Root Border Cells and Their Role in Plant Defense

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V I E Review in Advance first posted online E W on May 23, 2016. (Changes may R S still occur before final publication online and in print.)

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Root Border Cells and Their Role in Plant Defense

Martha Hawes,1,∗ Caitilyn Allen,2 B. Gillian Turgeon,3 Gilberto Curlango-Rivera,1 Tuan Minh Tran,2 David A. Huskey,1 and Zhongguo Xiong4

1Department of Soil, Water and Environmental Sciences, Bio5 Institute, University of Arizona, Tucson, AZ 85721; email: [email protected], [email protected], [email protected] 2Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706; email: [email protected], [email protected] 3School of Integrative Plant Science, Plant Pathology & Plant-Microbe Biology Section, Cornell University, Ithaca, New York 14853; email: [email protected] 4School of Plant Science, Bio5 Institute, University of Arizona, Tucson, AZ 85721; email: [email protected]

Annu. Rev. Phytopathol. 2016. 54:5.1–5.19 Keywords The Annual Review of Phytopathology is online at rhizosphere, extracellular traps, root cap slime, exDNA, exDNase phyto.annualreviews.org

This article’s doi: Abstract 10.1146/annurev-phyto-080615-100140 Root border cells separate from plant root tips and disperse into the soil en- Copyright c 2016 by Annual Reviews.

Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org vironment. In most species, each root tip can produce thousands of metabol- All rights reserved ically active cells daily, with specialized patterns of gene expression. Their function has been an enduring mystery. Recent studies suggest that border cells operate in a manner similar to mammalian neutrophils: Both cell types export a complex of extracellular DNA (exDNA) and antimicrobial proteins that neutralize threats by trapping pathogens and thereby prevent- ing invasion of host tissues. Extracellular DNases (exDNases) of pathogens promote virulence and systemic spread of the microbes. In plants, adding

Access provided by Chinese Academy of Agricultural Sciences (Agricultural Information Institute) on 05/25/16. For personal use only. DNase I to root tips eliminates border cell extracellular traps and abolishes root tip resistance to infection. Mutation of genes encoding exDNase activity in plant-pathogenic bacteria (Ralstonia solanacearum) and fungi (Cochliobolus heterostrophus) results in reduced virulence. The study of exDNase activities in plant pathogens may yield new targets for disease control.

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INTRODUCTION The story of root border cells and their role in plant defense begins with a conundrum: If carbon balance is important to life on earth, why do plants waste energy producing cells destined to be sloughed into the soil every day? Daily carbon release from roots of some species has been measured at 40 to 90 percent of the total carbon fixed by leaves (70, 77). Up to 98 percent of such root exudates are made up of the sloughed root cap cells and associated extracellular material (35). In plants, including legumes, cereals, cucurbits, cotton (Gossypium hirsutum), and gymnosperms, this carbon dump includes thousands of cells produced in a single day (Figure 1) (23, 53). Upon dispersal of the cells in response to water or mechanical forces, mitosis in the root cap meristem is activated, root cap turnover is initiated, and within minutes a new population of cells begins to emerge from the cap periphery to replace the departed population (7). The apparent illogic in such extravagance may have contributed to the long-standing presumption that the detached cells are moribund even before dispersal, as the waste that would be involved in the release of so many metabolically active cells would be hard to accept (41). Thus, the scientific community (e.g., 31, 81) did not assimilate the discovery by Knudson (67, 68) nearly 100 years ago that pea (Pisum sativum)andcorn(Zea mays) root cap cell populations can remain 100% viable and capable of active enzyme secretion for months after dispersal. In the 1980s, interest in sloughed root cap cells stemmed from the discovery that the cells from oats (Avena sativa) and corn expressed the same sensitivity as whole plants to host-specific toxins from Cochliobolus victoriae and Cochliobolus heterostrophus, the causal agents of Victoria blight of oats and Southern corn leaf blight, respectively (46, 56, 57). The potential to use root border cells as a convenient model system to define cellular responses to virulence factors as they Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org

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Figure 1 Border cell dispersal from root tips is initiated instantly upon exposure to water and is complete within minutes, leaving the root tip surface free of cells. Mitosis within the root cap meristem is evident within 5–10 minutes, and a new set of border cells is produced within 20 hours (7). Reproduced with permission from http://www.plantphysiol.org, Copyright American Society of Plant Biologists.

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occur has therefore been explored (47, 54). This review summarizes the ensuing chronology of discoveries about border cell properties whose signiﬁcance in plant defense ﬁnally came to light with the coincidental recognition of a similar property in human immune systems: In mammals as well as plants, extracellular DNA (exDNA)-based extracellular traps are previously unrecognized components of early immune responses (8–10, 13, 17, 23, 30, 50, 51, 72).

BORDER CELLS AND PLANT PATHOGENS The complex developmental dynamics of pathogen invasion of intact plant tissue make it difficult to observe the sequence of events in situ and define the steps needed for infection to occur (88). Studies therefore were designed to exploit developmentally uniform, viable populations of border cells as a model system to track infection at the cellular level (49). Host-microbe–specific recognition of border cells observed with bacteria, nematodes, zoospores, and fungi were consistent with a logical model for the effect of root exudates on soilborne pathogens: Contents released from the sloughed cells provide nutrition needed to stimulate growth and thereby facilitate infection of the host. Host-specific chemotaxis and binding yielded promising evidence that the cell populations would facilitate tracking experiments that allow direct observation of the cellular invasion as it happens and thereby yield new insights into the infection process (48, 52, 54). Thus, for example, Agrobacterium tumefaciens populations were chemotactically attracted to host border cells, where they accumulated in large numbers within minutes (Figure 2). No attraction or binding occurred on border cells of nonhost species. Despite thousands of replicates, however, in no case was infection of a single border cell observed. Root-infecting fungi also responded rapidly and in a host-specific manner to border cells, as predicted if the detached cells, like intact root tissue, were susceptible to infection. Spores of Nectria haematococca (Fusarium solani f. sp. pisi ) were stimulated to undergo rapid germination in response to border cells of host species, but in no case was penetration of individual border cells observed (38). The predicted stimulation of growth in response to nutrients from the border cells also did not occur. Instead, fungal growth ceased and remained static (39). Similar results occurred with nematodes (116): Rapid chemotaxis to border cells resulted in masses of aggregated nematodes within minutes, suggesting that an invasion was in progress Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org

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Figure 2 Agrobacterium tumefaciens binding to pea border cells (54). Chemotaxis of bacteria toward the plant cells (black arrow) is initiated instantaneously, and most cells are aggregated with bacteria into masses over time (white arrows).

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Figure 3 Nematode (white arrow) chemotaxis to border cells (blue arrow). Within 5 min of adding Meloidogyne incognita to pea roots, all the nematodes aggregate within the mass of border cells (116). No chemotaxis to root tips occurs if border cells are removed (59).

(Figure 3). However, this was followed by an induced state of quiescence lasting for days (59). No border cell penetration was observed. Zoospores of Pythium dissotocum and Pythium catenulatum were chemotactically attracted to border cells of their hosts, cotton (Gossypium barbadense and G. hirsutum) and cucumber (Cucumus sativus), respectively, but were not responsive to nonhost species (34). Invasion of the single border cells did occur (Figure 4). However, growth of the pathogen ceased after penetration and did not resume. Most surprising was the observation that chemotaxis was speciﬁc to border cells: When border cells were removed prior to adding bacteria, zoospores, or nematodes, no attraction to the root tip occurred. These ﬁndings did not support the premise that border cells stimulate pathogens in the soil, activate virulence factors, and thereby facilitate root infection. Instead, they suggested that border cells act as a decoy, releasing signals that quickly reverse quiescence of diverse soilborne pathogens but then inhibit their growth (48). Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org

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Figure 4 Zoospore chemotaxis and penetration of host border cells. Pythium dissotocum zoospores (arrow)are instantaneously attracted to cotton border cells, and invasion of the cells occurs within 10–15 min, but little additional growth occurs (34).

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667767 SD-PAGE kD 97

29 a b c Root tip proteins Border cell proteins Exported proteins

Figure 5 Fluorographs of in vivo [35S]Met-labeled proteins separated by two-dimensional gel electrophoresis employing isoelectric focusing (IEF) in the ﬁrst dimension and 5% to 20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. (a) Proteins from root tips. (b) Proteins from border cells. (c) Extracellular proteins synthesized by border cells. Open triangles indicate polypeptides seen only in root-tip samples; closed triangles indicate polypeptides seen only in root border-cell samples. Arrows indicate two polypeptides observed in both samples (6). Reproduced with permission from http://www.plantphysiol. org, Copyright American Society of Plant Biologists.

Taken together, these data were not consistent with the hypothesis that border cells reﬂect the genotype of the whole plant with regard to pathogen recognition and signal response, and thereby provide a reliable model to study infection processes at the cellular level (6). Indeed, the results indicated that border cell responses were distinct even from cells in the root cap from which they recently had been generated. A comparison of proteins and mRNAs supported that hypothesis: Labeled proteins synthesized by root caps and border cells during a one-hour period when no cell death occurred yielded markedly distinct proﬁles (Figure 5a,b). Many of the proteins expressed only in border cells were secreted during the one-hour period of study (Figure 5c). mRNA differential display revealed many quantitative and qualitative differences when mRNA from border cells was compared with that from root caps (6). Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org

PLANT GENES CONTROLLING BORDER CELL PRODUCTION AND PROPERTIES If border cells are uniquely differentiated to act as decoys to prevent root invasion in a host-speciﬁc manner, then gene expression changes resulting in altered border cell production and properties may yield new avenues for disease control (7). Plants with altered border cell numbers, border

Access provided by Chinese Academy of Agricultural Sciences (Agricultural Information Institute) on 05/25/16. For personal use only. cell delivery, or border cell gene expression would be predicted to exhibit altered susceptibility to infection. Experiments therefore were designed to develop tools to test the impact of border cells on root infection using plants with altered expression of genes controlling border cell development (103–105, 109–114). Altered border cell development was achieved in transgenic plants, but pleiotropic effects precluded their use in ﬁeld studies. For example, a pectin methylesterase gene was expressed in cells at the root cap periphery during cell separation, and when the gene’s

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expression was inhibited by gene silencing in transgenic pea hairy roots, border cell dispersal was inhibited (109). However, the transgenic hairy roots could not be used as a tool to examine the impact of border cells on root tip infection because the pectin methylesterase–silenced cells were also altered in multiple traits that could affect infection, including extracellular pH, cell morphology, and root growth. Alfalfa plants with altered border cell production were developed by inhibiting expression of UDP-glucuronosyl transferase localized within the root cap meristem during the first moments when mitosis is induced by border cell dispersal (112). Border cell production in the transgenic plants was reduced by >99%, and root tip infection by F. solani increased, but a reduced rate of root growth also occurred in the transgenic plants. Curiously, transgenic roots with altered expression of a flavin-binding protein gene that is expressed only in border cells markedly altered root morphology as well as rate and direction of growth (103). A second border cell–specific gene was found to encode a galactosidase that func- tioned outside the cell at only the low pH characteristic of the root tip extracellular environment (105). This seemed to be a likely prospect for altered gene expression without pleiotropic effects on root development. However, inhibition of its expression was lethal; not a single plant clone could be obtained. This unexpected result instigated a renewed look at the proteins secreted from border cells (Figure 5c) to determine how they could have such dramatic effects on overall plant growth and development (106). Proteins secreted from root tips and border cells during a 1-hour period when no cell death occurred were analyzed (106). Analysis of this secretome identified enzymes and other proteins long known to play a role in cell wall metabolism and plant defense (106). Knudson’s (68) report that invertase is secreted from viable sloughed root cap cells was also confirmed. A previously unreported and unexpected finding was that histone H4 is among the proteins synthesized and exported from root tips during a one-hour period when no cell death occurs (106). This proved to be a key discovery because the coincident discovery of extracellular histone associated with human neutrophils revealed strong parallels with newly discovered defense processes in animal systems.

EXTRACELLULAR DNA AND MICROBIAL DNases IN ANIMAL DEFENSE In 2004, Brinkmann et al. (9) reported for the first time that exDNA associated with human neutrophils is not necessarily a product of dead cells leaking their contents or artifacts of sample Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org preparation, but instead is actively exported as a component of the immune response. Subsequent discoveries (Table 1) have confirmed that exDNA together with extracellular proteins forms a neutrophil extracellular trap (NET) that inhibits pathogen invasion. The first functional study establishing the role of extracellular DNases (exDNases) in bacterial virulence revealed that group A Streptococcus (GAS) depends on the prophage-encoded nuclease Sda1 to invade host tissues during infection (92). Shortly thereafter, nucleases from several important streptococcal species were shown to facilitate escape from NETs (2, 12, 15, 26, 29, 40, 73). A GAS strain that causes necrotizing fasciitis in a mouse model is less virulent when lacking secreted Access provided by Chinese Academy of Agricultural Sciences (Agricultural Information Institute) on 05/25/16. For personal use only. nuclease Sda1 (12). Additional studies have confirmed that exDNases are key virulence factors in pathogenesis (72, 84, 100). Thus, the discovery of a major new eukaryotic defense mechanism against microbes was quickly followed by the discovery of a specific microbial counter-defense strategy. A short summary of microbial DNases in the context of defense against NETs trapping is shown in Table 2.

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Table 1 Extracellular trapping in eukaryotic defense: timeline 2004–2015 Year Notable Findings 2004: Discovery of NETs in mammalian immune Neutrophil extracellular traps (NETs) contain DNA, histones, and responses antimicrobial proteins. Pathogens are attracted to NETs, where they aggregate and are killed. Adding extracellular DNase inhibits killing, frees bacterial pathogens from NETs, and allows systemic dispersal (9). 2005–2006: Reduced virulence in bacterial exDNase mutant group A streptococcus (GAS) strains exhibit impaired pathogens with reduced exDNase virulence in association with reduced degradation of NETs (12, 92). 2006–2011: exDNA-based traps discovered in Candida albicans (96), protozoan pathogens (37), and plant pathogens (107) are response to fungal, protozoan and plant pathogens immobilized by host exDNA-based trapping. 2011–2014: exDNA trapping in autoimmune exDNA trapping is involved in pathogenesis of autoimmune and infectious diseases diseases, with proposed involvement in sepsis, cystic ﬁbrosis, lupus, vascular disorders, rheumatoid arthritis, and cancer (16, 55, 64, 115). 2015: Mechanisms Mechanisms of neutrophil extracellular trap function remain under investigation; DNA itself may play a role in microbial killing (45).

Table 2 Current knowledge on nucleases as virulence factors in major animal pathogens Organism Major ﬁndings Year Reference Group A Streptococcus Extracellular DNase SdaD2 protected group A streptococci (GAS) from 2005 95 polymorphonuclear neutrophil (PMN) killing Group A Streptococcus Nuclease Sda1 facilitates degradation of neutrophil extracellular traps (NETs) and 2006 12 increased bacterial survival as well as virulence Streptococcus Surface endonuclease EndA promoted escape from trapping from NETs but did 2006 2 pneumoniae not affect PMN killing. Staphylococcus aureus nuc-deﬁcient mutant failed to degrade NETs and is more susceptible to neutrophil 2010 3 killing Streptococcus pyogenes Cell wall–anchored SpnA degraded NETs and enhanced bacterial survival in whole 2011 15 blood Vibrio cholerae Xps and Dns nucleases were induced as a response to NETs and required for 2013 86 bacterial colonization and survival Streptococcus agalactiae NucA is a respiration-induced nuclease and protected S. agalactiae from clearance 2013 29 by NETs

Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org S. aureus Nuc degrades NETs to produce products to be converted to cytotoxic dAdo, which 2013 93 could promote macrophage cell death Streptococcus sanguinis Cell wall–anchored nuclease degraded NETs and contributed to bacterial evasion 2014 73 of NET killing S. aureus Membrane-bound Nuc2 is produced during mouse infection; no link to interaction 2014 65 with NETs yet Streptococcus suis Cell wall–anchored nuclease SsnA degrades NETs and protected bacterial cells 2014 26 from NET killing Access provided by Chinese Academy of Agricultural Sciences (Agricultural Information Institute) on 05/25/16. For personal use only. Leishmania infantum Leishmania 3 -nucleotidase/nuclease is required for nutritional acquirement and 2014 36 survival from NET trapping/killing Aeromonas hydrophila ahn nuclease-deﬁcient mutant was more susceptible to clearance by macrophages 2015 61 in ﬁsh and murine models Neisseria gonorrhoeae exDNase expression allowed the bacterium to escape and survive human NETs 2015 63

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Indeed, a recent study tracking the evolutionary history of GAS highlighted the importance of exDNases by showing that transfer of an exDNase-encoding phage is the ﬁrst step toward signiﬁcantly increased virulence of this dangerous bacterial pathogen (76, pE1768):

We sequenced the genomes of 3,615 strains of serotype Emm protein 1 (M1) group A Streptococcus to unravel the nature and timing of molecular events contributing to the emergence, dissemination, and genetic diversiﬁcation of an unusually virulent clone that now causes epidemic human infections worldwide. We discovered that the contemporary epidemic clone emerged in stepwise fashion from a precursor cell that ﬁrst contained the phage encoding an extracellular DNase virulence factor.

The best-studied nucleases in pathogenic bacteria are probably those of the widespread human and veterinary pathogen Staphylococcus aureus (4, 79). Although nuclease activity was identiﬁed in this pathogen in the 1960s and nuclease production was linked to bacterial survival and mor- tality (99), the biological function of these enzymes in bacterial pathogenesis was unveiled only recently.S.aureussecretes two distinct nucleases (18, 78). The secreted nuclease Nuc enhances bacterial tolerance of NET bactericidal activity; an isogenic nuclease-deﬁcient S. aureus strain was cleared from mouse lungs faster than its wild-type parent and was less virulent (3). This discovery demonstrated that digesting host NETs with secreted nucleases is a widespread adaptation to exDNA-based defense by animal immune systems. A second surface-attached S. aureus nuclease, Nuc2, is expressed during infection, but it is not clear whether Nuc2 also contributes to a delay of NET clearance of S. aureus (65). In addition to S. aureus and GAS, the last few years have seen an increasing number of studies on nucleases relating to pathogenesis in other bacteria. Vibrio cholerae nucleases, which were known to inhibit natural transformation, also degrade NET DNA, thereby helping the bacteria to survive and colonize animal hosts (86). The human pathogen Neisseria gonorrhoeae produces a thermostable nuclease, Nuc, that degrades NETs and helps the bacterium evade neutrophil killing (63). Neutrophils also produce NETs in response to fungi, such as Aspergillus fumigatus (11), Candida albicans (96), Cryptococcus neoformans,andCryptococcus gattii (85), and to less-aggressive pathogens, such as Paracoccidioides brasiliensis (28). A. fumigatus, for example, causes life threatening infections of immunocompromised patients. In 2010, McCormick and colleagues (71) demonstrated that a subpopulation of all human neutrophils produced NETs in vitro, and also in vivo, as assayed in a mouse model, when confronted with the fungus. The authors concluded that NET formation

Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org may have a fungistatic, rather than a killing, effect (as it does in C. albicans) on the pathogen and that this prevents systemic dispersal. A survey of exDNase activity among a large collection of C. neoformans and C. gattii strains indicated that DNase production was higher in clinical isolates than in environmental isolates (85). On the basis of these data, the authors concluded that exDNase could be considered a virulence factor. In general, to our knowledge, there are no published reports of the manipulation of genes encoding exDNases in fungal pathogens of humans that conﬁrm a role of these enzymes in virulence.

Access provided by Chinese Academy of Agricultural Sciences (Agricultural Information Institute) on 05/25/16. For personal use only. Extracellular nucleases also contribute to pathogenesis by higher organisms other than fungi. The trypanosome Leishmania (causal agent of leishmaniasis, affecting millions of people around the world) can also be trapped and killed by NETs (37). In response, the parasite expresses a 3-nucleotidase/nuclease that is essential for meeting the parasite’s nutritional requirements (90) and also reverses exDNA trapping and increases Leishmania survival in vivo (36).

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EXTRACELLULAR DNA AND MICROBIAL DNases IN PLANT DEFENSE Emerging data support the hypothesis that exDNA-based immunity also functions in plant tissues (root border cells, leaves, and vascular tissue) and that exDNases act as virulence factors for both bacterial and fungal plant pathogens (30, 60, 95, 98). Plant-microbe recognition events leading to defense have long been known to occur outside the cell (1). Our past failure to recognize the importance of exDNA and exDNases in immune responses likely stems in part from the fact that DNA is ubiquitous outside cells in many human and plant tissues, especially when dissected for analysis. As noted above, it is difficult to distinguish the products that leak from damaged cells from those exported by living cells. In response to our discovery that the maize and pea root cap secretomes include the DNA-packaging protein histone H4, we examined the hypothesis that DNA is also a component of root cap slime (106, 107). Direct electrophoretic analysis confirmed that this material contains DNA and that newly synthesized DNA (identified by labeling with [32P]dCTP) is released into the extracellular matrix of the root cap during a 1-h period when secretome proteins are also synthesized and secreted (6, 107, 108). The presence of exDNA in root exudates and exDNases in pathogenic bacteria has been documented since the 1950s but there was no basis for interpretation at that time (5, 14, 19, 82, 87, 89, 91, 97). We now know that, as with NETs, border cells trap and immobilize plant pathogens, including nematodes, fungi, and bacteria (Figures 2–5) (13, 49). As with NETs, the plant trapping response exhibits host-microbe specificity and occurs within minutes of adding the pathogen (30, 38, 49, 50, 51). Trapping of bacteria can be reversed in 2–3 minutes by adding DNase I, at which point bacteria disperse; a new slime layer forms within 30 minutes, and bacterial trapping resumes (23). A growth pouch assay that allows direct observation of root disease development without cell or tissue damage over time was used to measure the impact of exDNase on infection (50, 107). Direct tests of exDNA production and its role in fungal infection revealed three core observations: (a) ExDNA is synthesized and exported from plant cells at the root tip during a 1-h period of labeling with [32P]dCTP. (b) When the exDNA is degraded by adding DNase I at the time of inoculation with the pea pathogen F. solani f. sp. pisi or Phoma medicaginis, root tip infection increases from <6% of inoculated roots showing mild local lesions to 100% of roots developing root rot within 48–72 h (107). (c) The increased fungal infection in response to DNase I treatment of root tips is correlated with increased fungal growth, which does not appear to be a nutritional effect (Figure 6). In the presence of border cells, hyphae appeared truncated, fragmented, and separated from the root tip surface (Figure 6a) (50). No growth inhibition occurred when roots were inoculated with spores plus DNase I (Figure 6b). In control experiments, germination and growth of spores treated with DNase I alone were indistinguishable from that of water-treated Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org spores, and adding heat-killed exudates from treated seedlings did not reproduce the enhanced susceptibility phenotype, as would be predicted if a simple nutritional effect were the underlying mechanism of altered infection. Conversely, adding exogenous foreign DNA to root tips together with DNase 1 and/or fungal spores did not alter disease responses of the treated roots (107). In control experiments, roots treated with nucleases exhibited normal growth, development, and gravity sensing. The results suggest that, as in animals, the border cell exDNA together with secreted proteins form a trap that attracts, immobilizes, and inhibits infection. How have pathogens adapted to overcome this host defense? A straightforward counter-defense Access provided by Chinese Academy of Agricultural Sciences (Agricultural Information Institute) on 05/25/16. For personal use only. strategy is direct digestion of the traps’ DNA backbone, and this approach is demonstrably effective in experimental conditions. Treatment with commercial DNase I releases microbes that have been caught or excluded by root border cell extracellular traps (9, 23, 25, 107). Moreover, as described below, microbes have the capacity to digest trapping DNA because many of them produce exDNases. Together, these results strongly support the hypothesis that, as in humans, exDNA protects plants from pathogen attack by forming extracellular traps.

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Root tip Root tip

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Figure 6 Early changes in Nectria haematococca (Fusarium solani f. sp. pisi; green)-root tip (red ) interactions in response to DNase I. (a) At 36 h after inoculation, fungal hyphae (double white arrow) are truncated and fragmented, and remain separate from root tips, among detached border cells (small white arrow). (b) In the presence of DNase I, hyphae proliferate and penetrate the root tip (black arrows) despite increased border cell production (50). Reproduced with permission from http://www.plantphysiol.org, Copyright American Society of Plant Biologists.

Bacterial exDNases and Virulence In contrast to those of animal pathogens, little is known about the role of extracellular nucleases produced by plant pathogens (32). One example of a nuclease from a plant-pathogenic bacterium is NucM, which is produced by the soft-rot bacterium Dickeya dadantii (74). Although this enzyme is very similar to an exDNase, termed Dns, produced by Vibrio cholerae, and was thought to hinder DNA uptake by D. dadantii, experiments revealed no role for NucM in transformation or bacterial virulence (75). Accumulating sequence data reveal that the genomes of many plant-pathogenic bacteria encode putative nucleases and some of those nucleases contain predicted signal peptides, indicating that they may translocate across the bacterial membrane (Table 3). This trait is not unique to pathogens; nonpathogenic soil bacteria also encode nucleases with signal sequences. The genome of Ralstonia solanacearum, a soilborne pathogen that infects the roots of more than 200 plants in 50 different botanical families, encodes two putative secreted DNases (Table 3).

Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org R. solanacearum’s two putative extracellular nucleases are expressed during pathogenesis, suggesting the hypothesis that the ability to degrade and escape from border cell traps contributes to virulence. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Obvious changes in trapping responses on corn border cells were seen with wild type compared with ΔnucA, ΔnucB,andΔnucA/B mutants (Figure 7a). Functional studies with puriﬁed proteins revealed that NucA and NucB are distinct nonspeciﬁc endonucleases. Single ΔnucA and ΔnucB mutants and a ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay (95). Com-

Access provided by Chinese Academy of Agricultural Sciences (Agricultural Information Institute) on 05/25/16. For personal use only. pared to its wild-type parent strain, the ΔnucA/B mutant was less able to stunt growth of plant roots or colonize plant stems. In addition, the double mutant was outcompeted by the wild-type strain in planta. Further, the ΔnucA/B mutant was also defective in attachment to roots of pea, a nonhost, although it attached normally to roots of the host plant tomato. Exposing pea and tomato (Solanum lycopersicum) border cells to R. solanacearum triggered release of DNA-containing extracellular traps (Figure 7b). This response occurred in a ﬂagellin-dependent manner, suggesting

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Table 3 Predicted DNases with secretory peptides in selected soil bacteria Bacterial genomes Nonsecreted Secreteda Total Plant-pathogenic bacteria Agrobacterium tumefaciens F2 22 0 22 Dickeya dadantii 3937 11 1 12 Erwinia amylovora CFBP1430 17 3 20 Pseudomonas syringae pv. phaseolicola 1448A 24 3 27 P. syringae pv. syringae B728a 25 3 28 P. syringae pv. tomato DC3000 25 4 29 Pectobacterium carotovorum subsp. carotovorum PC1 6 0 6 P. syringae pv. oryzae str. 1.6 27 6 33 Ralstonia solanacearum GMI1000 9 2 11 Streptomyces scabiei 87.22 15 2 17 Xanthomonas campestris pv. vesicatoria 85–10 15 7 22 Xanthomonas oryzae pv. oryzae KACC10331 11 2 13 X. campestris pv. campestris 8004 16 3 19 Xylella fastidiosa 9a5c 10 0 10 Non-plant-pathogenic soil bacteria Azotobacter vinelandii DJ 18 3 21 Burkholderia cepacia GG4 8 4 12 Klebsiella pneumoniae subsp. pneumoniae HS11286 38 4 42 Pseudomonas chlororaphis subsp. aureofaciens 30–84 16 1 17 Pseudomonas ﬂuorescens F113 15 1 16 Ralstonia eutropha JMP134 23 1 24 Sinorhizobium meliloti 1021 57 2 59

aSecretion peptide was predicted with Signal P 3.0.

that trap release (called NETosis in animal systems) could be an element of PAMP (pathogen- associated molecular pattern)-triggered immunity (PTI) (8, 80). These traps rapidly immobilized the pathogen, but the bacteria could be released by treatment with puriﬁed DNase. Taken together, these results indicate that extracellular DNase is a novel virulence factor that helps

Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org R. solanacearum successfully infect plant roots and cause bacterial wilt disease (T. Tran & C. Allen, unpublished results).

Fungal exDNases and Virulence Little is known about the role, if any, that exDNases of plant fungal pathogens play in virulence to their hosts. The ﬁrst report of a nuclease produced by a fungal pathogen (Ustilago maydis, causal agent of smut of maize) was published in the early 1970s (58). Nucleases have also been detected in

Access provided by Chinese Academy of Agricultural Sciences (Agricultural Information Institute) on 05/25/16. For personal use only. ﬁltrates from cultures of other pathogenic fungi. Several isolates of the soilborne vascular fungus F. solani produced a 22-kDa heat-stable exDNase at substantially higher concentrations when macroconidia were exposed to pea pod endocarp tissue than when in growth medium, suggesting that this nuclease was induced by the presence of the plant (33, 66). A recent study by Hadwiger et al. (43) revealed that several other fungal phytopathogens, including Alternaria limicola, Ascochita rabei, Helminthosporium solani, Phytophthora infestans, Pythium

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ab 30 μm 30 μm

WT ΔnucA

30 μm 30 μm

50 μm ΔnucB ΔnucA/B

Figure 7 Border cell trapping responses to Ralstonia solanacearum.(a) Altered corn border cell trapping of R. solanacearum wild type (WT) compared with nuclease (ΔnucA, ΔnucB,andΔnucA/B)mutants. R. solanacearum cultures were grown in CPG medium and added to corn border cells in suspension. Trapping of bacteria was observed by direct microscopic observation. The presence of trapping was evident as a layer of bacteria immobilized at the edge of the border cell trap. Photos by D.A. Huskey. (b) Pea root border cell extracellular trap released in response to R. solanacearum GMI1000. SYTOX-Green was used to visualize DNA in the border cell–bacteria suspension. Image was taken approximately 30 min postinoculation with a ﬂuorescence compound microscope (Olympus BX60F5) equipped with a FITC ﬁlter set. Some partially decondensed nuclei are still visible in the trap, together with many interconnecting threads.

irregulare, Gaeumannomyces graminis, Puccinia striiformis, Colletotrichum coccodes,andVerticillium dahliae also secrete exDNases into growth medium. Further characterization of a V. dahliae– exDNase showed that fungal nuclease activity coincides with the timing of both pisatin production and PR gene expression by the plant host, suggesting the possibility that, like Fusarium, Verticillium exDNase could elicit plant defenses, possibly through chromatin remodeling, as DNA damage was visible shortly after inoculation (42, 43). Surprisingly, an exDNase from C. coccodes with very similar biochemical properties did not trigger any defense response in a nonhost plant, suggesting that this enzyme may take part in nutrient scavenging rather than participate in signaling during interaction with plants (101). It is now apparent that DNase-encoding genes are plentiful in fungal genomes and that at Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org least some are predicted to be secreted. With this in mind, we extracted all proteins annotated as DNases from the genome of the filamentous maize pathogen C. heterostrophus and identified 31 such proteins. In addition, a blast search of the C. heterostrophus genome was conducted with previously identified fungal DNases (including, e.g., the F. solani sequence mentioned above) as queries. No additional proteins beyond the 31 were identified. Importantly, there was no hit when thesequencefromF. solani identified as an exDNase by Hadwiger et al. (42) was used as a query. Furthermore, this protein does not have domains identifying it as a DNase.

Access provided by Chinese Academy of Agricultural Sciences (Agricultural Information Institute) on 05/25/16. For personal use only. Five of the thirty-one extracted C. heterostrophus proteins had secretion signals and the corresponding genes were deleted (Table 4). One of the ﬁve [ JGI ( Joint Genome Institute) protein ID 144206], a Mg-dependent, TatD-related deoxyribonuclease, appears to be important for virulence to maize because the corresponding mutant is reduced in virulence compared with the wild type (98). When used as a query against JGI protein catalogs, protein 144206 was determined to be highly conserved in Dothideomycetes, Eurotiomycetes, and Lecanoromycetes, but it is

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Table 4 Candidate exDNase encoding proteins in Cochliobolus heterostrophus strain C4 Characteristics Protein IDa Annotation NNb score SignalP 33717 Deoxyribonuclease, TatD-related (Spombe SCN1) 0.890 no G-protein coupled receptor (GPCR), rhodopsin-like 83474 DNase I-like 0.627 yes Endonuclease/exonuclease/phosphatase 122478 DNase I-like 0.775 yes Endonuclease/exonuclease/phosphatase 144206 TatD deoxyribonuclease (Mg) 0.456 no 149183 TatD-related deoxyribonuclease 0.529 no

aJoint Genome Institute protein IDs in Cochliobolus heterostrophus strain C4. bNeural network prediction of signal peptides.

missing from most Leotiomycetes and Sordariomycetes. A phylogenetic reconstruction (using JGI resources) indicates that the gene cluster in which it resides appears to have been duplicated. The studies described above suggest that plant-associated microbes have evolved exDNases in response to the selection pressures exerted by microbe-trapping exDNA from plant cells. Under- standing how these enzymes beneﬁt microbes that live in or traverse the rhizosphere could lead to improved control strategies for soilborne pathogens. Examples include selecting crop plants that have more root border cells, release border cells or exDNA faster in response to pathogen signals, or secrete effective inhibitors of microbial exDNases.

CONCLUDING REMARKS The significance of the evolutionary parallels between plant and animal trapping has been noted in recent articles (10, 17). The trapping process in root caps and detached root border cells can be induced and synchronized in situ, in minutes, in response to specific pathogenic microorganisms and elicitors (20–22, 24, 49–51). This together with the extended survival of border cells after separation from the root provides an important experimental advantage over neutrophils, which are produced and mature in bone marrow and then delivered progressively over time through the blood system to the site of injury (9). Defining extracellular traps at the cellular and molecular level in plants, and parallel studies to characterize their specific interactions with pathogen exDNases,

Annu. Rev. Phytopathol. 2016.54. Downloaded from www.annualreviews.org will provide insights into understanding and controlling this immune response in agriculture and medicine (21). The plant model could yield mechanistic insights to complement ongoing medical work in exDNA-driven human immune responses (55). Research to expand our understanding of this antimicrobial defense into the realm of plants and crops may serve as a conceptual bridge between medicine and agriculture. As such, it has the potential to directly shape the implementation of current methods and the design of novel strategies to prevent plant diseases. Possible practical applications for agriculture of exDNA and plant extracellular traps range from breeding plants with more effective resistance to root-infecting pathogens via enhanced pathogen trapping to

Access provided by Chinese Academy of Agricultural Sciences (Agricultural Information Institute) on 05/25/16. For personal use only. understanding the antimicrobial properties of compost in the soil (24, 94). In addition, ongoing efforts to engineer the plant rhizosphere may be facilitated by a better understanding of the ways plants already engineer the rhizosphere (44, 117). The importance of soil health and the rhizosphere environment has been long recognized but deﬁning it has been a challenge (62, 69, 83). New insights into specialized metabolism in border cells may yield tools that will facilitate moving toward an integration of ideas and applications (27, 102).

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SUMMARY POINTS 1. exDNA is produced by both plants and animals, and it does much more than carry genetic information. Plant and animal immune systems both use exDNA as an antimicrobial defense, in a striking example of convergent evolution. 2. exDNA is an integral structural and possibly bactericidal component of the extracellular matrix that surrounds root border cells and the root cap, protecting root tips from infection by trapping or blocking pathogenic microbes. 3. Some animal pathogens produce exDNases to counteract extracellular traps cast by immune cells. Mounting evidence suggests that plant pathogens have evolved a similar counter-defense strategy. 4. exDNases are abundant in soilborne pathogens, and preliminary evidence indicates that these enzymes could also act as virulence factors during infection. 5. Engineering the rhizosphere to increase plant resistance to pathogens could be achieved through breeding, which potentially selects for novel root and root border cells’ architec- ture and function. Alternatively, engineering or selecting benign rhizosphere microbes that secrete microbial nuclease inhibitors could offer a novel form of biocontrol for root-infecting pathogens.

DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENT We thank the National Science Foundation for funding to support research on extracellular DNA in plant defense.

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