Extracellular DNA secreted in yeast cultures is metabolism-specific and inhibits cell proliferation.

Extracellular DNA (exDNA) can be actively released by living cells and different putative functions have been attributed to it. Further, homologous exDNA has been reported to exert species-specific inhibitory effects on several organisms. Here, we demonstrate by different experimental evidence, including 1H-NMR metabolomic fingerprint, that the growth rate decline in Saccharomyces cerevisiae fed-batch cultures is determined by the accumulation of exDNA in the medium. Sequencing of such secreted exDNA represents a portion of the entire genome, showing a great similarity with extrachromosomal circular DNA (eccDNA) already reported inside yeast cells. The recovered DNA molecules were mostly single strands and specifically associated to the yeast metabolism displayed during cell growth. Flow cytometric analysis showed that the observed growth inhibition by exDNA corresponded to an arrest in the S phase of the cell cycle. These unprecedented findings open a new scenario on the functional role of exDNA produced by living cells.

Metabolic Responses of the Microalga Neochloris oleoabundans to Extracellular Self- and Nonself-DNA.

Stressed organisms identify intracellular molecules released from damaged cells due to trauma or pathogen infection as components of the innate immune response. These molecules called DAMPs (Damage-Associated Molecular Patterns) are extracellular ATP, sugars, and extracellular DNA, among others. Animals and plants can recognize their own DNA applied externally (self-exDNA) as a DAMP with a high degree of specificity. However, little is known about the microalgae responses to damage when exposed to DAMPs and specifically to self-exDNAs. Here we compared the response of the oilseed microalgae Neochloris oleoabundans to self-exDNA, with the stress responses elicited by nonself-exDNA, methyl jasmonate (MeJA) and sodium bicarbonate (NaHCO3). We analyzed the peroxidase enzyme activity related to the production of reactive oxygen species (ROS), as well as the production of polyphenols, lipids, triacylglycerols, and phytohormones. After 5 min of addition, self-exDNA induced peroxidase enzyme activity higher than the other elicitors. Polyphenols and lipids were increased by self-exDNA at 48 and 24 h, respectively. Triacylglycerols were increased with all elicitors from addition and up to 48 h, except with nonself-exDNA. Regarding phytohormones, self-exDNA and MeJA increased gibberellic acid, isopentenyladenine, and benzylaminopurine at 24 h. Results show that Neochloris oleoabundans have self-exDNA specific responses.

Arabidopsis thaliana Response to Extracellular DNA: Self Versus Nonself Exposure.

The inhibitory effect of extracellular DNA (exDNA) on the growth of conspecific individuals was demonstrated in different kingdoms. In plants, the inhibition has been observed on root growth and seed germination, demonstrating its role in plant-soil negative feedback. Several hypotheses have been proposed to explain the early response to exDNA and the inhibitory effect of conspecific exDNA. We here contribute with a whole-plant transcriptome profiling in the model species Arabidopsis thaliana exposed to extracellular self- (conspecific) and nonself- (heterologous) DNA. The results highlight that cells distinguish self- from nonself-DNA. Moreover, confocal microscopy analyses reveal that nonself-DNA enters root tissues and cells, while self-DNA remains outside. Specifically, exposure to self-DNA limits cell permeability, affecting chloroplast functioning and reactive oxygen species (ROS) production, eventually causing cell cycle arrest, consistently with macroscopic observations of root apex necrosis, increased root hair density and leaf chlorosis. In contrast, nonself-DNA enters the cells triggering the activation of a hypersensitive response and evolving into systemic acquired resistance. Complex and different cascades of events emerge from exposure to extracellular self- or nonself-DNA and are discussed in the context of Damage- and Pathogen-Associated Molecular Patterns (DAMP and PAMP, respectively) responses.

Green pesticide: Tapping to the promising roles of plant secreted small RNAs and responses towards extracellular DNA.

The diverse roles of non-coding RNA and DNA in cross-species communication is yet to be revealed. Once thought to only involve intra-specifically in regulating gene expression, the evidence that these genetic materials can also modulate gene expression between species that belong to different kingdoms is accumulating. Plants send small RNAs to the pathogen or parasite when they are being attacked, targeting essential mRNAs for infection or parasitism of the hosts. However, the same survival mechanism is also deployed by the pathogen or parasite to destabilize plant immune responses. In plants, it is suggested that exposure to extracellular self-DNA impedes growth, while to extracellular non-self-DNA induces the modulation of reactive oxygen species, expression of resistance related genes, epigenetic mechanism, or suppression of disease severity. Exploring the potential of secreted RNA and extracellular DNA as a green pesticide could be a promising alternative if we are to provide food for the future global population without further damaging the environment. Hence, some studies on plant secreted RNA and responses towards extracellular DNA are discussed in this review. The precise mode of action of entry and the following cascade of signaling once the plant cell is exposed to secreted RNA or extracellular DNA could be an interesting topic for future research.

New Insights into Plant Extracellular DNA. A Study in Soybean Root Extracellular Trap.

exDNA is found in various organisms, including plants. However, plant exDNA has thus far received little attention related to its origin and role in the RET (root extracellular trap). In this study, we performed the first high-throughput genomic sequencing of plant exDNA from a Fabaceae with worldwide interest: soybean (Glycine max (L.) Merr.). The origin of this exDNA was first investigated in control condition, and the results show high-coverage on organelles (mitochondria/plastid) DNA relative to nuclear DNA, as well as a mix of coding and non-coding sequences. In the second part of this study, we investigated if exDNA release was modified during an elicitation with PEP-13 (a peptide elicitor from oomycete genus Phytophthora). Our results show that treatment of roots with PEP-13 does not affect the composition of exDNA.

The masking effect of extracellular DNA and robustness of intracellular DNA in anaerobic digester NGS studies: A discriminatory study of the total DNA pool.

Most commonly, next generation sequencing-based microbiome studies are performed on the total DNA (totDNA) pool; however, this consists of extracellular- (exDNA) and intracellular (iDNA) DNA fractions. By investigating the microbiomes of different anaerobic digesters over time, we found that totDNA suggested lower species richness considering all and/or only common species and yielded fewer unique reads as compared to iDNA. Additionally, exDNA-derived sequences were more similar to those from totDNA than from iDNA and, finally, iDNA showed the best performance in tracking temporal changes in microbial communities. We postulate that abundant sequences present within the exDNA fraction mask the overall results of totDNA and provide evidence that exDNA has the potential to qualitatively bias microbiome studies at least in the anaerobic digester environment as it contains information about cells that were lysed hours or days ago. iDNA, however, was found to be more appropriate in providing reliable genetic information about potentially alive as well as rare microbes within the target habitat.

Root Border Cells and Mucilage Secretions of Soybean, Glycine Max (Merr) L.: Characterization and Role in Interactions with the Oomycete Phytophthora Parasitica.

Root border cells (BCs) and their associated secretions form a protective structure termed the root extracellular trap (RET) that plays a major role in root interactions with soil borne microorganisms. In this study, we investigated the release and morphology of BCs of Glycine max using light and cryo-scanning electron microscopy (SEM). We also examined the occurrence of cell-wall glycomolecules in BCs and secreted mucilage using immunofluorescence microscopy in conjunction with anti-glycan antibodies. Our data show that root tips released three populations of BCs defined as spherical, intermediate and elongated cells. The mechanism of shedding seemed to be cell morphotype-specific. The data also show that mucilage contained pectin, cellulose, extracellular DNA, histones and two hemicellulosic polysaccharides, xyloglucan and heteromannan. The latter has never been reported previously in any plant root secretions. Both hemicellulosic polysaccharides formed a dense fibrillary network embedding BCs and holding them together within the mucilage. Finally, we investigated the effect of the RET on the interactions of root with the pathogenic oomycete Phytophthora parasitica early during infection. Our findings reveal that the RET prevented zoospores from colonizing root tips by blocking their entry into root tissues and inducing their lysis.

The Role of DNA in the Extracellular Environment: A Focus on NETs, RETs and Biofilms.

The capacity to actively release genetic material into the extracellular environment has been reported for bacteria, archaea, fungi, and in general, for microbial communities, but it is also described in the context of multicellular organisms, animals and plants. This material is often present in matrices that locate outside the cells. Extracellular matrices have important roles in defense response and disease in microbes, animal and plants cells, appearing as barrier against pathogen invasion or for their recognition. Specifically, neutrophils extracellular traps (NETs) in animals and root extracellular traps (RETs) in plants, are recognized to be important players in immunity. A growing amount of evidence revealed that the extracellular DNA, in these contexts, plays an active role in the defense action. Moreover, the protective role of extracellular DNA against antimicrobials and mechanical stress also appears to be confirmed in bacterial biofilms. In parallel, recent efforts highlighted different roles of self (homologous) and non-self (heterologous) extracellular DNA, paving the way to discussions on its role as a "Damage-associated molecular pattern" (DAMP). We here provide an evolutionary overview on extracellular DNA in extracellular matrices like RETs, NETs, and microbial biofilms, discussing on its roles and inferring on possible novel functionalities.

Root extracellular traps versus neutrophil extracellular traps in host defence, a case of functional convergence?

The root cap releases cells that produce massive amounts of mucilage containing polysaccharides, proteoglycans, extracellular DNA (exDNA) and a variety of antimicrobial compounds. The released cells - known as border cells or border-like cells - and mucilage secretions form networks that are defined as root extracellular traps (RETs). RETs are important players in root immunity. In animals, phagocytes are some of the most abundant white blood cells in circulation and are very important for immunity. These cells combat pathogens through multiple defence mechanisms, including the release of exDNA-containing extracellular traps (ETs). Traps of neutrophil origin are abbreviated herein as NETs. Similar to phagocytes, plant root cap-originating cells actively contribute to frontline defence against pathogens. RETs and NETs are thus components of the plant and animal immune systems, respectively, that exhibit similar compositional and functional properties. Herein, we describe and discuss the formation, molecular composition and functional similarities of these similar but different extracellular traps.

Use of rhodizonic acid for rapid detection of root border cell trapping of lead and reversal of trapping with DNase.

PREMISE OF THE STUDY: Lead (Pb) is a contaminant whose removal from soil remains a challenge. In a previous study, border cells released from root tips were found to trap Pb, alter its chemistry, and prevent root uptake. Rhodizonic acid (RA) is a forensic tool used to reveal gunshot residue, and also to detect Pb within plant tissues. Here we report preliminary observations to assess the potential application of RA in exploring the dynamics of Pb accumulation at the root tip surface. METHODS AND RESULTS: Corn root tips were immersed in Pb solution, stained with RA, and observed microscopically. Pb trapping by border cells was evident within minutes. The role of extracellular DNA was revealed when addition of nucleases resulted in dispersal of RA-stained Pb particles. CONCLUSIONS: RA is an efficient tool to monitor Pb-root interactions. Trapping by border cells may control Pb levels and chemistry at the root tip surface. Understanding how plants influence Pb distribution in soil may facilitate its remediation.

The use of extracellular DNA as a proxy for specific microbial activity.

The ubiquity and relevance of extracellular DNA (exDNA) are well-known and increasingly gaining importance in many fields of application such as medicine and environmental microbiology. Although sources and types of exDNA are manifold, ratios of specific DNA-molecules inside and outside of living cells can give reliable information about the activity of entire systems and of specific microbial groups or species. Here, we introduce a method to discriminate between internal (iDNA), as well as bound and free exDNA, and evaluate various DNA fractions and related ratios (ex:iDNA) regarding their applicability to be used as a fast, convenient, and reliable alternative to more tedious RNA-based activity measurements. In order to deal with microbial consortia that can be regulated regarding their activity, we tested and evaluated the proposed method in comparison to sophisticated dehydrogenase- and RNA-based activity measurements with two anaerobic microbial consortia (anaerobic fungi and syntrophic archaea and a microbial rumen consortium) and three levels of resolution (overall activity, total bacteria, methanogenic archaea). Furthermore, we introduce a 28S rRNA gene-specific primer set and qPCR protocol, targeting anaerobic fungi (Neocallimastigomycota). Our findings show that the amount of actively released free exDNA (fDNA) strongly correlates with different activity measurements and is thus suggested to serve as a proxy for microbial activity.

Visualization of extracellular DNA released during border cell separation from the root cap.

PREMISE OF THE STUDY: Root border cells are programmed to separate from the root cap as it penetrates the soil environment, where the cells actively secrete >100 extracellular proteins into the surrounding mucilage. The detached cells function in defense of the root tip by an extracellular trapping process that also requires DNA, as in mammalian white blood cells. Trapping in animals and plants is reversed by treatment with DNase, which results in increased infection. The goal of this study was to evaluate the role of DNA in the structural integrity of extracellular structures released as border cells disperse from the root tip upon contact with water. METHODS: DNA stains including crystal violet, toluidine blue, Hoechst 33342, DAPI, and SYTOX green were added to root tips to visualize the extracellular mucilage as it absorbed water and border cell populations dispersed. DNase I was used to assess structural changes occurring when extracellular DNA was degraded. KEY RESULTS: Complex masses associated with living border cells were immediately evident in response to each stain, including those that are specific for DNA. Treating with DNase I dramatically altered the appearance of the extracellular structures and their association with border cells. No extracellular DNA was found in association with border cells killed by freezing or high-speed centrifugation. This observation is consistent with the hypothesis that, as with border cell extracellular proteins, DNA is secreted by living cells. CONCLUSION: DNA is an integral component of border cell extracellular traps.

Root Border Cells and Their Role in Plant Defense.

Root border cells separate from plant root tips and disperse into the soil environment. In most species, each root tip can produce thousands of metabolically 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 preventing invasion of host tissues. Extracellular DNases (exDNases) of pathogens promote virulence and systemic spread of the microbes. In plants, adding 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.

Inhibitory effects of extracellular self-DNA: a general biological process?

Self-inhibition of growth has been observed in different organisms, but an underlying common mechanism has not been proposed so far. Recently, extracellular DNA (exDNA) has been reported as species-specific growth inhibitor in plants and proposed as an explanation of negative plant-soil feedback. In this work the effect of exDNA was tested on different species to assess the occurrence of such inhibition in organisms other than plants. Bioassays were performed on six species of different taxonomic groups, including bacteria, fungi, algae, plants, protozoa and insects. Treatments consisted in the addition to the growth substrate of conspecific and heterologous DNA at different concentration levels. Results showed that treatments with conspecific DNA always produced a concentration dependent growth inhibition, which instead was not observed in the case of heterologous DNA. Reported evidence suggests the generality of the observed phenomenon which opens new perspectives in the context of self-inhibition processes. Moreover, the existence of a general species-specific biological effect of exDNA raises interesting questions on its possible involvement in self-recognition mechanisms. Further investigation at molecular level will be required to unravel the specific functioning of the observed inhibitory effects.