Roles of root border cells in plant defense and regulation of rhizosphere microbial populations by extracellular DNA ‘trapping’
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- Hawes, M.C., Curlango-Rivera, G., Xiong, Z. et al. Plant Soil (2012) 355: 1. doi:10.1007/s11104-012-1218-3
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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.
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.
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.
KeywordsRoot border cellsMucilageRoot capExtracellular DNA (exDNA)Root exudatesRhizosphere colonization
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.
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 field-grown 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 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.’
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). 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).
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 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
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 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).
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 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.
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 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).
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.