Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Piriformospora indica mycorrhization increases grain yield by accelerating early development of barley plants

Root colonization by the basidiomycete fungus Piriformospora indica induces host plant tolerance against abiotic and biotic stress, and enhances growth and yield. As P. indica has a broad host range, it has been established as a model system to study beneficial plant-microbe interactions. Moreover, its properties led to the assumption that P. indica shows potential for application in crop plant production. Therefore, possible mechanisms of P. indica improving host plant yield were tested in outdoor experiments: Induction of higher grain yield in barley was independent of elevated pathogen levels and independent of different phosphate fertilization levels. In contrast to the arbuscular mycorrhiza fungus Glomus mosseae total phosphate contents of host plant roots and shoots were not significantly affected by P. indica. Analysis of plant development and yield parameters indicated that positive effects of P. indica on grain yield are due to accelerated growth of barley plants early in development.Key words: mycorrhiza, barley development, Piriformospora indica, phosphate uptake, grain yield, pathogen resistanceThe wide majority of plant roots in natural ecosystems is associated with fungi, which very often play an important role for the host plants'' fitness.¹ The widespread arbuscular mycorrhizal (AM) symbiosis formed by fungi of the phylum Glomeromycota is mainly characterized by providing phosphate to their host plant in exchange for carbohydrates.^2,3 Fungi of the order Sebacinales also form beneficial interactions with plant roots and Piriformospora indica is the best-studied example of this group.⁴ This endophyte was originally identified in the rhizosphere of shrubs in the Indian Thar desert,⁵ but it turned out that the fungus colonizes roots of a very broad range of mono- and dicotyledonous plants,⁶ including major crop plants.^7–9 Like other mutualistic endophytes, P. indica colonizes roots in an asymptomatic manner¹⁰ and promotes growth in several tested plant species.^6,11,12 The root endophyte, moreover, enhances yield in barley and tomato and increases in both plants resistance against biotic stresses,^7,9 suggesting that application in agri- and horticulture could be successful.     

Roles of calcineurin B-like protein-interacting protein kinases in innate immunity in rice

Cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-asssociated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca²⁺ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed.Key words: PAMPs/MAMPs, calcium signaling, CBL-CIPK, hypersensitive cell death, reactive oxygen speciesCa²⁺ plays an essential role as an intracellular second messenger in plants as well as in animals. Several families of Ca²⁺ sensor proteins have been identified in higher plants, which decode spatiotemporal patterns of intracellular Ca²⁺ concentration.^1,2 Calcineurin B-Like Proteins (CBLs) comprise a family of Ca²⁺ sensor proteins similar to both the regulatory β-subunit of calcineurin and neuronal Ca²⁺ sensors of animals.^3,4 Unlike calcineurin B that regulates protein phosphatases, CBLs specifically target a family of protein kinases referred to as CIPKs (CBL-Interacting Protein Kinases).⁵ The CBL-CIPK system has been shown to be involved in a wide range of signaling pathways, including abiotic stress responses such as drought and salt, plant hormone responses and K+ channel regulation.^6,7Following the recognition of pathogenic signals, plant cells initiate the activation of a widespread signal transduction network that trigger inducible defense responses, including the production of reactive oxygen species (ROS), biosynthesis of phytoalexins, expression of pathogenesis-related (PR) genes and reorganization of cytoskeletons and the vacuole,⁸ followed by a form of programmed cell death known as hypersensitive response (HR).^9,10 Because complexed spatiotemporal patterns of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been suggested to play pivotal roles in defense signaling,^1,9 multiple Ca²⁺ sensor proteins and their effectors should function in defense signaling pathways. Although possible involvement of some calmodulin isoforms^11–13 and the calmodulin-domain/calcium-dependent protein kinases (CDPKs)^14–19 has been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs had so far been implicated as signaling components in innate immunity.     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

R type anion channel: A multifunctional channel seeking its molecular identity

Plant genomes code for channels involved in the transport of cations, anions and uncharged molecules through membranes. Although the molecular identity of channels for cations and uncharged molecules has progressed rapidly in the recent years, the molecular identity of anion channels has lagged behind. Electrophysiological studies have identified S-type (slow) and R-type (rapid) anion channels. In this brief review, we summarize the proposed functions of the R-type anion channels which, like the S-type, were first characterized by electrophysiology over 20 years ago, but unlike the S-type, have still yet to be cloned. We show that the R-type channel can play multiple roles.Key words: R-type anion channel, nitrate, sulphate, guard cell, action potentialAnion channels play a central role in signal transduction, nutrient transport and cell turgor regulation.¹ By far, their function was particularly well investigated in the guard cells of stomata using a combination of electrophysiological, pharmacological and genetic tools. In this system, anion channel activation was shown to be one of the limiting steps in the loss of cell turgor leading to stomatal closure.² In algal cells, anion channels were shown to contribute to membrane excitability through the generation of action potential.^1,3With the burst of molecular biology in the nineties, the genes coding for plant ion channels started to be unveiled. The first channel gene to be cloned in plant was the shaker-like potassium channel identified in a yeast functional expression screen.^4,5 More than ten years later, TaALMT1 and AtCLCa were characterized as the first members of two important anion channel families.^6,7 This growing group of newly identified channels, accounting for electrophysiological activity described long ago, includes the MSLs anion selective mechanosensitive channels.⁸ Recently, the well known S-type channel has been finally recognized to be encoded by members of the SLAC1 (and other SLAH) family (Slow Anion Channel-Associated 1).⁹ In agreement with electrophysiological data,^10–13 it requires phosphorylation by a Protein Kinase in order to be functional.^14,15 In contrast, the molecular identity of the R-type anion channel remains unknown. Therefore, this candidate, which has been functionally known since twenty years, remains the next challenge for plant channel physiologists.     

Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

Human non-CG methylation: Are human stem cells plant-like?

Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.¹ DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.²Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.³ Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,^4,5 or transposons and repetitive sequences such as the human L1 retrotransposon⁶ in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.⁶Non-CG methylation has also been reported at origins of replication^7,8 and a region of the human myogenic gene Myf3.⁹ The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.¹⁰ The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.¹⁰ The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.¹¹ Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.¹² Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.¹³ Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.¹³ This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.¹⁴ confirmed a much earlier report by Ramsahoye et al.¹⁵ suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,¹⁵ thus global methylation patterning was assessed. Lister et al.¹⁴ extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report¹⁴ that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,¹⁴ one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.¹⁴Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,^14,15 raises the possibility that cancer stem cells¹⁶ carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing⁸ or ligation mediated PCR¹⁷ methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster¹⁸ or human breast cancer DNA¹³ treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand¹⁸ or at a symmetrical CWG site.¹³ In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.¹³In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,^14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family¹⁹ and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.¹⁵ DRM proteins however, possess a unique permuted domain structure found exclusively in plants¹⁹ and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published^20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies²⁴ or find that CG methylation seen in plants but not non-CG methylation is detected.^21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,²⁷ and is consistent with the recent report that cultured stem cells are epigenetically unstable.²⁸The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.^9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes²⁹ even though the promoters were symmetrically de novo methylated at ^mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.^9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.¹⁴ Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,²⁹ however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.^30–33 However, depletion of CWG sites is not observed in the human genome.³⁴ Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome¹⁴ where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome¹⁴ raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.¹⁴ This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.¹⁴By analogy with the well known methylated DNA binding proteins specific for CG methylation,³⁵ methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.^36–38 While Dnmt1 has been proposed to function stoichiometrically³⁹ and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,⁴⁰ a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design^41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.^14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR⁴³ the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche^44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,⁴⁶ COMPARE-MS⁴⁷) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP⁴⁸) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.¹⁴In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.     

Complete Genome Sequence of Croceibacter atlanticus HTCC2559T

Here we announce the complete genome sequence of Croceibacter atlanticus HTCC2559^T, which was isolated by high-throughput dilution-to-extinction culturing from the Bermuda Atlantic Time Series station in the Western Sargasso Sea. Strain HTCC2559^T contained genes for carotenoid biosynthesis, flavonoid biosynthesis, and several macromolecule-degrading enzymes. The genome confirmed physiological observations of cultivated Croceibacter atlanticus strain HTCC2559^T, which identified it as an obligate chemoheterotroph.The phylum Bacteroidetes comprises 6 to ∼30% of total bacterial communities in the ocean by fluorescence in situ hybridization (8-10). Most marine Bacteroidetes are in the family Flavobacteriaceae, most of which are aerobic respiratory heterotrophs that form a well-defined clade by 16S rRNA phylogenetic analyses (4). The members of this family are well known for degrading macromolecules, including chitin, DNA, cellulose, starch, and pectin (17), suggesting their environmental roles as detritus decomposers in the ocean (6). Marine Polaribacter and Dokdonia species in the Flavobacteriaceae have also shown to have photoheterotrophic metabolism mediated by proteorhodopsins (11, 12).Several strains of the family Flavobacteriaceae were isolated from the Sargasso Sea and Oregon coast, using high-throughput culturing approaches (7). Croceibacter atlanticus HTCC2559^T was cultivated from seawater collected at a depth of 250 m from the Sargasso Sea and was identified as a new genus in the family Flavobacteriaceae based on its 16S rRNA gene sequence similarities (6). Strain HTCC2559^T met the minimal standards for genera of the family Flavobacteriaceae (3) on the basis of phenotypic characteristics (6).Here we report the complete genome sequence of Croceibacter atlanticus HTCC2559^T. The genome sequencing was initiated by the J. Craig Venter Institute as a part of the Moore Foundation Microbial Genome Sequencing Project and completed in the current announcement. Gaps among contigs were closed by Genotech Co., Ltd. (Daejeon, Korea), using direct sequencing of combinatorial PCR products (16). The HTCC2559^T genome was analyzed with a genome annotation system based on GenDB (14) at Oregon State University and with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (15, 16).The HTCC2559^T genome is 2,952,962 bp long, with 33.9 mol% G+C content, and there was no evidence of plasmids. The number of protein-coding genes was 2,715; there were two copies of the 16S-23S-5S rRNA operon and 36 tRNA genes. The HTCC2559^T genome contained genes for a complete tricarboxylic acid cycle, glycolysis, and a pentose phosphate pathway. The genome also contained sets of genes for metabolic enzymes involved in carotenoid biosynthesis and also a serine/glycine hydroxymethyltransferase, which is often associated with the assimilatory serine cycle (13). The potential for HTCC2559^T to use bacterial type III polyketide synthase (PKS) needs to be confirmed because this organism had a naringenin-chalcone synthase (CHS) or chalcone synthase (EC 2.3.1.74), a key enzyme in flavonoid biosynthesis. CHS initiates the addition of three molecules of malonyl coenzyme A (malonyl-CoA) to a starter CoA ester (e.g., 4-coumaroyl-CoA) (1) and takes part in a few bacterial type III polyketide synthase systems (1, 2, 5, 18).The complete genome sequence confirmed that strain HTCC2559^T is an obligate chemoheterotroph because no genes for phototrophy were found. As expected from physiological characteristics (6), the HTCC2559^T genome contained a set of genes coding for enzymes required to degrade high-molecular-weight compounds, including peptidases, metallo-/serine proteases, pectinase, alginate lyases, and α-amylase.     

Strigolactones as mediators of plant growth responses to environmental conditions

Strigolactones (SLs) have been recently identified as a new group of plant hormones or their derivatives thereof, shown to play a role in plant development. Evolutionary forces have driven the development of mechanisms in plants that allow adaptive adjustments to a variety of different habitats by employing plasticity in shoot and root growth and development. The ability of SLs to regulate both shoot and root development suggests a role in the plant''s response to its growth environment. To play this role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward increased adaptive adjustment. Here, the effects of SLs on shoot and root development are presented, and possible feedback loops between SLs and two environmental cues, light and nutrient status, are discussed; these might suggest a role for SLs in plants'' adaptive adjustment to growth conditions.Key words: strigolactones, light, nutrient status, root, shoot, branching, lateral roots, root hairsStrigolactones (SLs) are carotenoid-derived terpenoid lactones suggested to stem from the carotenoid pathway¹ via the activity of various oxygenases.^2,3 SLs production has been demonstrated in both monocotyledons and eudicotyledons (reviewed in ref. ⁴), suggesting their presence in many plant species.⁵ SLs are synthesized mainly in the roots and in some parts of the stem and then move towards the shoot apex (reviewed ref. ⁷).^6,8,9SLs were first characterized more than 40 years ago as germination stimulants of the parasitic plants Striga and Orobanche and later, as stimulants of arbuscular mycorrhiza hyphal branching as well (reviewed in ref. ^{4, 10–13}). Recently, SLs or derivatives thereof, have been identified as a new group of plant hormones, shown to play a role in inhibition of shoot branching,^2,3,8,9 thereby affecting shoot architecture; more recently they have also been shown to affect root growth by affecting auxin efflux.¹⁴Plants have developed mechanisms that allow adaptive adjustments to a variety of different habitats by employing plasticity in their growth and development.¹⁵ Shoot architecture is affected by environmental cues, such as light quality and quantity and nutrient status.^16–19 Root-system architecture and development are affected by environmental conditions such as nutrient availability (reviewed in ref. ^{20, 21}). At the same time, plant hormones are known to be involved in the regulation of plant growth, development and architecture (reviewed in ref. ^22–24) and to be mediators of the effects of environmental cues on plant development; one classic example is auxin''s role in the plant''s shade-avoidance response (reviewed in ref. ²⁵).The ability of SLs to regulate shoot and root development suggests that these phytohormones also have a role in the plant''s growth response to its environment. To play this putative role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward enhancing its adaptive adjustment. The present review examines the SLs'' possible role in adaptive adjustment of the plant''s response to growth conditions, by discussing their effect on plant development and the possible associations and feedback loops between SLs and two environmental cues: light and nutrient status.     

Neutralization of Clostridium difficile toxin A using antibody combinations

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.^1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.^3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.⁵ Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A⁻ B⁺”) suggest a critical role for toxin B in pathogenicity.^6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,^8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.^9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,^11–14 and is responsible for cell surface recognition and endocytosis.^12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.^2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue^19,20 and various non-antibiotic treatments are under investigation.^20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,^26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.^20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.³¹ Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.^33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis.