Gilberto Curlango-Rivera

Roles of root border cells in plant defense and regulation of rhizosphere microbial populations by extracellular DNA 'trapping'

BackgroundAs 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.ScopeHistones 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.ConclusionsResearch 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.

Transient exposure of root tips to primary and secondary metabolites: Impact on root growth and production of border cells

Here we describe the use of Pisum sativum L. as a model system to measure how short-term treatment of root tips with soluble metabolites can influence root growth and release of root exudates. The results revealed that even a 3-minute exposure of root tips to metabolites normally released from roots into the rhizosphere (e.g. rhamnose, ferulic acid, salicylic acid) can significantly influence root growth without affecting production of border cells and associated exudates. Conversely, products including caffeine, saccharide lactone, and pisatin alter production of border cells, without affecting root growth. Understanding how root-derived and exogenous metabolites can selectively impact root function may yield benefits in crop production, especially in greenhouse agriculture systems where growing roots can be exposed to a significant accumulation of plant exudates.

Measuring root disease suppression in response to a compost water extract.

Commercial application of compost to prevent plant disease is hindered by variable performance. Here, we describe the use of a growth pouch assay to measure impact of a compost water extract (CWE) on root infection under controlled conditions. Most pea roots (≥95%) inoculated with Fusarium solani or Phoma pinodella spores rapidly develop a single local lesion in the region of elongation. In the presence of CWE, infection of pea roots grown in pouches was reduced by 93 to 100%. CWE used as a drench on pea seedlings grown in sand also resulted in 100% protection but, in a heavy clay soil, infection was reduced by <50%. CWE filtered to remove microorganisms did not inhibit frequency of F. solani infection, and resulted in increased local lesion development on individual roots. CWE inhibited mycelial growth of both pea- and cucumber-infecting isolates of F. solani in culture but exerted <40% protection against cucumber root infection. CWE treatment of pea but not cucumber was associated with retention of a sheath of border cells interspersed with bacteria covering the region of elongation. Growth pouch assays may provide a system to monitor effects of specific compost mixtures on root-rhizosphere interactions, and to identify variables influencing disease control.

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.

Root tips moving through soil: an intrinsic vulnerability.

Root elongation occurs by the generation of new cells from meristematic tissue within the apical 1-2 mm region of root tips. Therefore penetration of the soil environment is carried out by newly synthesized plant tissue, whose cells are inherently vulnerable to invasion by pathogens. This conundrum, on its face, would seem to reflect an intolerable risk to the successful establishment of root systems needed for plant life. Yet root tip regions housing the meristematic tissues repeatedly have been found to be free of microbial infection and colonization. Even when spore germination, chemotaxis, and/or growth of pathogens are stimulated by signals from the root tip, the underlying root tissue can escape invasion. Recent insights into the functions of root border cells, and the regulation of their production by transient exposure to external signals, may shed light on long-standing observations.

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.

Signals Controlling Extracellular Trap Formation in Plant and Animal Immune Responses

Newly characterized defence processes based on extracellular DNA-based trapping in mammals point to new targets for control of diseases ranging from lupus to sepsis to cancer. Emerging evidence that this is an ancient underpinning of immune systems includes the observation that DNA-based extracellular traps also operate in plants. Potential clinical applications include the use of plant metabolites as signals to modulate plant and animal extracellular trapping responses.

Altered growth and root tip morphology in Pisum sativum L. in response to altered expression of a gene expressed in border cells

Background and aimsRecent studies suggest that root border cells function in defense of the root by an extracellular DNA-based trapping mechanism similar to that described in mammalian white blood cells. Genes controlling the specialized properties of border cells as they detach from the root tip therefore are of interest.MethodsmRNA differential display was used to identify Brd13, a sequence expressed in border cells but not other root tissues. RNase protection and mRNA Northern blot analyses, and reporter gene expression under the control of the Brd13 promoter in transgenic hairy roots were used to confirm localized expression. Phenotype analysis of transgenic hairy roots expressing Brd13 antisense mRNA was carried out.ResultsBrd13 was expressed constitutively in border cells but not in leaves, stems, or roots without border cells. The predicted protein shares sequence similarity with flavin-binding proteins. Transgenic hairy roots expressing Brd13 antisense mRNA exhibited abnormal growth and morphology.ConclusionsWe report here that altered expression of a putative flavin-binding protein in border cells resulted in altered root development. Flavin-binding proteins play key roles in development, defense, and local auxin biosynthesis. The Brd13 gene and its promoter may be useful in creating defined changes in root development and defense.

Trapping of lead (Pb) by corn and pea root border cells

AimsMost plants produce a root tip extracellular matrix that includes viable border cell populations programmed to disperse into soil. Like neutrophils, border cells export structures that trap pathogens and prevent root tip infection. Border cells also trap metals. The goal of this study was to determine if border cells trap Pb.MethodsBorder cell responses to Pb were observed microscopically. Border cell impact on Pb-induced injury to roots was assessed using root growth assays. Pb removal from solution was measured using inductively coupled plasma mass spectrometry (ICP-MS). Speciation of Pb associated with border cells was evaluated by synchrotron X-ray absorption spectroscopy (XAS).ResultsIncreased border cell trap size and number occurred within minutes in response to Pb but not silicon (Si). Transient immersion of root tips into Pb after border cells were removed resulted in growth inhibition. Immersion of root tips and border cells into Pb solution resulted in significant removal of Pb. Si levels in the presence of root tips remained unchanged. The Pb speciation, measured with Pb LIII XAS, altered when reacted with border cells, indicating that direct binding by extracellular traps occurred.ConclusionsBorder cells can trap Pb and prevent damage to the root tip.