathogenicity of Agrobacterium tumefaciens on roots of soil-grown pea plants. J Bacteriol 171:5668– 5671 13 Hawes MC, Brigham LA (1992) Impact of root border cells on microbial populations in the rhizosphere. Adv Plant Pathol 8:119–148 Hawes MC, Smith LY, Howarth AJ (1988) Agrobacterium tumefaciens mutants deficient in chemotaxis to root exudates. Mol Plant Microbe Int 1:182–186 Hawes MC, Brigham LA, Wen F, Woo HH, Zhu Y (1998) Function of root border cells in plant health: pioneers in the rhizosphere. Ann Rev Phytopathol 36:311–327 Hawes MC, Gunawardena U, Miyasaka S, Zhao X (2000) The role of root border cells in plant defense. Trends Plant Sci 5:128–133 Hawes MC, Bengough G, Cassab G, Ponce G (2003) Root caps and rhizosphere. J Plant Growth Regul 21:352–367 Hawes MC, Curlango-Rivera G, Wen F et al (2011) Extracellular DNA: the tip of root defenses. Plant Sci 180:741–745 Hinsinger P (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil 237:173–195 Hinsinger P, Brauman A, Devau N et al (2011) Acquisition of phosphorus and other poorly mobile nutrients by roots. Where do plant nutrition models fail? Plant Soil 348:29–61 Hirsch AM (2004) Plant-microbe symbioses: a continuum from commensalism to parasitism. Symbiosis 37:345–363 Humphris SN, Bengough AG, Griffiths BS et al (2005) Root cap influences root colonisation by Pseudomonas fluorescens SBW25 on maize. FEMS Microbiol Ecol 54:123–130 Iijima M, Griffiths B, Bengough AG (2000) Sloughing of cap cells and carbon exudation from maize seedling roots in compacted sand. New Phytol 145:477–482 Iijima M, Sako Y, Rao TP (2003) A new approach for the quantification of root cap mucilage exudation in the soil. Plant Soil 255:399–407 Izano EA, Amarante MA, Kher WB, Kaplan JB (2008) Differential roles of poly-N-acetylglucosamine surface polysaccharide and extracellular DNA in Staphylococcus aureus and S. epidermidis biofilms. Appl Environ Microbiol 74:470–476 Jaroszuk-Scisel J, Kurek E, Rodzik B, Winiarczyk K (2009) Interactions between rye (Secale cereale) root border cells (RBCs) and pathogenic and nonpathogenic rhizosphere strains of Fusarium culmorum. Mycol Res 113:1053–1061 Jones DD, Morre DJ (1973) Golgi apparatus mediated polysaccharide secretion by outer root cap cells of Zea mays. III. Control by exogenous sugars. Physiol Plant 29:68–75 Jones DL, Nguyen C, Finlay RD (2009) Carbon flow in the rhizosphere: carbon trading at the soil-root interface. Plant Soil 321:5–33 Kawasaki H, Iwamuro S (2008) Potential roles of histones in host defense as antimicrobial agents. Infect Disord Drug Targets 8:195–205 Knee EM, Gong FC, Gao MS et al (2001) Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source. Mol Plant Microbe Int 14:775–784 Knox OGG, Vadakattu GVSR (2005) Evaluation of border cell number and cry protein expression from root tips of Gossypium hirsutum. In: Cote JC, Otvos IS, Schwartz JL (eds) Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact Knox OGG, Gupta VVSR, Nehl DB, Stiller WN (2007) Constitutive expression of cry proteins in roots and border cells of transgenic cotton. Euphytica 154:83–90 14 Knox OGG, Gupta VVSR, Lardner R (2009) Cotton cultivar selection impacts on microbial diversity and function. Aspects Appl Biol 98:1–8 Knudson L (1917) The toxicity of galactose and mannose for green plants and the antagonistic action of other sugars toward these. Am J Bot 4:430–437 Knudson L (1919) Viability of detached root cap cells. Am J Bot 6:309–310 Kraszewska EK, Bjerknes CA, Lamm SS, Van’t Hopf J (1985) Extrachromosomal DNA of pea-root (Pisum sativum) has repeated sequences and ribosomal genes. Plant Mol Biol 5:353–361 Kubista M, Akerman B, Norden B (1987) Characterization of interaction between DNA and 4,6-diamidino- 2- phenylindole by optical spectroscopy. Biochemistry 26:4545–4553 Kuzyakov YV (2001) Tracer studies of carbon translocation by plants from the atmosphere into the soil. Eurasian Soil Sci 34:28–42 Kwon C, Bednarek P, Schulze-Lefert P (2008) Secretory pathways in plant immune responses. Plant Physiol 147:1575–1583 Lagopodi AL, Ram AFJ, Lamers GEM et al (2002) Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker. Mol Plant Microbe Int 15:172–179 Lee A, Hirsch AM (2006) Signals and responses: choreographing the complex interaction between legumes and alphaand beta-rhizobia. Plant Signal Behavior 1:161–168 Levy-Booth DJ, Campbell RG, Gulden RH et al (2007) Cycling of extracellular DNA in the soil environment. Soil Biol Biochem 39:2977–2991 Liu B, Zeng Q, Yan F et al (2005) Effects of transgenic plants on soil microorganisms. Plant Soil 271:1–13 Liu J, Yu M, Wang W, Feng Y (2007) Influence of boron and aluminum on production and viability of root border cells of pea. Adv Plant Animal Boron Nutrition, pp 67–74 Loh J, Pierson EA, Pierson LS III, Stacy G, Chatterjee A (2002) Quorum sensing in plant-associated bacteria. Curr Opin Plant Biol 5:1–5 Lundegarth H, Stenlid G (1944) On the exudation of nucleotides and flavonone from living roots. Arkiv f Bot 31A:10 Luster J, Gottlein A, Nowack B, Sarret G (2009) Sampling, defining, characterizing and modeling the rhizosphere— the soil science tool box. Plant Soil 321:457–482 Lynch JM, Whipps JM (1990) Substrate flow in the rhizosphere. Plant Soil 129:1–10 Lynch MA, Staehelin LA (1995) Immunocytochemical localization of cell wall polysaccharides in the root tip of Avena sativa. Protoplasma 188:115–127 Marschner P, Crowley D, Rengel Z (2011) Rhizosphere interactions between microorganisms and plants govern iron phosphorus acquisition along the root axis—model and research methods. Soil Biol Biochem 43:883–894 Matsuyama T, Satoh H, Yamada Y, Hashimoto T (1999) A maize glycine-rich protein is synthesized in the lateral root cap and accumulates in the mucilage. Plant Physiol 120:665–674 Maxwell CA, Phillips DA (1990) Concurrent synthesis and release of nod gene inducing flavonoids from alfalfa roots. Plant Physiol 93:1552–1558 Plant Soil (2012) 355:1–16 McDougall BM, Rovira AD (1970) Sites of exudation of C-14labelled compounds from wheat roots. New Phytol 69:999 Medina E (2009) Neutrophil extracellular traps: a strategic tactic to defeat pathogens with potential consequenes for the host. J Innate Immun 1:176–179 Miki NK, Clarke KJ, McCully ME (1980) A histological and histochemical comparison of the mucilages on the root tips of several grasses. Can J Bot 58:2581–2593 Mitroulis I, Kambas K, Chrysanthopoulou A et al (2011) Neutrophil extracellular trap formation is associated with IL1beta and autophagy-related signaling in gout. PLoS One 6:e29318 Miyasaka S, Hawes MC (2001) Possible role of root border cells in detection and avoidance of aluminum toxicity. Plant Physiol 125:1978–1987 Moody SF, Clarke AE, Bacic A (1988) Structural analysis of secreted slime from wheat and cowpea roots. Phytochemical 27:2857–2861 Moore R, Fondren WM (1986) The possible involvement of root-cap mucilage in gravitropism and calcium movement across root tips of Allium cepa L. Ann Bot 58:381–387 Morris CE, Monier J-M (2003) The ecological significance of biofilm formation by plant-associated bacteria. Ann Rev Phytopathol 41:429–453 Newcomb EH (1967) Fine structure of protein-storing plastids in bean root tips. J Cell Biol 33:143–163 Oades JM (1978) Mucilage at the root surface. J Soil Sci 29:1– 16 Odell RE, Dumlao MR, Samar D, Silk WK (2008) Stagedependent border cell and carbon flow from roots to rhizosphere. Am J Bot 95:441–446 Pan J, Ye D, Wang L et al (2004) Root border cell development is a temperature-insensitive and Al-sensitive process in barley. Plant Cell Physiol 45:751–760 Park SJ, Pai KS, Kim JH, Shin JI (2012) ANCA-associated glomerulonephritis in a patient with infections endocarditis: the role of neutrophil extracellular traps? Revue Med Int 33:57 Patat SA, Carnegie RB, Kingsbury C et al (2004) Antimicrobial activity of histones from hemocytes of the pacific white shrimp. Eur J Biochem 271:4825–4833 Patel S, Kumar S, Jyoti A et al (2010) Nitric oxide donors release extracellular traps from human neutrophils by augmenting free radical generation. Nitric Oxide 22:226–234 Peters NK, Long SR (1988) Alfalfa root exudates and compounds which promote or inhibit induction of Rhizobium meliloti nodulation genes. Plant Physiol 88:396–400 Phillips HL, Torrey JG (1971) Deoxyribonucleic acid synthesis in root cap cells of cultured roots of Convolvulus. Plant Physiol 48:213–218 Pierson LS III, Pierson EA (2007) Roles of diffusible signals in communication among plant-associated bacteria. Phytopathology 97:227–232 Pietramellara G, Ascher J, Borgogni F et al (2009) Extracellular DNA in soil and sediment: fate and ecological relevance. Biol Fertil Soils 45:219–235 Pilszik FH, Salina D, Poon KK et al (2010) A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol 185:7413–7425 Pinton R, Varanini Z, Nanipieri P, eds (2007) The rhizosphere: biochemistry and organic substances at the soil-plant interface. Marcel Dekker, Inc. New York, Basel Plant Soil (2012) 355:1–16 Ponce G, Barlow PW, Feldman LJ, Cassab GI (2005) Auxin and ethylene interactions control mitotic activity of the quiescent centre, root cap size, and pattern of cap cell differentiation in maize. Plant Cell Environ 28:719–732 Read DB, Gregory PJ, Bell AE (1999) Physiological properties of axenic maize root mucilage. Plant Soil 211:87091 Rogers HT, Pearson RW, Pierre WH (1942) The source and phosphatase activity of exoenzyme systems of corn and tomato roots. Soil Sci 54:353–365 Rovira AD (1969) Plant root exudates. Bot Rev 35:35–57 Rovira AD (1991) Rhizosphere research: 85 years of progress and frustration. In: Keister DL, Cregan PB (eds) The rhizosphere and plant growth. Kluwer, Dordrecht, pp 3–13 Saxena D, Stotzky G (2001) Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. Soil Biol Biochem 33:1225–1230 Schroth MN, Snyder WC (1961) Efect of host exudates on chlamydospore germination of the bean root rot fungus Fusarium solani f sp phaseoli in soil. Phytopathology 52:279–285 Sealey LJ, McCully ME, Canny MJ (1995) The expansion of maize root cap mucilage during hydration. I. Kinetics. Physiol Plant 93:38–46 Sherwood RT (1987) Papilla formation in corn root cap cells and leaves inoculated with Colletotrichum graminicola. Phytopathology 77:930–934 Smucker AJM, Erickson AE (1987) Anaerobic stimulation of root exudates and disease of peas. Plant Soil 99:423–433 Somasundaram S, Fukuzono S, Iijima M (2008) Dynamics of root border cells in rhizosphere soil of Zea mays L.: crushed cells during root penetration, survival in soil, and long term soil compaction effect. Plant Prod Sci 11:440–446 Stenlid G (1944) Physicochemical properties of the surface of growing plant cells. Nature 153:618–619 Stubbs VEC, Standing D, Knox OGG et al (2004) Root border cells take up and release glucose-C. Ann Bot 93:221–224 Sylvia DM, Fuhrmann JJ, Hartel PG, Zuberer DA (1998) Principles and applications of soil microbiology. Prentice Hall, USA Tamas L, Budikova S, Huttova J et al (2005) Aluminuminduced cell death of barley root border cells is correlated with peroxidase and oxalate oxidase-mediated hydrogen peroxide production. Plant Cell Rep 24:189–194 Tapp H, Stotzky G (1997) Monitoring the fate of insecticidal toxins from Bacillus thuringiensis in soil with flow cytometry. Can J Microbiol 43:1074–1078 Urban CF, Lourido S, Zychlinsky A (2006) How do microbes evade neutrophil killing? Cell Microbiol 8:1687–1696 Uren NC (2001) Types, amounts, and possible functions of compounds released into the rhizosphere by soil-grown plants. In: Pinton R, Varanini P, Nannipieri P (eds) The rhizosphere: biochemistry and organic substances at the soil-plant interface. Marcel Dekker, Inc., New York, Basel. pp 19–40 VanEgeraat AWSM (1975) Exudation of ninhydrin-positive compounds by pea seedling roots: a study of the sites of exudation and of the composition of the exudate. Plant Soil 42:37–47 Van’t Hopf J, Bjerknes CA (1982) Cells of pea (Pisum sativum) that differentiate from G2 phase have extrachromosomal DNA. Mol Cell Biol 2:339–345 15 Vermeer J, McCully ME (1982) The rhizosphere in Zea: new insight into its structure and development. Planta 156:45– 61 Vicre M, Santaella C, Blanchet S, Gateau A, Driouich A (2005) Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with rhizobacteria. Plant Physiol 138:998–1008 Vlassov VV, Laktionov PP, Rykova EY (2007) Extracellular nucleic acids. Bioessays 29:654–667 Voeller BR, Ledbetter MC, Porter KR (1964) The plant cell: aspects of its form and function. In: Bracket J, Minsky EE (eds) The cell, vol 6. Academic, London, pp 245–312 Wang Y, Li M, Stadler S, Correll S et al (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 184:205– 213 Wardini AB, Guimaraes-Costa AB, Nascimento MT et al (2010) Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus. J Gen Virol 91:259–264 Watt M, McCully ME, Jeffree CE (1993) Plant and bacterial mucilages of the maize rhizosphere: comparison of their soil binding properties and histochemistry in a model system. Plant Soil 151:151–165 Watt M, Silk WK, Passioura J (2006) Rates of root and organism growth, soil conditions and temporal and spatial development of the rhizosphere. Ann Bot 97:839–855 Weller DM (1988) Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu Rev Phytopathol 26:379–407 Wen F, Zhu Y, Brigham LA, Hawes MC (1999) Expression of an inducible pectinmethylesterase gene is required for border cell separation from roots of pea. Plant Cell 11:1129–1140 Wen F, Curlango-Rivera G, Hawes MC (2007a) Proteins among the polysaccharides: a new perspective on root cap slime. Plant Signal Behavior 2:1–3 Wen F, VanEtten HD, Tsaprailis G, Hawes MC (2007b) Extracellular proteins in pea root tip and border cell exudates. Plant Physiol 143:773–783 Wen F, Woo HH, Pierson EA et al (2008) Synchronous elicitation of development in root caps induces transient gene expression changes common to legume and gymnosperm species. Plant Mol Biol Rep 27:58–68 Wen F, White GJ, VanEtten HD et al (2009) Extracellular DNA is required for root tip resistance to fungal infection. Plant Physiol 151:820–829 Wen F, Shen A, Choi A, Shi J (2012) A xenograft pancreatic cancer mouse model to study the function of extracellular DNA in metastasis. Proceedings, American Association of Cancer Research Whipps J, Lynch JM (1983) Substrate flow and utilization in the rhizosphere of cereals. New Phytol 95:605–623 Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295:1487 Woo HH, Hirsch AM, Hawes MC (2004) Altered susceptibility to infection by Sinorhizobium meliloti and Nectria haematococca in alfalfa roots with altered cell cycle. Plant Cell Rep 12:967–973 Wood RKS (1967) Physiological plant pathology. Blackwell, Oxford 16 Wuyts N, Maung ZTZ, Swennen R, De Waele D (2006) Banana rhizodeposition: characterization of root border cell production and effects on chemotaxis and motility of the parasitic nematode Radopholus similis. Plant Soil 283:217–228 Xu J, Zhang X, Pelayo R, Monestier M et al (2009) Extracellular histones are major mediators of death in sepsis. Nat Med 15:1318–1321 Yost CC, Cody MJ, Harris ES et al (2009) Impaired NET formation: a novel innate immune deficiency of human neonates. Blood 113:6419–6427 Young RL, Malcolm KC, Kret JE, Caceres SM et al (2011) Neutrophil extracellular trap (NET)-mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the cystic fibrosis airway, independent of CFTR. PLoS One 6:e23637 Plant Soil (2012) 355:1–16 Zentmyer G (1963) Biological control of Phytophthora root rot of avocado with alfalfa meal. Phytopathology 53:1383 Zhao X, Misaghi IJ, Hawes MC (2000) Stimulation of border cell production in response to increased carbon dioxide levels. Plant Physiol 122:181–188 Zhu M, Ahn S, Matsumoto H (2003) Inhibition of growth and development of root border cells in wheat by Al. Physiol Plant 117:359–367 Zhu Y, Pierson LS, Hawes MC (1997) Induction of microbial genes for pathogenesis and symbiosis by chemicals from root border cells. Plant Physiol 115:1691–1698 Zobel RW, Wright SF (2005) Roots and soil management: interactions between roots and the soil. American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc. Madison WI, USA