Dirigent isoflavene-forming PsPTS2: 3D structure, stereochemical, and kinetic characterization comparison with pterocarpan-forming PsPTS1 homolog in pea.

Pea phytoalexins (-)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity toward four possible 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers was investigated. Stereoisomer configurations were determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF 20-fold faster than the trans-(3R,4S)-isomer. The 4R-configured substrate's near ß-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4S-isomer's α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured ß-axial OH was closest to Asp51, whereas 4S-isomer's α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S,4R)-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin. PsPTS2 used cis-(3R,4R)-7,2'-dihydroxy-4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and C3/C4 stereoisomers to give 2',7-dihydroxy-4'-methoxyisoflav-3-ene (DMIF). DP homologs may exist in licorice (Glycyrrhiza pallidiflora) and tree legume Bolusanthus speciosus, as DMIF occurs in both species. PsPTS1 utilized cis-(3R,4R)-DMDI to give (-)-maackiain 2200-fold more efficiently than with cis-(3R,4R)-DMI to give (-)-medicarpin. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, reflecting the better 4R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a configurations in (+)-pisatin and (-)-maackiain, via identical DP-engendered mono-QM bound intermediate generation, which PsPTS2 either re-aromatizes to give DMDIF or PsPTS1 intramolecularly cyclizes to afford (-)-maackiain. Substrate docking simulations using PsPTS2 and PsPTS1 indicate cis-(3R,4R)-DMDI binds in the anti-configuration in PsPTS2 to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain.

Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum.

Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease.

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.

Extracellular DNases of Ralstonia solanacearum modulate biofilms and facilitate bacterial wilt virulence.

Ralstonia solanacearum is a soil-borne vascular pathogen that colonizes plant xylem vessels, a flowing, low-nutrient habitat where biofilms could be adaptive. Ralstonia solanacearum forms biofilm in vitro, but it was not known if the pathogen benefits from biofilms during infection. Scanning electron microscopy revealed that during tomato infection, R. solanacearum forms biofilm-like masses in xylem vessels. These aggregates contain bacteria embedded in a matrix including chromatin-like fibres commonly observed in other bacterial biofilms. Chemical and enzymatic assays demonstrated that the bacterium releases extracellular DNA in culture and that DNA is an integral component of the biofilm matrix. An R. solanacearum mutant lacking the pathogen's two extracellular nucleases (exDNases) formed non-spreading colonies and abnormally thick biofilms in vitro. The biofilms formed by the exDNase mutant in planta contained more and thicker fibres. This mutant was also reduced in virulence on tomato plants and did not spread in tomato stems as well as the wild-type strain, suggesting that these exDNases facilitate biofilm maturation and bacterial dispersal. To our knowledge, this is the first demonstration that R. solanacearum forms biofilms in plant xylem vessels, and the first documentation that plant pathogens use DNases to modulate their biofilm structure for systemic spread and virulence.

Intraspecies variation in cotton border cell production: rhizosphere microbiome implications.

PREMISE OF THE STUDY: Border cells, which separate from the root cap, can comprise >90% of carbon-based exudates released into the rhizosphere, but may not provide a general source of nutrients for soil microorganisms. Instead, this population of specialized cells appears to function in defense of the root tip by an extracellular trapping process similar to that of mammalian white blood cells. Border cell production is tightly regulated, and direct tests of their impact on crop production have been hindered by lack of intraspecies variation. • METHODS: Border cell number, viability, and clumping were compared among 22 cotton cultivars. Slime layer "extracellular trap" production by border cells in response to copper chloride, an elicitor of plant defenses, was compared in two cultivars with divergent border cell production. Trapping of bacteria by border cells in these lines also was measured. • KEY RESULTS: Emerging roots of some cultivars produced more than 20000 border cells per root, a 100% increase over previously reported values for this species. No differences in border cell morphology, viability, or clumping were found. Copper chloride-induced extracellular trap formation by border cells from a cultivar that produced 27921 ± 2111 cells per root was similar to that of cells from a cultivar with 10002 ± 614 cells, but bacterial trapping was reduced. • CONCLUSIONS: Intraspecific variation in border cell production provides a tool to measure their impact on plant development in the laboratory, greenhouse, and field. Further research is needed to determine the basis for this variation, and its impact on rhizosphere community structure.

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.

Association between border cell responses and localized root infection by pathogenic Aphanomyces euteiches.

BACKGROUND AND AIMS: The oomycete Aphanomyces euteiches causes up to 80 % crop loss in pea (Pisum sativum). Aphanomyces euteiches invades the root system leading to a complete arrest of root growth and ultimately to plant death. To date, disease control measures are limited to crop rotation and no resistant pea lines are available. The present study aims to get a deeper understanding of the early oomycete-plant interaction at the tissue and cellular levels. METHODS: Here, the process of root infection by A. euteiches on pea is investigated using flow cytometry and microscopic techniques. Dynamic changes in secondary metabolism are analysed with high-performance liquid chromatography with diode-array detection. KEY RESULTS: Root infection is initiated in the elongation zone but not in the root cap and border cells. Border-cell production is significantly enhanced in response to root inoculation with changes in their size and morphology. The stimulatory effect of A. euteiches on border-cell production is dependent on the number of oospores inoculated. Interestingly, border cells respond to pathogen challenge by increasing the synthesis of the phytoalexin pisatin. CONCLUSIONS: Distinctive responses to A. euteiches inoculation occur at the root tissue level. The findings suggest that root border cells in pea are involved in local defence of the root tip against A. euteiches. Root border cells constitute a convenient quantitative model to measure the molecular and cellular basis of plant-microbe interactions.

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.

A DNase from a Fungal Phytopathogen Is a Virulence Factor Likely Deployed as Counter Defense against Host-Secreted Extracellular DNA.

Histone-linked extracellular DNA (exDNA) is a component of neutrophil extracellular traps (NETs). NETs have been shown to play a role in immune response to bacteria, fungi, viruses, and protozoan parasites. Mutation of genes encoding group A Streptococcus extracellular DNases (exDNases) results in reduced virulence in animals, a finding that implies that exDNases are deployed as counter defense against host DNA-containing NETs. Is the exDNA/exDNase mechanism also relevant to plants and their pathogens? It has been demonstrated previously that exDNA is a component of a matrix secreted from plant root caps and that plants also carry out an extracellular trapping process. Treatment with DNase I destroys root tip resistance to infection by fungi, the most abundant plant pathogens. We show that the absence of a single gene encoding a candidate exDNase results in significantly reduced virulence of a fungal plant pathogen to its host on leaves, the known infection site, and on roots. Mg2+-dependent exDNase activity was demonstrated in fungal culture filtrates and induced when host leaf material was present. It is speculated that the enzyme functions to degrade plant-secreted DNA, a component of a complex matrix akin to neutrophil extracellular traps of animals.IMPORTANCE We document that the absence of a single gene encoding a DNase in a fungal plant pathogen results in significantly reduced virulence to a plant host. We compared a wild-type strain of the maize pathogen Cochliobolus heterostrophus and an isogenic mutant lacking a candidate secreted DNase-encoding gene and demonstrated that the mutant is reduced in virulence on leaves and on roots. There are no previous reports of deletion of such a gene from either an animal or plant fungal pathogen accompanied by comparative assays of mutants and wild type for alterations in virulence. We observed DNase activity, in fungal culture filtrates, that is Mg2+ dependent and induced when plant host leaf material is present. Our findings demonstrate not only that fungi use extracellular DNases (exDNases) for virulence, but also that the relevant molecules are deployed in above-ground leaves as well as below-ground plant tissues. Overall, these data provide support for a common defense/counter defense virulence mechanism used by animals, plants, and their fungal and bacterial pathogens and suggest that components of the mechanism might be novel targets for the control of plant disease.

A century of spine surgery: what can patients expect?

PURPOSE: To evaluate the hypothesis that spinal fusion surgery is an effective method to address spinal deformity-associated clinical problems, including magnitude of curvature (Cobb angle), pulmonary dysfunction, and pain. METHOD: A systematic review was carried out using Science Citation Index (SCI) Expanded (1900 - present), Social Sciences Citation Index (1956 - present), Arts and Humanities Citation Index (1965 - present), Medline (1950 - present) and PubMed Central databases (1887 - present) to access information regarding efficacy of spine surgery in preventing or improving the health and function of patients diagnosed with scoliosis in adolescence. RESULTS: Since 1950, more than 12,600 articles on scoliosis have been published, and nearly 50% (5721) focus on methods, rationale, outcome, and complications of surgical intervention. Among these, 82 articles have documented outcome for groups of > or =10 patients, treated for adolescent idiopathic scoliosis, and followed for at least 2 years after treatment. These data provide an overview of the impact of spine surgery on scoliosis for 5780 patients as surgery methods and approaches have evolved. CONCLUSIONS: For most patients, a reduced magnitude of spinal curvature can be achieved through one or more spinal fusion surgeries. There is no evidence to support the premise that this result is correlated with improved pulmonary function or reduced pain.

Scoliosis and the human genome project.

Understanding the cause of a disease or disorder is key to developing effective and humane strategies for early intervention and treatment. School screening programs have made it possible to demonstrate the high prevalence of childhood scoliosis, worldwide, and to reliably identify spinal curvatures early in the disease process before progression to a fixed structural deformity. Unfortunately, effective early interventions have not been established. Developing strategies to prevent scoliosis has been compromised, in general, by lack of understanding of its causes on a case by case basis. Information about genetic loci associated with disorders including scoliosis is emerging rapidly, since completion of the human genome sequence in 2003. These data can be used to identify children at high risk for developing spinal deformities and to design strategies for prevention.

Indications for conservative management of scoliosis (SOSORT guidelines).

This guideline has been discussed by the SOSORT guideline committee prior to the SOSORT consensus meeting in Milan, January 2005 and published in its first version on the SOSORT homepage: http://www.sosort.org/meetings.php. After the meeting it again has been discussed by the members of the SOSORT guideline committee to establish the final 2005 version submitted to Scoliosis, the official Journal of the society, in December 2005. This chapter is a republication from the original paper published in "Scoliosis" BioMed journal and it is included in this book due to its high importance.

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.

Extracellular DNA: A Bridge to Cancer.

DNase I is a secreted enzyme whose function has been presumed to control "waste management" in the human system, by degrading DNA that leaks from dead and dying cells. Emerging studies have instead yielded evidence that DNase I plays a central role in newly defined dynamics of immune and autoimmune diseases, as well as cancer and vascular disorders, including thrombosis. Cancer cells have been reported to be associated with distinctive extracellular structures that facilitate aggregation and implantation. The fact that DNA is a component of such structures and that it plays a role in cancer development is illustrated by direct evidence: DNase I added to tumor cells eliminates the structures and inhibits tumorigenicity of some cancer cell lines. DNase I injected into experimental animals, moreover, results in significant inhibition of metastasis. Despite independent observations of such phenomena in diverse cancers for over 50 years, the potential for using DNase I as a clinical tool to prevent or treat cancer remains unexplored. The discovery of neutrophil extracellular traps has yielded a conceptual framework for interpreting how extracellular DNA may function in cancer development and why it may prove to be an important clinical target in stopping cancer outside the cell.

Tissue specific localization of root infection by fungal pathogens: role of root border cells.

When roots of pea seedlings were inoculated uniformly with spores of Nectria haematocca or other pea pathogenic fungi, more than 90% developed lesions in the region of elongation within 3 days. More mature regions of most roots as well as the tip showed no visible signs of infection. Yet, microscopic observation revealed that 'mantles,' comprised of fungal hyphae intermeshed with populations of border cells, covered the tips of most roots. After physical detachment of the mantle, the underlying tip of most roots was found to be free of infection. Mantle-covered root tips did not respond to invasion of their border cells by activation of known defense genes unless there was invasion of the tip itself, as revealed by the presence of a lesion. Concomitant with the activation of defense genes was the induction of a cell-wall degrading enzyme whose expression is a marker for renewed production of border cells. Mantle formation did not occur in response to nonpathogens. The data are consistent with the hypothesis that border cells serve as a host-specific 'decoy' that protects root meristems by inhibiting fungal infection of the root tip.

Reversal of the signs and symptoms of moderately severe idiopathic scoliosis in response to physical methods.

This report describes improved signs and symptoms of previously untreated symptomatic spinal deformity in an adult female diagnosed with moderately severe thoracic scoliosis at the age of .7 years. Current treatment initiated at the age of forty included massage therapy, manual traction, ischemic pressure, and comprehensive manipulative medicine (CMM). A left-right chest circumference inequity was reduced by >10 cm, in correlation with improved appearance of the ribcage deformity and a 40% reduction in magnitude of Cobb angle, which had been stable for 30 years. The changes occurred gradually over an eight-year period, with the most rapid improvement occurring during two periods when CMM was employed.

Root border cells and secretions as critical elements in plant host defense.

Border cells and border-like cells are released from the root tip as individual cells and small aggregates, or as a group of attached cells. These are viable components of the root system that play a key role in controlling root interaction with living microbes of the rhizosphere. As their separation from root tip proceeds, the cells synthesize and secrete a hydrated mucilage that contains polysaccharides, secondary metabolites, antimicrobial proteins and extracellular DNA (exDNA). This exDNA-based matrix seems to function in root defense in a way similar to that of recently characterized neutrophil extracellular traps (NETs) in mammalian cells. This review discusses the role of the cells and secreted compounds in the protection of root tip against microbial infections.

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. 

Extracellular DNA: the tip of root defenses?

This review discusses how extracellular DNA (exDNA) might function in plant defense, and at what level(s) of innate immunity this process might operate. A new role for extracellular factors in mammalian defense has been described in a series of studies. These studies reveal that cells including neutrophils, eosinophils, and mast cells produce 'extracellular traps' (ETs) consisting of histone-linked exDNA. When pathogens are attracted to such ETs, they are trapped and killed. When the exDNA component of ETs is degraded, trapping is impaired and resistance against invasion is reduced. Conversely, mutation of microbial genes encoding exDNases that degrade exDNA results in loss of virulence. This discovery that exDNases are virulence factors opens new avenues for disease control. In plants, exDNA is required for defense of the root tip. Innate immunity-related proteins are among a group of >100 proteins secreted from the root cap and root border cell populations. Direct tests revealed that exDNA also is rapidly synthesized and exported from the root tip. When this exDNA is degraded by the endonuclease DNase 1, root tip resistance to fungal infection is lost; when the polymeric structure is degraded more slowly, by the exonuclease BAL31, loss of resistance to fungal infection is delayed accordingly. The results suggest that root border cells may function in a manner analogous to that which occurs in mammalian cells.