Genomics spurs rapid advances in our understanding of the biology of vascular wilt pathogens in the genus Verticillium
Klimes, A. ; Dobinson, K.F. ; Thomma, B. ; Klosterman, S.J. - \ 2015
Annual Review of Phytopathology 53 (2015). - ISSN 0066-4286 - p. 181 - 198.
protein-kinase gene - molecular characterization - functional-analysis - microsclerotia development - ethylene perception - hydrophobin gene - dahliae kleb - tomato ve1 - resistance - expression
The availability of genomic sequences of several Verticillium species triggered an explosion of genome-scale investigations of mechanisms fundamental to the Verticillium life cycle and disease process. Comparative genomics studies have revealed evolutionary mechanisms, such as hybridization and interchromosomal rearrangements, that have shaped these genomes. Functional analyses of a diverse group of genes encoding virulence factors indicate that successful host xylem colonization relies on specific Verticillium responses to various stresses, including nutrient deficiency and host defense–derived oxidative stress. Regulatory pathways that control responses to changes in nutrient availability also appear to positively control resting structure development. Conversely, resting structure development seems to be repressed by pathways, such as those involving effector secretion, which promote responses to host defenses. The genomics-enabled functional characterization of responses to the challenges presented by the xylem environment, accompanied by identification of novel virulence factors, has rapidly expanded our understanding of niche adaptation in Verticillium species.
Genome mining and metabolic profiling of the rhizosphere bacterium Pseudomonas sp. SH-C52 for antimicrobial compounds
Voort, M. van der; Meijer, H.J.G. ; Schmidt, Y. ; Watrous, J. ; Dekkers, E. ; Mendes, R. ; Dorrestein, P.C. ; Gross, H. ; Raaijmakers, J.M. - \ 2015
Frontiers in Microbiology 6 (2015). - ISSN 1664-302X - 14 p.
ii secretion system - phytophthora-infestans - biological-activity - functional-analysis - mass-spectrometry - natural functions - phospholipase-d - corrugata - biocontrol - lipopeptides
The plant microbiome represents an enormous untapped resource for discovering novel genes and bioactive compounds. Previously, we isolated Pseudomonas sp. SH-C52 from the rhizosphere of sugar beet plants grown in a soil suppressive to the fungal pathogen Rhizoctonia solani and showed that its antifungal activity is, in part, attributed to the production of the chlorinated 9-amino-acid lipopeptide thanamycin (Mendes et al., 2011). To get more insight into its biosynthetic repertoire, the genome of Pseudomonas sp. SH-C52 was sequenced and subjected to in silico, mutational and functional analyses. The sequencing revealed a genome size of 6.3 Mb and 5579 predicted ORFs. Phylogenetic analysis placed strain SH-C52 within the Pseudomonas corrugata clade. In silico analysis for secondary metabolites revealed a total of six non-ribosomal peptide synthetase (NRPS) gene clusters, including the two previously described NRPS clusters for thanamycin and the 2-amino acid antibacterial lipopeptide brabantamide. Here we show that thanamycin also has activity against an array of other fungi and that brabantamide A exhibits anti-oomycete activity and affects phospholipases of the late blight pathogen Phytophthora infestans. Most notably, mass spectrometry led to the discovery of a third lipopeptide, designated thanapeptin, with a 22-amino-acid peptide moiety. Seven structural variants of thanapeptin were found with varying degrees of activity against P. infestans. Of the remaining four NRPS clusters, one was predicted to encode for yet another and unknown lipopeptide with a predicted peptide moiety of 8-amino acids. Collectively, these results show an enormous metabolic potential for Pseudomonas sp. SH-C52, with at least three structurally diverse lipopeptides, each with a different antimicrobial activity spectrum.
Catalytic and hydrodynamic properties of styrene monooxygenases from Rhocodoccus opacus 1CP are modulated by cofactor binding.
Riedel, A. ; Heine, T. ; Westphal, A.H. ; Conrad, C. ; Rathsack, P. ; Berkel, W.J.H. van; Tischler, D. - \ 2015
AMB Express 5 (2015). - ISSN 2191-0855 - 11 p.
recombinant escherichia-coli - pseudomonas-fluorescens st - functional-analysis - crystal-structure - catabolism genes - strain vlb120 - putida ca-3 - degradation - mechanism - oxide
Styrene monooxygenases (SMOs) are flavoenzymes catalyzing the epoxidation of styrene into styrene oxide. SMOs are composed of a monooxygenase (StyA) and a reductase (StyB). The latter delivers reduced FAD to StyA on the expense of NADH. We identified Rhodococcus opacus 1CP as the first microorganism to possess three different StyA isoforms occurring in two systems StyA1/StyA2B and StyA/StyB, respectively. The hydrodynamic properties of StyA isozymes were found to be modulated by the binding of the (reduced) FAD cofactor. StyA1 and SyA2B mainly occur as dimers in their active forms while StyA is a monomer. StyA1 showed the highest epoxidation activity and excellent enantioselectivity in aromatic sulfoxidation. The hydrodynamic and biocatalytic properties of SMOs from strain 1CP are of relevance for investigation of possible industrial applications.
Oral administration of Lactobacillus plantarum 299v modulates gene expression in the ileum of pigs: prediction of crosstalk between intestinal immune cells and sub-mucosal adipocytes
Hulst, M.M. ; Gross, G. ; Liu, Yapin ; Hoekman, A.J.W. ; Niewold, T. ; Meulen, J. van der; Smits, M.A. - \ 2015
Genes & Nutrition 10 (2015)3. - ISSN 1555-8932 - 13 p.
kappa-b - functional-analysis - in-vivo - inhibition - immunoglobulins - identification - adipogenesis - macrophages - metabolism - activation
To study host–probiotic interactions in parts of the intestine only accessible in humans by surgery (jejunum, ileum and colon), pigs were used as model for humans. Groups of eight 6-week-old pigs were repeatedly orally administered with 5 × 1012 CFU Lactobacillus plantarum 299v (L. plantarum 299v) or PBS, starting with a single dose followed by three consecutive daily dosings 10 days later. Gene expression was assessed with pooled RNA samples isolated from jejunum, ileum and colon scrapings of the eight pigs per group using Affymetrix porcine microarrays. Comparison of gene expression profiles recorded from L. plantarum 299v-treated pigs with PBS-treated pigs indicated that L. plantarum 299v affected metabolic and immunological processes, particularly in the ileum. A higher expression level of several B cell-specific transcription factors/regulators was observed, suggesting that an influx of B cells from the periphery to the ileum and/or the proliferation of progenitor B cells to IgA-committed plasma cells in the Peyer’s patches of the ileum was stimulated. Genes coding for enzymes that metabolize leukotriene B4, 1,25-dihydroxyvitamin D3 and steroids were regulated in the ileum. Bioinformatics analysis predicted that these metabolites may play a role in the crosstalk between intestinal immune cells and sub-mucosal adipocytes. Together with regulation of genes that repress NFKB- and PPARG-mediated transcription, this crosstalk may contribute to tempering of inflammatory reactions. Furthermore, the enzyme adenosine deaminase, responsible for the breakdown of the anti-inflammatory mediator adenosine, was strongly down-regulated in response to L. plantarum 299v. This suggested that L. plantarum 299v-regulated production of adenosine by immune cells like regulatory T cells may also be a mechanism that tempers inflammation in the ileum, and perhaps also in other parts of the pig’s body.
Identifying the core microbial community in the gut of fungus-growing termites
Otani, S. ; Mikaelyan, A. ; Nobre, T. ; Hansen, L.H. ; Kone, N.A. ; Sorensen, S.J. ; Aanen, D.K. ; Boomsma, J.J. ; Brune, A. ; Poulsen, M. - \ 2014
Molecular Ecology 23 (2014)18. - ISSN 0962-1083 - p. 4631 - 4644.
feeding higher termite - bacterial community - phylogenetic analysis - functional-analysis - macrotermes-gilvus - lignin degradation - nasutitermes spp. - sp-nov. - diversity - hindgut
Gut microbes play a crucial role in decomposing lignocellulose to fuel termite societies, with protists in the lower termites and prokaryotes in the higher termites providing these services. However, a single basal subfamily of the higher termites, the Macrotermitinae, also domesticated a plant biomass-degrading fungus (Termitomyces), and how this symbiont acquisition has affected the fungus-growing termite gut microbiota has remained unclear. The objective of our study was to compare the intestinal bacterial communities of five genera (nine species) of fungus-growing termites to establish whether or not an ancestral core microbiota has been maintained and characterizes extant lineages. Using 454-pyrosequencing of the 16S rRNA gene, we show that gut communities have representatives of 26 bacterial phyla and are dominated by Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria and Synergistetes. A set of 42 genus-level taxa was present in all termite species and accounted for 56–68% of the species-specific reads. Gut communities of termites from the same genus were more similar than distantly related species, suggesting that phylogenetic ancestry matters, possibly in connection with specific termite genus-level ecological niches. Finally, we show that gut communities of fungus-growing termites are similar to cockroaches, both at the bacterial phylum level and in a comparison of the core Macrotermitinae taxa abundances with representative cockroach, lower termite and higher nonfungus-growing termites. These results suggest that the obligate association with Termitomyces has forced the bacterial gut communities of the fungus-growing termites towards a relatively uniform composition with higher similarity to their omnivorous relatives than to more closely related termites
Apoplastic Venom Allergen-like Proteins of Cyst Nematodes Modulate the Activation of Basal Plant Innate Immunity by Cell Surface Receptors
Lozano Torres, J.L. ; Wilbers, R.H.P. ; Warmerdam, S. ; Finkers-Tomczak, A.M. ; Diaz Granados Muñoz, A. ; Schaik, C.C. van; Helder, J. ; Bakker, J. ; Goverse, A. ; Schots, A. ; Smant, G. - \ 2014
PLoS Pathogens 10 (2014)12. - ISSN 1553-7366 - 18 p.
root-knot nematode - cf-2-dependent disease resistance - pamp-triggered immunity - arabidopsis-thaliana - globodera-rostochiensis - heterodera-schachtii - ancylostoma-caninum - parasitic nematodes - extracellular-matrix - functional-analysis
Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize venom allergen-like proteins to suppress the activation of defenses by immunogenic breakdown products in damaged host tissue.
Discovery of new regulatory genes of lipopeptide biosynthesis in Pseudomonas fluorescens
Song, C. ; Aundy, K. ; Mortel, J.E. van de; Raaijmakers, J.M. - \ 2014
FEMS Microbiology Letters 356 (2014). - ISSN 0378-1097 - p. 166 - 175.
syringae pv.-syringae - biosurfactant production - functional-analysis - natural functions - putida pcl1445 - putisolvin-ii - diversity - cluster - identification - antibiotics
Pseudomonas fluorescens SS101 produces the cyclic lipopeptide massetolide with diverse functions in antimicrobial activity, motility, and biofilm formation. To understand how massetolide biosynthesis is genetically regulated in SS101, c. 8000 random plasposon mutants were screened for reduced or loss of massetolide production. Of a total of 58 putative mutants, 45 had a mutation in one of the three massetolide biosynthesis genes massA, massB, or massC. For five mutants, the insertions were located in the known regulatory genes gacS, gacA, and clpP. For the remaining eight mutants, insertions were located in clpA, encoding the ClpP chaperone, in phgdh, encoding D-3-phosphoglycerate dehydrogenase, in the heat shock protein-encoding dnaK, or in the transmembrane regulatory gene prtR. Genetic, chemical, and phenotypic analyses showed that phgdh, dnaK, and prtR are indeed involved in the regulation of massetolide biosynthesis, most likely by transcriptional repression of the LuxR-type regulator genes massAR and massBCR. In addition to their role in massetolide biosynthesis, dnaK and prtR were found to affect siderophore and extracellular protease(s) production, respectively. The identification of new regulatory genes substantially extended insights into the signal transduction pathways of lipopeptide biosynthesis in P. fluorescens and into regulation of other traits that may contribute to its life-style in the rhizosphere.
Genomic Characterization of Non-Mucus Adherent Derivatives of Lactobacillus rhamnosus GG Reveals Genes Affecting Pilus Biogenesis
Rasinkangas, P. ; Reunanen, J. ; Douillard, F.P. ; Ritari, J. ; Uotinen, V. ; Palva, A. ; Vos, W.M. de - \ 2014
Applied and Environmental Microbiology 80 (2014)22. - ISSN 0099-2240 - p. 7001 - 7009.
intestinal epithelial-cells - placebo-controlled trial - gram-positive bacteria - functional-analysis - atopic disease - strain gg - adhesion - probiotics - surface - prevention
Lactobacillus rhamnosus GG is one of the best-characterized lactic acid bacteria and can be considered a probiotic paradigm. Comparative and functional genome analysis showed that L. rhamnosus GG harbors a genomic island including the spaCBA-srtC1 gene cluster, encoding the cell surface-decorating host-interacting pili. Here, induced mutagenesis was used to study pilus biogenesis in L. rhamnosus GG. A combination of two powerful approaches, mutation selection and next-generation sequencing, was applied to L. rhamnosus GG for the selection of pilus-deficient mutants from an enriched population. The isolated mutants were first screened by immuno-dot blot analysis using antiserum against pilin proteins. Relevant mutants were selected, and the lack of pili was confirmed by immunoelectron microscopy. The pilosotype of 10 mutant strains was further characterized by analyzing pilin expression using Western blot, dot blot, and immunofluorescence methods. A mucus binding assay showed that the mutants did not adhere to porcine intestinal mucus. Comparative genome sequence analysis using the Illumina MiSeq platform allowed us to determine the nature of the mutations in the obtained pilus-deficient derivatives. Three major classes of mutants with unique genotypes were observed: class I, with mutations in the srtC1 gene; class II, with a deletion containing the spaCBA-srtC1 gene cluster; and class III, with mutations in the spaA gene. Only a limited number of collateral mutations were observed, and one of the pilus-deficient derivatives with a deficient srtC1 gene contained 24 other mutations. This strain, PB12, can be considered a candidate for human trials addressing the impact of the absence of pili.
Metabolomic engineering for the microbial production of cartenoids and related products with a focus on the rare C50 carotenoids
Heider, S.A.E. ; Peters-Wendisch, P. ; Wendisch, V.F. ; Beekwilder, M.J. - \ 2014
Applied Microbiology and Biotechnology 98 (2014)10. - ISSN 0175-7598 - p. 4355 - 4368.
alga haematococcus-pluvialis - blue-light photoreception - escherichia-coli - bacterial carotenoids - biosynthetic-pathway - bacillus-subtilis - astaxanthin biosynthesis - functional-analysis - beta-carotene - corynebacterium-glutamicum
Carotenoids, a subfamily of terpenoids, are yellowtored-colored pigments synthesized by plants, fungi, algae, and bacteria. They are ubiquitous in nature and take over crucial roles in many biological processes as for example photosynthesis, vision, and the quenching of free radicals and singlet oxygen. Due to their color and their potential beneficial effects on human health, carotenoids receive increasing attention. Carotenoids can be classified due to the length of their carbon backbone. Most carotenoids have a C40 backbone, but also C30 and C50 carotenoids are known. All carotenoids are derived fromisopentenyl pyrophosphate (IPP) as a common precursor. Pathways leading to IPP as well as metabolic engineering of IPP synthesis and C40 carotenoid production have been reviewed expertly elsewhere. Since C50 carotenoids are synthesized from the C40 carotenoid lycopene, we will summarize common strategies for optimizing lycopene production and we will focus our review on the characteristics, biosynthesis, glycosylation, and overproduction of C50 carotenoids.
Modelling cell division and endoreduplication in tomato fruit pericarp
Apri, M. ; Kromdijk, J. ; Visser, P.H.B. de; Gee, M. de; Molenaar, J. - \ 2014
Journal of Theoretical Biology 349 (2014). - ISSN 0022-5193 - p. 32 - 43.
cyclin-dependent kinase - transcriptional adapter protein - arabidopsis-thaliana trichomes - anaphase-promoting complex - f-box protein - solanum-lycopersicon - fission yeast - functional-analysis - endocycle onset - auxin
In many developing plant tissues and organs, differentiating cells switch from the classical cell cycle to an alternative partial cycle. This partial cycle bypasses mitosis and allows for multiple rounds of genome duplication without cell division, giving rise to cells with high ploidy numbers. This partial cycle is referred to as endoreduplication. Cell division and endoreduplication are important processes for biomass allocation and yield in tomato. Quantitative trait loci for tomato fruit size or weight are frequently associated with variations in the pericarp cell number, and due to the tight connection between endoreduplication and cell expansion and the prevalence of polyploidy in storage tissues, a functional correlation between nuclear ploidy number and cell growth has also been implicated (karyoplasmic ratio theory). In this paper, we assess the applicability of putative mechanisms for the onset of endoreduplication in tomato pericarp cells via development of a mathematical model for the cell cycle gene regulatory network. We focus on targets for regulation of the transition to endoreduplication by the phytohormone auxin, which is known to play a vital role in the onset of cell expansion and differentiation in developing tomato fruit. We show that several putative mechanisms are capable of inducing the onset of endoreduplication. This redundancy in explanatory mechanisms is explained by analysing system behaviour as a function of their combined action. Namely, when all these routes to endoreduplication are used in a combined fashion, robustness of the regulation of the transition to endoreduplication is greatly improved
Fragmentation of an aflatoxin-like gene cluster in a forest pathogen
Bradshaw, R.E. ; Slot, J.C. ; Moore, G.G. ; Chettri, P. ; Wit, P.J.G.M. de; Ehrlich, K.C. ; Ganley, A.R.D. ; Olson, M.A. ; Rokas, A. ; Carbone, I. ; Cox, M.P. - \ 2013
New Phytologist 198 (2013)2. - ISSN 0028-646X - p. 525 - 535.
aspergillus-parasiticus - dothistroma-septosporum - phylogenetic analyses - biosynthetic-pathway - recombination events - secondary metabolism - functional-analysis - horizontal transfer - filamentous fungi - evolution
Plant pathogens use a complex arsenal of weapons, such as toxic secondary metabolites, to invade and destroy their hosts. Knowledge of how secondary metabolite pathways evolved is central to understanding the evolution of host specificity. The secondary metabolite dothistromin is structurally similar to aflatoxins and is produced by the fungal pine pathogen Dothistroma septosporum. Our study focused on dothistromin genes, which are widely dispersed across one chromosome, to determine whether this unusual distributed arrangement evolved from an ancestral cluster. We combined comparative genomics and population genetics approaches to elucidate the origins of the dispersed arrangement of dothistromin genes over a broad evolutionary time-scale at the phylum, class and species levels. Orthologs of dothistromin genes were found in two major classes of fungi. Their organization is consistent with clustering of core pathway genes in a common ancestor, but with intermediate cluster fragmentation states in the Dothideomycetes fungi. Recombination hotspots in a D.septosporum population matched sites of gene acquisition and cluster fragmentation at higher evolutionary levels. The results suggest that fragmentation of a larger ancestral cluster gave rise to the arrangement seen in D.septosporum. We propose that cluster fragmentation may facilitate metabolic retooling and subsequent host adaptation of plant pathogens.
Genetic Analysis of Health-Related Secondary Metabolites in a Brassica rapa Recombinant Inbred Line Population
Bagheri, H. ; Soda, M. El; Kim, H.K. ; Fritsche, S. ; Jung, C. ; Aarts, M.G.M. - \ 2013
International Journal of Molecular Sciences 14 (2013)8. - ISSN 1661-6596 - p. 15561 - 15577.
synechocystis sp pcc-6803 - quantitative trait loci - vitamin-e content - arabidopsis-thaliana - hydroxyphenylpyruvate dioxygenase - tocopherol biosynthesis - plastoquinone synthesis - functional-analysis - natural variation - alpha-tocopherol
The genetic basis of the wide variation for nutritional traits in Brassica rapa is largely unknown. A new Recombinant Inbred Line (RIL) population was profiled using High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR) analysis to detect quantitative trait loci (QTLs) controlling seed tocopherol and seedling metabolite concentrations. RIL population parent L58 had a higher level of glucosinolates and phenylpropanoids, whereas levels of sucrose, glucose and glutamate were higher in the other RIL population parent, R-o-18. QTL related to seed tocopherol (-, -, -, -, -/- and total tocopherol) concentrations were detected on chromosomes A3, A6, A9 and A10, explaining 11%-35% of the respective variation. The locus on A3 co-locates with the BrVTE1gene, encoding tocopherol cyclase. NMR spectroscopy identified the presence of organic/amino acid, sugar/glucosinolate and aromatic compounds in seedlings. QTL positions were obtained for most of the identified compounds. Compared to previous studies, novel loci were found for glucosinolate concentrations. This work can be used to design markers for marker-assisted selection of nutritional compounds in B. rapa.
Comparative genome analysis of Lactobacillus casei strains isolated from Actimel and Yakult products reveals marked similarities and points to a common origin
Douillard, F.P. ; Kant, R. ; Ritari, J. ; Paulin, L. ; Palva, A. ; Vos, W.M. de - \ 2013
Microbial Biotechnology 6 (2013)5. - ISSN 1751-7907 - p. 576 - 587.
lactic-acid bacteria - gram-positive bacteria - rhamnosus gg - functional-analysis - cell-wall - surface-proteins - staphylococcus-aureus - controlled-trial - binding-protein - sequence
The members of the Lactobacillus genus are widely used in the food and feed industry and show a remarkable ecological adaptability. Several Lactobacillus strains have been marketed as probiotics as they possess health-promoting properties for the host. In the present study, we used two complementary next-generation sequencing technologies to deduce the genome sequences of two Lactobacillus casei strains LcA and LcY, which were isolated from the products Actimel and Yakult, commercialized as probiotics. The LcA and LcY draft genomes have, respectively, an estimated size of 3067 and 3082 Mb and a G+ C content of 46.3%. Both strains are close to identical to each other and differ by no more than minor chromosomal re-arrangements, substitutions, insertions and deletions, as evident from the verified presence of one insertion-deletion (InDel) and only 29 single-nucleotide polymorphisms (SNPs). In terms of coding capacity, LcA and LcY are predicted to encode a comparable exoproteome, indicating that LcA and LcY are likely to establish similar interactions with human intestinal cells. Moreover, both L. casei LcA and LcY harboured a 59.6 kb plasmid that shared high similarities with plasmids found in other L. casei strains, such as W56 and BD-II. Further analysis revealed that the L. casei plasmids constitute a good evolution marker within the L. casei species. The plasmids of the LcA and LcY strains are almost identical, as testified by the presence of only three verified SNPs, and share a 3.5 kb region encoding a remnant of a lactose PTS system that is absent from the plasmids of W56 and BD-II but conserved in another smaller L. casei plasmid (pLC2W). Our observations imply that the results obtained in animal and human experiments performed with the Actimel and Yakult strains can be compared with each other as these strains share a very recent common ancestor.
Dendritic cells and their role in tumor immunosurveillance
Strioga, M.M. ; Schijns, V.E.J.C. ; Powell, D.J. ; Pasukoniene, V. ; Dobrovolskiene, N.T. ; Michalek, J. - \ 2013
Innate Immunity 19 (2013)1. - ISSN 1753-4259 - p. 98 - 111.
regulatory t-cells - epidermal langerhans cells - ovarian-cancer progression - antigen-presenting cells - in-vivo - peripheral tolerance - adaptive immunity - functional-analysis - colorectal-cancer - apoptotic cells
Dendritic cells (DCs) comprise a heterogeneous population of cells that play a key role in initiating, directing and regulating adaptive immune responses, including those critically involved in tumor immunosurveillance. As a riposte to the central role of DCs in the generation of antitumor immune responses, tumors have developed various mechanisms which impair the immunostimulatory functions of DCs or even instruct them to actively contribute to tumor growth and progression. In the first part of this review we discuss general aspects of DC biology, including their origin, subtypes, immature and mature states, and functional plasticity which ensures a delicate balance between active immune response and immune tolerance. In the second part of the review we discuss the complex interactions between DCs and the tumor microenvironment, and point out the challenges faced by DCs during the recognition of tumor Ags. We also discuss the role of DCs in tumor angiogenesis and vasculogenesis.
Ve1-mediated resistance against Verticillium does not involve a hypersensitive response in Arabidopsis
Zhang, Z. ; Esse, H.P. van; Damme, M. van; Fradin, E.F. ; Liu, Chun-Ming ; Thomma, B.P.H.J. - \ 2013
Molecular Plant Pathology 14 (2013)7. - ISSN 1464-6722 - p. 719 - 727.
ethylene-inducing xylanase - receptor-like proteins - gated ion-channel - disease resistance - rhynchosporium-secalis - functional-analysis - defense responses - gene family - tomato ve1 - cell-death
The recognition of pathogen effectors by plant immune receptors leads to the activation of immune responses that often include a hypersensitive response (HR): rapid and localized host cell death surrounding the site of attempted pathogen ingress. We have demonstrated previously that the recognition of the Verticillium dahliae effector protein Ave1 by the tomato immune receptor Ve1 triggers an HR in tomato and tobacco. Furthermore, we have demonstrated that tomato Ve1 provides Verticillium resistance in Arabidopsis upon Ave1 recognition. In this study, we investigated whether the co-expression of Ve1 and Ave1 in Arabidopsis results in an HR, which could facilitate a forward genetics screen. Surprisingly, we found that the co-expression of Ve1 and Ave1 does not induce an HR in Arabidopsis. These results suggest that an HR may occur as a consequence of Ve1/Ave1-induced immune signalling in tomato and tobacco, but is not absolutely required for Verticillium resistance.
System-Wide Hypersensitive Response-Associated Transcriptome and Metabolome Reprogramming in Tomato
Etalo, D.W. ; Stulemeijer, I.J.E. ; Esse, H.P. van; Vos, R.C.H. de; Bouwmeester, H.J. ; Joosten, M.H.A.J. - \ 2013
Plant Physiology 162 (2013)3. - ISSN 0032-0889 - p. 1599 - 1617.
programmed cell-death - pathogen pseudomonas-syringae - campestris pv. vesicatoria - glutathione s-transferases - amino-acid catabolism - leaf rust resistance - higher-plant cells - mass-spectrometry - cladosporium-fulvum - functional-analysis
The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR.
Temperature-dependent regulation of flowering by antagonistic FLM variants
Posé, D. ; Verhage, D.S.L. ; Ott, F. ; Yant, L. ; Mathieu, J. ; Angenent, G.C. ; Immink, G.H. ; Schmid, M. - \ 2013
Nature 503 (2013)7476. - ISSN 0028-0836 - p. 414 - 417.
mads-box gene - arabidopsis-thaliana - transcription factor - circadian clock - functional-analysis - floral transition - identification - repressor - time - vernalization
The appropriate timing of flowering is crucial for plant reproductive success. It is therefore not surprising that intricate genetic networks have evolved to perceive and integrate both endogenous and environmental signals, such as carbohydrate and hormonal status, photoperiod and temperature1,2. In contrast to our detailed understanding of the vernalization pathway, little is known about how flowering time is controlled in response to changes in the ambient growth temperature. In Arabidopsis thaliana, the MADSbox transcription factor genesFLOWERING LOCUSM (FLM) and SHORTVEGETATIVEPHASE (SVP)have key roles in this process3,4. FLM is subject to temperature-dependent alternative splicing3. Here we report that the two mainFLMprotein splice variants,FLM-b and FLM-d, compete for interaction with the floral repressor SVP. The SVP–FLM-b complex is predominately formed at low temperatures and prevents precocious flowering. By contrast, the competingSVP–FLM-d complex is impaired in DNA binding and acts as a dominant-negative activator of flowering at higher temperatures. Our results show a new mechanism that controls the timing of the floral transition in response to changes in ambient temperature. A better understanding of how temperature controls the molecular mechanismsof flowering will be important to cope with current changes in global climate5,6.
Regulation of intestinal homeostasis and immunity with probiotic lactobacilli
Baarlen, P. van; Wells, J. ; Kleerebezem, M. - \ 2013
Trends in Immunology 34 (2013)5. - ISSN 1471-4906 - p. 208 - 215.
wall teichoic-acid - gut microbiota - rhamnosus gg - t-cells - lipoteichoic acid - functional-analysis - epithelial barrier - controlled-trials - dendritic cell - in-vivo
The gut microbiota provide important stimuli to the human innate and adaptive immune system and co-mediate metabolic and immune homeostasis. Probiotic bacteria can be regarded as part of the natural human microbiota, and have been associated with improving homeostasis, albeit with different levels of success. Composition of microbiota, probiotic strain identity, and host genetic differences may account for differential modulation of immune responses by probiotics. Here, we review the mechanisms of immunomodulating capacities of specific probiotic strains, the responses they can induce in the host, and how microbiota and genetic differences between individuals may co-influence host responses and immune homeostasis.
Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG
Tripathi, P. ; Beaussart, A. ; Alsteens, D. ; Dupres, V. ; Claes, I. ; Ossowski, I. von; Vos, W.M. de; Palva, A. ; Lebeer, S. ; Vanderleyden, J. ; Dufrene, Y.F. - \ 2013
ACS Nano 7 (2013)4. - ISSN 1936-0851 - p. 3685 - 3697.
atomic-force-microscopy - coli p-pili - functional-analysis - escherichia-coli - stabilizing isopeptide - streptococcus-pyogenes - gastrointestinal-tract - mechanical force - binding-protein - antigen-i/ii
Knowledge of the mechanisms by which bacterial pili adhere to host cells and withstand external forces is critical to our understanding of their functional roles and offers exciting avenues in biomedicine for controlling the adhesion of bacterial pathogens and probiotics. While much progress has been made in the nanoscale characterization of pili from Gram-negative bacteria, the adhesive and mechanical properties of Gram-positive bacterial pili remain largely unknown. Here, we use single-molecule atomic force microscopy to unravel the binding mechanism of pili from the probiotic Gram-positive bacterium Lactobacillus rhamnosus GG (LGG). First, we show that SpaC, the key adhesion protein of the LGG pilus, is a multifunctional adhesin with broad specificity. SpaC forms homophilic trans-interactions engaged in bacterial aggregation and specifically binds mucin and collagen, two major extracellular components of host epithelial layers. Homophilic and heterophilic interactions display similar binding strengths and dissociation rates. Next, pulling experiments on living bacteria demonstrate that LGG pili exhibit two unique mechanical responses, that is, zipper-like adhesion involving multiple SpaC molecules distributed along the pilus length and nanospring properties enabling pili to resist high force. These mechanical properties may represent a generic mechanism among Gram-positive bacterial pili for strengthening adhesion and withstanding shear stresses in the natural environment. The single-molecule experiments presented here may help us to design molecules capable of promoting or inhibiting bacterial-host interactions
Using recombinant Lactococci as an approach to dissect the immunomodulating capacity of surface piliation in probiotic Lactobacillus rhamnosus GG
Ossowski, I. von; Pietilä, T.E. ; Rintahaka, J. ; Nummenmaa, E. ; Mäkinen, V.M. ; Reunanen, J. ; Satokari, R.M. ; Vos, W.M. de; Palva, I. ; Palva, A. - \ 2013
PLoS ONE 8 (2013)5. - ISSN 1932-6203
lactic-acid bacteria - intestinal epithelial-cells - functional-analysis - dendritic cells - gastrointestinal-tract - dependent mechanism - protein-production - oral consumption - adhesion - pili
Primarily arising from their well understood beneficial health effects, many lactobacilli strains are considered good candidates for use as probiotics in humans and animals. Lactobacillar probiosis can itself be best typified by the Lactobacillus rhamnosus GG strain, which, with its well-documented clinical benefits, has emerged as one of the most widely used probiotics in the food and health-supplement industries. Even so, many facets of its molecular mechanisms and limitations as a beneficial commensal bacterium still remain to be thoroughly explored and dissected. Because L. rhamnosus GG is one of only a few such strains exhibiting surface piliation (called SpaCBA), we sought to examine whether this particular type of cell-surface appendage has a discernible immunomodulating capacity and is able to trigger targeted responses in human immune-related cells. Thus, presented herein for this study, we recombinantly engineered Lactococcus lactis to produce native (and pilin-deleted) SpaCBA pili that were assembled in a structurally authentic form and anchored to the cell surface, and which had retained mucus-binding functionality. By using these recombinant lactococcal constructs, we were able to demonstrate that the SpaCBA pilus can be a contributory factor in the activation of Toll-like receptor 2-dependent signaling in HEK cells as well as in the modulation of pro- and anti-inflammatory cytokine (TNF-a, IL-6, IL-10, and IL-12) production in human monocyte-derived dendritic cells. From these data, we suggest that the recombinant-expressed and surface-anchored SpaCBA pilus, given its projected functioning in the gut environment, might be viewed as a new microbe-associated molecular pattern (MAMP)-like modulator of innate immunity. Accordingly, our study has brought some new insight to the molecular immunogenicity of the SpaCBA pilus, thus opening the way to a better understanding of its possible role in the multifaceted nature of L. rhamnosus GG probiosis within the human gut
The NADPH Oxidase Complexes in Botrytis cinerea: Evidence for a Close Association with the ER and the Tetraspanin Pls1
Siegmund, U. ; Heller, J. ; Kan, J.A.L. van; Tudzynski, P. - \ 2013
PLoS ONE 8 (2013)2. - ISSN 1932-6203 - 16 p.
protein-disulfide-isomerase - appressorium-mediated penetration - smooth-muscle-cells - gray mold fungus - reactive oxygen - endoplasmic-reticulum - secretory pathway - ascospore germination - functional-analysis - perennial ryegrass
NADPH oxidases (Nox) are major enzymatic systems that generate reactive-oxygen species (ROS) in multicellular eukaryotes. In several fungi they have been shown to be involved in sexual differentiation and pathogenicity. However, in contrast to the well characterized mammalian systems, basic information on the composition, recruitment, and localization of fungal Nox complexes and on the molecular mechanisms of their cellular effects are still lacking. Here we give a detailed analysis of components of the Nox complexes in the gray mold fungus Botrytis cinerea. It had previously been shown that the two catalytic transmembrane subunits BcNoxA and B are important for development of sclerotia and for full virulence, with BcNoxA being involved in spreading of lesions and BcNoxB in penetration; BcNoxR functions as a regulator of both subunits. Here we present evidence (using for the first time a functional GFP fusion able to complement the ¿bcnoxA mutant) that BcNoxA localizes mainly to the ER and at the plasma membrane; BcNoxB shows a similar localization pattern, while the regulator BcNoxR is found in vesicles throughout the hyphae and at the hyphal tip. To identify possible interaction partners, which could be involved in the localization or recruitment of the Nox complexes, we functionally characterized the tetraspanin Pls1, a transmembrane protein, which had been suggested to be a NoxB-interacting partner in the saprophyte Podospora anserina. Knock-out experiments and GFP fusions substantiate a link between BcNoxB and BcPls1 because both deletion mutants have overlapping phenotypes (especially a defect in penetration), and the proteins show a similar localization pattern (ER). However, in contrast to the corresponding protein in P. anserina BcPls1 is important for female fertility, but not for ascospore germination.
Impact of Lactobacillus plantarum sortase on target-protein sorting, gastrointestinal persistence, and host immune response modulation
Remus, D.M. ; Bongers, R.S. ; Meijerink, M. ; Fusetti, F. ; Poolman, B. ; Vos, P. de; Wells, J. ; Kleerebezem, M. ; Bron, P.A. - \ 2013
Journal of Bacteriology 195 (2013)3. - ISSN 0021-9193 - p. 502 - 509.
surface-associated proteins - gene-expression omnibus - cell-wall - staphylococcus-aureus - srta gene - streptococcus-gordonii - listeria-monocytogenes - functional-analysis - lipoteichoic acid - binding-protein
Sortases are transpeptidases that couple surface proteins to the peptidoglycan of Gram-positive bacteria, and several sortase-dependent proteins (SDPs) have been demonstrated to be crucial for the interactions of pathogenic and nonpathogenic bacteria with their hosts. Here, we studied the role of sortase A (SrtA) in Lactobacillus plantarum WCFS1, a model Lactobacillus for probiotic organisms. An isogenic srtA deletion derivative was constructed which did not show residual SrtA activity. DNA microarray-based transcriptome analysis revealed that the srtA deletion had only minor impact on the full-genome transcriptome of L. plantarum, while the expression of SDP-encoding genes remained completely unaffected. Mass spectrometry analysis of the bacterial cell surface proteome, which was assessed by trypsinization of intact bacterial cells and by LiCl protein extraction, revealed that SrtA is required for the appropriate subcellular location of specific SDPs and for their covalent coupling to the cell envelope, respectively. We further found that SrtA deficiency did not affect the persistence and/or survival of L. plantarum in the gastrointestinal tract of mice. In addition, an in vitro immature dendritic cell (iDC) assay revealed that the removal of surface proteins by LiCl strongly affected the proinflammatory signaling properties of the SrtA-deficient strain but not of the wild type, which suggests a role of SDPs in host immune response modulation.
Optimized agroinfiltration and virus-induced gene silencing to study Ve1-mediated Verticillium resistance in tobacco
Zhang, Z. ; Fradin, E. ; Jonge, R. de; Esse, P. van; Smit, P. ; Liu, C.M. ; Thomma, B.P.H.J. - \ 2013
Molecular Plant-Microbe Interactions 26 (2013)2. - ISSN 0894-0282 - p. 182 - 190.
receptor-like proteins - transient expression system - mediated plant transformation - functional-analysis - disease resistance - albo-atrum - hypersensitive response - nicotiana-benthamiana - arabidopsis-thaliana - binary vectors
Recognition of pathogen effectors by plant immune receptors often leads to the activation of a hypersensitive response (HR), which is a rapid and localized cell death of plant tissue surrounding the site at which recognition occurs. Due to its particular amenability to transient assays for functional genetics, tobacco is a model for immune signaling in the Solanaceae plant family. Here, we show that coexpression of the tomato (Solanum lycopersicum) immune receptor Ve1 and the corresponding Verticillium effector protein Ave1 leads to HR only in particular tobacco species. Whereas HR is obtained in Nicotiana tabacum, no such response is obtained in N. benthamiana. Furthermore, our analysis revealed an endogenous Ve1 ortholog in Nicotiana glutinosa, as expression of Ave1 in absence of Ve1 induced a HR, and N. glutinosa was found to be resistant against race 1 Verticillium dahliae. We furthermore report the establishment of virus-induced gene silencing in N. tabacum for functional analysis of Ve1 signaling. Collectively, our data show that N. tabacum can be used as a model plant to study Ve1-mediated immune signaling.
Epigenetic Remodeling of Meiotic Crossover Frequency in Arabidopsis thaliana DNA Methyltransferase Mutants
Yelina, N.E. ; Choi, K. ; Chelysheva, L. ; Macaulay, M. ; Snoo, B. de; Wijnker, T.G. ; Miller, N. ; Drouaud, J. ; Grelon, M. ; Copenhaver, G.P. ; Mezard, C. ; Kelly, K.A. ; Henderson, I.R. - \ 2012
Plos Genetics 8 (2012)8. - ISSN 1553-7404
double-strand breaks - genome-wide analysis - yeast saccharomyces-cerevisiae - mouse recombination hotspots - h3 lysine 4 - crossing-over - synaptonemal complex - functional-analysis - chiasma formation - cpg methylation
Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes.
The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG
Lebeer, S. ; Claes, I.J. ; Balog, C.I. ; Schoofs, G. ; Verhoeven, T.L.A. ; Nys, K. ; Ossowski, I. von; Vos, W.M. de - \ 2012
Microbial Cell Factories 11 (2012)15. - ISSN 1475-2859
functional-analysis - glycoproteins - biosynthesis - molecules - bacteria - host - pathogens - cell
BACKGROUND: Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes. RESULTS: Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein. CONCLUSIONS: In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the serine residues at 106 and 107. Glycosylation is not required for the peptidoglycan hydrolase activity of Msp1 nor for Akt activation capacity in epithelial cells, but appears to be important for its stability and protection against proteases
Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG
Claes, I.J. ; Schoofs, G. ; Regulski, K. ; Vos, W.M. de - \ 2012
PLoS ONE 7 (2012)2. - ISSN 1932-6203
lactococcus-lactis - functional-analysis - peptidoglycan - reveals - growth - enzyme - bl23
Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG) by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG
A multicomponent sugar phosphate sensor system specifically induced in Bacillus cereus during infection of the insect gut
Song, F. ; Peng, Q. ; Brillard, J. ; Buisson, C. ; Been, M.W.H.J. de; Abee, T. ; Broussolle, V. ; Huang, D. ; Zhang, J. ; Lereclus, D. ; Nielsen-LeRoux, C. - \ 2012
FASEB Journal 26 (2012)8. - ISSN 0892-6638 - p. 3336 - 3350.
2-component signal-transduction - gram-positive bacteria - plcr virulence regulon - low-gc-content - escherichia-coli - in-vivo - functional-analysis - histidine kinases - genome sequence - gene-expression
Using a previously developed Bacillus cereus in vivo expression technology (IVET) promoter trap system, we showed that spsA, a gene of unknown function, was specifically expressed in the larval gut during infection. Search for gut-related compounds inducing spsA transcription identified glucose-6-phosphate (G6P) as an activation signal. Analysis of the spsA-related 5-gene cluster indicated that SpsA is part of a new sugar phosphate sensor system composed of a 2-component system (TCS) encoded by spsR and spsK, and 2 additional downstream genes, spsB and spsC. In B. cereus, American Type Culture Collection (ATCC) 14579, spsRK, and spsABC are separate transcriptional units, of which only spsABC was activated by extracellular G6P. lacZ transcriptional fusions tested in mutant and complemented strains showed that SpsRK, SpsA, and SpsB are essential for the transcription of spsABC. Deletion mutant analysis showed that SpsC is essential for the G6P uptake. gfp-transcriptional fusions showed that these genes are required for host-activated expression, as well. This sugar phosphate sensor and transport system is found in pathogenic Bacillus group and Clostridia bacteria and may be important for host adaptation. Our findings provide new insights into the function of 2-component sensor systems in host-pathogen interactions, specifically in the gut.—Song, F., Peng, Q., Brillard, J., Buisson, C., de Been, M., Abee, T., Broussolle, V., Huang, D., Zhang, J., Lereclus, D., Nielsen-LeRoux, C. A multicomponent sugar phosphate sensor system specifically induced in Bacillus cereus during infection of the insect gut.
StyA1 and StyA2B from Rhodococcus opacus 1CP: a Multifunctional Styrene Monooxygenase System
Tischler, D. ; Kermer, R. ; Groning, J.A.D. ; Kaschabek, S.R. ; Berkel, W.J.H. van; Schlomann, M. - \ 2010
Journal of Bacteriology 192 (2010)19. - ISSN 0021-9193 - p. 5220 - 5227.
whole-cell biocatalyst - (s)-styrene oxide - escherichia-coli - functional-analysis - strain vlb120 - flavin - degradation - regeneration - mechanism - cytochrome-p450
Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of FADH(2) provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when FADH(2) is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of FADH(2), resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH(2)-induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotechnological applications and is highly relevant from a structural and evolutionary point of view.
Identification of Genetic Loci in Lactobacillus plantarum That Modulate the Immune Response of Dendritic Cells Using Comparative Genome Hybridization
Meijerink, M. ; Hemert, S. van; Taverne, N. ; Wels, M.W.W. ; Bron, P.A. ; Vos, P. de; Savelkoul, H.F.J. ; Bilsen, J.G.P.M. van; Kleerebeezem, M. ; Wells, J. - \ 2010
PLoS ONE 5 (2010)5. - ISSN 1932-6203 - 12 p.
lactic-acid bacteria - necrosis-factor-alpha - regulatory t-cells - intestinal inflammation - functional-analysis - probiotic bacteria - lipoteichoic acid - rhamnosus gg - immunomodulatory properties - gastrointestinal-tract
Background - Probiotics can be used to stimulate or regulate epithelial and immune cells of the intestinal mucosa and generate beneficial mucosal immunomodulatory effects. Beneficial effects of specific strains of probiotics have been established in the treatment and prevention of various intestinal disorders, including allergic diseases and diarrhea. However, the precise molecular mechanisms and the strain-dependent factors involved are poorly understood. Methodology/Principal Findings - In this study, we aimed to identify gene loci in the model probiotic organism Lactobacillus plantarum WCFS1 that modulate the immune response of host dendritic cells. The amounts of IL-10 and IL-12 secreted by dendritic cells (DCs) after stimulation with 42 individual L. plantarum strains were measured and correlated with the strain-specific genomic composition using comparative genome hybridisation and the Random Forest algorithm. This in silico “gene-trait matching” approach led to the identification of eight candidate genes in the L. plantarum genome that might modulate the DC cytokine response to L. plantarum. Six of these genes were involved in bacteriocin production or secretion, one encoded a bile salt hydrolase and one encoded a transcription regulator of which the exact function is unknown. Subsequently, gene deletions mutants were constructed in L. plantarum WCFS1 and compared to the wild-type strain in DC stimulation assays. All three bacteriocin mutants as well as the transcription regulator (lp_2991) had the predicted effect on cytokine production confirming their immunomodulatory effect on the DC response to L. plantarum. Transcriptome analysis and qPCR data showed that transcript level of gtcA3, which is predicted to be involved in glycosylation of cell wall teichoic acids, was substantially increased in the lp_2991 deletion mutant (44 and 29 fold respectively). Conclusion - Comparative genome hybridization led to the identification of gene loci in L. plantarum WCFS1 that modulate the immune response of DCs
Flexible tools for gene expression and silencing in tomato
Fernandez, A.I. ; Viron, N. ; Alhagdow, M. ; Karimi, M. ; Jones, M. ; Amsellem, Z. ; Sicard, A. ; Czerednik, A. ; Angenent, G.C. ; Grierson, D. ; May, S. ; Seymour, G. ; Eshed, Y. ; Lemaire-Chamley, M. ; Rothan, C. ; Hilson, P. - \ 2009
Plant Physiology 151 (2009)4. - ISSN 0032-0889 - p. 1729 - 1740.
zinc-finger - artificial micrornas - functional-analysis - sequence tags - crabs-claw - fruit - arabidopsis - plants - polygalacturonase - transcription
As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources
Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples
Espley, R.V. ; Brendolise, C. ; Chagné, D. ; Kutty-Amma, S. ; Green, S. ; Volz, R. ; Putterill, J. ; Schouten, H.J. ; Gardiner, S.E. ; Hellens, R.P. ; Allan, A.C. - \ 2009
The Plant Cell 21 (2009). - ISSN 1040-4651 - p. 168 - 183.
common morning glory - genetic-linkage map - loop-helix domain - arabidopsis-thaliana - regulatory genes - functional-analysis - protein-binding - bhlh factors - human dna - myb
Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to color phenotypes. Generally, this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. Here, we describe a rearrangement in the upstream regulatory region of the gene encoding an apple (Malus x domestica) anthocyanin-regulating transcription factor, MYB10. We show that this modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. This rearrangement is a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23-bp sequence. This MYB10 rearrangement is present in all the red foliage apple varieties and species tested but in none of the white fleshed varieties. Transient assays demonstrated that the 23-bp sequence motif is a target of the MYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MYB10 protein. We show that the repeat motif is capable of binding MYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that an allelic rearrangement in the promoter of MYB10 has generated an autoregulatory locus, and this autoregulation is sufficient to account for the increase in MYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant
Quantitative phosphoproteomics of tomato mounting a hypersensitive response reveals a swift suppression of photosynthetic activity and a differential role for Hsp90 isoforms
Stulemeijer, I.J.E. ; Joosten, M.H.A.J. ; Jensen, O.N. - \ 2009
Journal of Proteome Research 8 (2009)3. - ISSN 1535-3893 - p. 1168 - 1182.
dependent protein-kinase - highly selective enrichment - plant-pathogen interactions - innate immune-responses - programmed cell-death - heat-shock-protein - cladosporium-fulvum - mass-spectrometry - plasma-membrane - functional-analysis
An important mechanism by which plants defend themselves against pathogens is the rapid execution of a hypersensitive response (HR). Tomato plants containing the Cf-4 resistance gene mount an HR that relies on the activation of phosphorylation cascades, when challenged with the Avr4 elicitor secreted by the pathogenic fungus Cladosporium fulvum. Phosphopeptides were isolated from tomato seedlings expressing both Cf-4 and Avr4 using titanium dioxide columns and LC-MS/MS analysis led to the identification of 50 phosphoproteins, most of which have not been described in tomato before. Phosphopeptides were quantified using a label-free approach based on the MS peak areas. We identified 12 phosphopeptides for which the abundance changed upon HR initiation, as compared to control seedlings. Our results suggest that photosynthetic activity is specifically suppressed in a phosphorylation-dependent way during the very early stages of HR development. In addition, phosphopeptides originating from four Hsp90 isoforms exhibited altered abundances in Cf-4/Avr4 seedlings compared to control seedlings, suggesting that the isoforms of this chaperone protein have a different function in defense signaling. We show that label-free relative quantification of the phosphoproteome of complex samples is feasible, allowing extension of our knowledge on the general physiology and defense signaling of plants mounting the HR.
Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1
Fradin, E.F. ; Zhang, Z. ; Juarez Ayala, J.C. ; Castroverde, C.C.M. ; Nazar, R.N. ; Robb, J. ; Liu, Chun-Ming ; Thomma, B.P.H.J. - \ 2009
Plant Physiology 150 (2009). - ISSN 0032-0889 - p. 320 - 332.
receptor-like proteins - plant-disease resistance - leucine-rich repeats - cladosporium-fulvum - hypersensitive response - fungal pathogen - functional-analysis - scab resistance - cell-death - defense
Vascular wilt diseases caused by soil-borne pathogens are among the most devastating plant diseases worldwide. The Verticillium genus includes vascular wilt pathogens with a wide host range. Although V. longisporum infects various hosts belonging to the Cruciferaceae, V. dahliae and V. albo-atrum cause vascular wilt diseases in over 200 dicotyledonous species, including economically important crops. A locus responsible for resistance against race 1 strains of V. dahliae and V. albo-atrum has been cloned from tomato (Solanum lycopersicum) only. This locus, known as Ve, comprises two closely linked inversely oriented genes, Ve1 and Ve2, that encode cell surface receptor proteins of the extracellular leucine-rich repeat receptor-like protein class of disease resistance proteins. Here, we show that Ve1, but not Ve2, provides resistance in tomato against race 1 strains of V. dahliae and V. albo-atrum and not against race 2 strains. Using virus-induced gene silencing in tomato, the signaling cascade downstream of Ve1 is shown to require both EDS1 and NDR1. In addition, NRC1, ACIF, MEK2, and SERK3/BAK1 also act as positive regulators of Ve1 in tomato. In conclusion, Ve1-mediated resistance signaling only partially overlaps with signaling mediated by Cf proteins, type members of the receptor-like protein class of resistance proteins.
Growth of Pseudomonas chloritidismutans AW-1(T) on n-alkanes with chlorate as electron acceptor
Mehboob, F. ; Junca, H. ; Schraa, G. ; Stams, A.J.M. - \ 2009
Applied Microbiology and Biotechnology 83 (2009)4. - ISSN 0175-7598 - p. 739 - 747.
sp strain m-1 - sulfate-reducing bacterium - functional-analysis - omega-hydroxylase - anoxic conditions - escherichia-coli - fatty-acids - genes - monooxygenase - oleovorans
Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1(T) grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1(T) also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 +/- 0.1 and 0.4 +/- 0.02 day(-1), respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes
NADPH oxidases are involved in differentiation and pathogenicity in Botrytis cinerea
Segmüller, N. ; Kokkelink, L. ; Giesbert, S. ; Odinius, D. ; Kan, J. van; Tudzynski, P. - \ 2008
Molecular Plant-Microbe Interactions 21 (2008)6. - ISSN 0894-0282 - p. 808 - 819.
respiratory burst oxidase - reactive oxygen - functional-analysis - oxidative burst - active oxygen - cell-growth - disease - localization - infection - virulence
Nicotinamide adenine dinucleotide (NADPH) oxidases have been shown to be involved in various differentiation processes in fungi. We investigated the role of two NADPH oxidases in the necrotrophic phytopathogenic fungus, Botrytis cinerea. The genes bcnoxA and bcnoxB were cloned and characterized; their deduced amino acid sequences show high homology to fungal NADPH oxidases. Analyses of single and double knock-out mutants of both NADPH oxidase genes showed that both bcnoxA and bcnoxB are involved in formation of sclerotia. Both genes have a great impact on pathogenicity: whereas bcnoxB mutants showed a retarded formation of primary lesions, probably due to an impaired formation of penetration structures, bcnoxA mutants were able to penetrate host tissue in the same way as the wild type but were much slower in colonizing the host tissue. Double mutants showed an additive effect: they were aberrant in penetration and colonization of plant tissue and, therefore, almost nonpathogenic. To study the structure of the fungal Nox complex in more detail, bcnoxR (encoding a homolog of the mammalian p67phox, a regulatory subunit of the Nox complex) was functionally characterized. The phenotype of ¿bcnoxR mutants is identical to that of ¿bcnoxAB double mutants, providing evidence that BcnoxR is involved in activation of both Bcnox enzymes.
RNA-mediated gene silencing of superoxide dismutase (bcsod1) in Botrytis cinerea
Patel, R.M. ; Kan, J.A.L. van; Bailey, A.M. ; Foster, G.D. - \ 2008
Phytopathology 98 (2008)12. - ISSN 0031-949X - p. 1334 - 1339.
fungicide resistance - functional-analysis - neurospora-crassa - expression - plants - transformation - cutinase - disease - fungus - tool
Gene silencing is a powerful tool utilized for identification of gene function and analysis in plants, animals, and fungi. Here, we report the silencing of superoxide dismutase (bcsod1) in Botrytis cinerea through sense and antisense-mediated silencing mechanisms. Because superoxide dismutase (SOD) is a virulence factor, transformants were tested for phenotypic silencing in vitro and reduction in pathogenicity in planta. Plate-based assays with and without paraquat were performed to screen initial silencing efficiency, and a subset of transformants was used for in planta studies of virulence. Transformants exhibiting strongly decreased transcripts levels were recovered with both constructs but none of those exhibited a reduction in virulence in planta. Our investigations may help optimize a high-throughput gene silencing system useful for identifying potential gene targets for future fungal control.
Efficient cloning system for construction of gene silencing vectors in Aspergillus niger
Oliveira, J.M. ; Veen, D. van der; Graaff, L.H. de; Qin Ling, - \ 2008
Applied Microbiology and Biotechnology 80 (2008)5. - ISSN 0175-7598 - p. 917 - 924.
transcriptional activator xlnr - double-stranded-rna - neurospora-crassa - caenorhabditis-elegans - functional-analysis - expression - interference - transformation - fungus - inactivation
An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two semi-quantitative plate assays testing different activities for xylan degradation. Upon induction on D-xylose, transcript levels of xlnR were decreased in the xlnR-silenced strains, compared to a wild-type background. Under these conditions, the transcript levels of xyrA and xynB (two genes regulated by XlnR) were also decreased for these xlnR-silenced strains. These results indicate that the newly developed system for rapid generation of silencing vectors is an effective tool for A. niger, and this can be used to generate strains with a tailored spectrum of enzyme activities or product formation by silencing specific genes encoding, e.g., regulators such as XlnR
Detailed Analysis of the Expression of an Alpha-gliadin Promoter and the Deposition of Alpha-gliadin Protein During Wheat Grain Development
Herpen, T.W.J.M. van; Riley, M. ; Sparks, C. ; Jones, H.D. ; Gritsch, C. ; Dekking, E.H. ; Hamer, R.J. ; Bosch, H.J. ; Salentijn, E.M.J. ; Smulders, M.J.M. ; Shewry, P.R. ; Gilissen, L.J.W.J. - \ 2008
Annals of Botany 102 (2008)3. - ISSN 0305-7364 - p. 331 - 342.
endosperm-specific expression - celiac-disease - activates transcription - transgenic wheat - gene-expression - functional-analysis - barley endosperm - seed development - gcn4-like motif - bzip protein
Background and Aims: Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (c¿liac) disease (CD). The B-genome-encoded -gliadin genes, however, contain very few epitopes. Controlling -gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of -gliadin protein during wheat grain development. Methods: A 592-bp fragment of the promotor of a B-genome-encoded -gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an -gliadin-specific antibody was used to detect -gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available -gliadin promoter sequences. Key Results: GUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The -gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An -gliadin-specific antibody detected -gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no -gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of -gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream. Conclusions: The results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the -gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how -gliadin expression can be controlled
Conserved leucines in N-terminal heptad repeat HR1 of envelope fusion protein F of group II nucleopolyhedroviruses are important for correct processing and essential for fusogenicity
Long, G. ; Pan, X. ; Vlak, J.M. - \ 2008
Journal of Virology 82 (2008)5. - ISSN 0022-538X - p. 2437 - 2447.
virus type-1 gp41 - viral membrane-fusion - coronavirus spike protein - crystal-structure - multicapsid nucleopolyhedrovirus - influenza hemagglutinin - mutational analysis - functional-analysis - structural basis - coiled coils
The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common features to class I fusion proteins, such as proteolytic cleavage and the presence of an N-terminal open fusion peptide and multiple HR domains on the transmembrane subunit F1. Similar to many vertebrate viral fusion proteins, a conserved leucine zipper motif is predicted in this HR region proximal to the fusion peptide in baculovirus F proteins. To facilitate our understanding of the functional role of this leucine zipper-like HR1 domain in baculovirus F protein synthesis, processing, and viral infectivity, key leucine residues (Leu209, Leu216, and Leu223) were replaced by alanine (A) or arginine (R), respectively. By using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a pseudotype expression system, we demonstrated that all mutant F proteins incorporated into budded virus, indicating that leucine substitutions did not affect intercellular trafficking of F. Furin-like protease cleavage was not affected by any of the leucine substitutions; however, the disulfide bridging and N-linked glycosylation patterns were partly altered. Single substitutions in HR1 showed that the three leucine residues were critical for F fusogenicity and the rescue of AcMNPV infectivity. Our results support the view that the leucine zipper-like HR1 domain is important to safeguard the proper folding, glycosylation, and fusogenicity of baculovirus F proteins.
The absence of histone H2B monoubiquitination in the Arabidopsis hub 1 (rdo4) mutant reveals a role for chromatin remodeling in seed dormancy
Liu, Y. ; Koornneef, M. ; Soppe, W.J.J. - \ 2007
The Plant Cell 19 (2007)2. - ISSN 1040-4651 - p. 433 - 444.
abscisic-acid - functional-analysis - gene-expression - h3 methylation - germination - thaliana - protein - aba - ubiquitination - yeast
Seed dormancy is defined as the failure of a viable seed to germinate under favorable conditions. Besides playing an adaptive role in nature by optimizing germination to the most suitable time, a tight control of dormancy is important in crop plants. Extensive genetic and physiological studies have identified the involvement of several factors, but the molecular mechanisms underlying this process are still largely unknown. Wecloned the HISTONE MONOUBIQUITINATION1 (HUB1) gene, of which the mutant ( previously identified as reduced dormancy4) has reduced seed dormancy and several pleiotropic phenotypes. HUB1 encodes a C3HC4 RING finger protein. The Arabidopsis thaliana genome contains one HUB1 homolog, which we named HUB2. The hub2 mutant also has reduced seed dormancy and is not redundant with hub1. Homologs of HUB1 and HUB2 in other species are required for histone H2B monoubiquitination. In agreement with this, the ubiquitinated form of histone H2B could not be detected in the hub1 and hub2 mutants. In yeast and human cells, histone H2B monoubiquitination is associated with actively transcribed genes. The hub1 mutant showed altered expression levels for several dormancy-related genes. We propose a role for chromatin remodeling in seed dormancy by H2B monoubiquitination through HUB1 and HUB2.
Dothideomycete-plant interactions illuminated by genome sequencing and EST analysis of the wheat pathogen Stagonospora nodorum
Hane, J.K. ; Lowe, R.G.T. ; Solomon, P.S. ; Tan, K.C. ; Schoch, C.L. ; Spatafora, J.W. ; Crous, P.W. ; Kodira, C. ; Birren, B.W. ; Galagan, J.E. ; Torriani, S.F.F. ; McDonald, B.A. ; Oliver, R.P. - \ 2007
The Plant Cell 19 (2007)11. - ISSN 1040-4651 - p. 3347 - 3368.
polyketide synthase genes - rice blast fungus - anamorph fusarium-graminearum - pyrenophora-tritici-repentis - peptide synthetase gene - complete dna-sequence - magnaporthe-grisea - leptosphaeria-maculans - mitochondrial genome - functional-analysis
Stagonospora nodorum is a major necrotrophic fungal pathogen of wheat (Triticum aestivum) and a member of the Dothideomycetes, a large fungal taxon that includes many important plant pathogens affecting all major crop plant families. Here, we report the acquisition and initial analysis of a draft genome sequence for this fungus. The assembly comprises 37,164,227 bp of nuclear DNA contained in 107 scaffolds. The circular mitochondrial genome comprises 49,761 bp encoding 46 genes, including four that are intron encoded. The nuclear genome assembly contains 26 classes of repetitive DNA, comprising 4.5% of the genome. Some of the repeats show evidence of repeat-induced point mutations consistent with a frequent sexual cycle. ESTs and gene prediction models support a minimum of 10,762 nuclear genes. Extensive orthology was found between the polyketide synthase family in S. nodorum and Cochliobolus heterostrophus, suggesting an ancient origin and conserved functions for these genes. A striking feature of the gene catalog was the large number of genes predicted to encode secreted proteins; the majority has no meaningful similarity to any other known genes. It is likely that genes for host-specific toxins, in addition to ToxA, will be found among this group. ESTs obtained from axenic mycelium grown on oleate (chosen to mimic early infection) and late-stage lesions sporulating on wheat leaves were obtained. Statistical analysis shows that transcripts encoding proteins involved in protein synthesis and in the production of extracellular proteases, cellulases, and xylanases predominate in the infection library. This suggests that the fungus is dependant on the degradation of wheat macromolecular constituents to provide the carbon skeletons and energy for the synthesis of proteins and other components destined for the developing pycnidiospores.
Botrytis cinerea: the cause of grey mould disease
Williamson, B. ; Tudzynski, B. ; Tudzynski, P. ; Kan, J.A.L. van - \ 2007
Molecular Plant Pathology 8 (2007)5. - ISSN 1464-6722 - p. 561 - 580.
fungus magnaporthe-grisea - polygalacturonase-inhibiting proteins - postharvest gray mold - in-field strains - botryotinia-fuckeliana - functional-analysis - map kinase - phaseolus-vulgaris - virulence factor - endopolygalacturonase genes
Introduction: Botrytis cinerea (teleomorph: Botryotinia fuckeliana) is an airborne plant pathogen with a necrotrophic lifestyle attacking over 200 crop hosts worldwide. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. It has become an important model for molecular study of necrotrophic fungi. Taxonomy: Kingdom: Fungi, phylum: Ascomycota, subphylum: Pezizomycotina, class: Leotiomycetes, order: Helotiales, family: Sclerotiniaceae, genus: Botryotinia. Host range and symptoms: Over 200 mainly dicotyledonous plant species, including important protein, oil, fibre and horticultural crops, are affected in temperate and subtropical regions. It can cause soft rotting of all aerial plant parts, and rotting of vegetables, fruits and flowers post-harvest to produce prolific grey conidiophores and (macro)conidia typical of the disease. Pathogenicity: B. cinerea produces a range of cell-wall-degrading enzymes, toxins and other low-molecular-weight compounds such as oxalic acid. New evidence suggests that the pathogen triggers the host to induce programmed cell death as an attack strategy. Resistance: There are few examples of robust genetic host resistance, but recent work has identified quantitative trait loci in tomato that offer new approaches for stable polygenic resistance in future.
The BRI1-associated kinase 1, BAK1, has a brassinolide-independent role in plant cell-death control
Kemmerling, B. ; Schwedt, A. ; Rodriguez, P. ; Mazzotta, S. ; Frank, M. ; Abu Qamar, S. ; Mengiste, T. ; Betsuyaku, S. ; Parker, J.E. ; Müssig, C. ; Thomma, B.P.H.J. ; Albrecht, C. ; Vries, S.C. de; Hirt, H. ; Nürnberger, T. - \ 2007
Current Biology 17 (2007)13. - ISSN 0960-9822 - p. 1116 - 1122.
receptor-like kinase - arabidopsis-thaliana - functional-analysis - plasma-membrane - protein - resistance - pathogen - gene - bri1 - brassinosteroids
Programmed cell death (PCD) is a common host response to microbial infection [1-3]. In plants, PCD is associated with immunity to biotrophic pathogens, but it can also promote disease upon infection by necrotrophic pathogens . Therefore, plant cell-suicide programs must be strictly controlled. Here we demonstrate that the Arabidopsis thaliana Brassinosteroid Insensitive 1 (BRI1)-associated receptor Kinase 1 (BAK1), which operates as a coreceptor of BRI1 in brassinolide (BL)-dependent plant development, also regulates the containment of microbial infection-induced cell death. BAK1-deficient plants develop spreading necrosis upon infection. This is accompanied by production of reactive oxygen intermediates and results in enhanced susceptibility to necrotrophic fungal pathogens. The exogenous application of BL rescues growth defects of bak1 mutants but fails to restore immunity to fungal infection. Moreover, BL-insensitive and -deficient mutants do not exhibit spreading necrosis or enhanced susceptibility to fungal infections. Together, these findings suggest that plant steroid-hormone signaling is dispensable for the containment of infection-induced PCD. We propose a novel, BL-independent function of BAK1 in plant cell-death control that is distinct from its BL-dependent role in plant development.
An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins
Gabriëls, S.H.E.J. ; Vossen, J.H. ; Ekengren, S.K. ; Ooijen, G. van; Abd-El-Haliem, A.M. ; Berg, G.C.M. van den; Rainey, D.Y. ; Martin, G.B. ; Takken, F.L.W. ; Wit, P.J.G.M. de; Joosten, M.H.A.J. - \ 2007
The Plant Journal 50 (2007)1. - ISSN 0960-7412 - p. 14 - 28.
plant-disease resistance - tobacco-mosaic-virus - cell-death - hypersensitive response - cladosporium-fulvum - functional-analysis - confers resistance - dna interactions - gene-products - arc domain
Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1D481V, which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein
Expression of plant flavour genes in Lactococcus lactis
Hernández, I. ; Molenaar, D. ; Beekwilder, M.J. ; Bouwmeester, H.J. ; Hylckama Vlieg, J.E.T. van - \ 2007
Applied and Environmental Microbiology 73 (2007)5. - ISSN 0099-2240 - p. 1544 - 1552.
alcohol acyl-transferase - escherichia-coli - acid bacteria - transfer-rna - heterologous expression - terpenoid metabolism - functional-analysis - molecular-cloning - dna microarrays - volatile esters
Lactic acid bacteria, such as Lactococcus lactis, are attractive hosts for the production of plant-bioactive compounds because of their food grade status, efficient expression, and metabolic engineering tools. Two genes from strawberry (Fragaria x ananassa), encoding an alcohol acyltransferase (SAAT) and a linalool/nerolidol synthase (FaNES), were cloned in L. lactis and actively expressed using the nisin-induced expression system. The specific activity of SAAT could be improved threefold (up to 564 pmol octyl acetate h¿1 mg protein¿1) by increasing the concentration of tRNA1Arg, which is a rare tRNA molecule in L. lactis. Fermentation tests with GM17 medium and milk with recombinant L. lactis strains expressing SAAT or FaNES resulted in the production of octyl acetate (1.9 µM) and linalool (85 nM) to levels above their odor thresholds in water. The results illustrate the potential of the application of L. lactis as a food grade expression platform for the recombinant production of proteins and bioactive compounds from plants
Characterization of oil palm MADS box genes in relation to the mantled flower abnormality
Syed Alwee, S. ; Linden, C.G. van der; Schoot, J. van der; Folter, S. de; Angenent, G.C. ; Cheah, S.C. ; Smulders, M.J.M. - \ 2006
Plant Cell, Tissue and Organ Culture: an international journal on in vitro culture of higher plants 85 (2006). - ISSN 0167-6857 - p. 331 - 344.
floral organ identity - temporally regulated expression - elaeis-guineensis - somaclonal variation - molecular characterization - functional-analysis - in-vitro - arabidopsis - maize - tissue
In vitro propagation of oil palm (Elaeis guineensis Jacq.) frequently induces a somaclonal variant called `mantled¿ abnormality, in which the stamens of both male and female flowers are transformed into carpels. This leads to a reduced yield or complete loss of the harvest of palm oil. The high frequency of the abnormality in independent lines and the high reversal rate suggest that it is due to an epigenetic change. The type of morphological changes suggest that it involves homeotic MADS box genes that regulate the identity of the flower whorls. We have isolated a number of MADS box genes from oil palm inflorescences by a MADS box-directed mRNA display approach. The isolated partial cDNAs included genes that were likely to function at the initial stages of flowering as well as genes that may function in determination of the inflorescence and the identity of the flower whorls. For four genes that were homologous to genes known to affect the reproductive parts of the flower, full length cDNAs were isolated. These were a B-type MADS box gene which may function in the determination of stamen formation, a C-type gene expected to be involved in stamen and carpel formation, and two putative SEP genes which act in concert with the A-, B- and C-type MADS box gene in determining flower whorl formation. The B-type gene EgMADS16 was functionally characterized as a PISTILLATA orthologue; it was able to complement an Arabidopsis thaliana pi mutant. Whether EgMADS16, or any of the other EgMADS genes, are functionally involved in the mantled condition remains to be established.
Licensed to kill: the lifestyle of a necrotrophic plant pathogen
Kan, J.A.L. van - \ 2006
Trends in Plant Science 11 (2006)5. - ISSN 1360-1385 - p. 247 - 253.
fungus botrytis-cinerea - agrobacterium-mediated transformation - programmed cell-death - sclerotinia-sclerotiorum - functional-analysis - phaseolus-vulgaris - endopolygalacturonase genes - arabidopsis-thaliana - magnaporthe-grisea - lipid-peroxidation
Necrotrophic plant pathogens have received an increasing amount of attention over the past decade. Initially considered to invade their hosts in a rather unsophisticated manner, necrotrophs are now known to use subtle mechanisms to subdue host plants. The gray mould pathogen Botrytis cinerea is one of the most comprehensively studied necrotrophic fungal plant pathogens. The genome sequences of two strains have been determined. Targeted mutagenesis studies are unraveling the roles played in the infection process by a variety of B. cinerea genes that are required for penetration, host cell killing, plant tissue decomposition or signaling. Our increasing understanding of the tools used by a necrotrophic fungal pathogen to invade plants will be instrumental to designing rational strategies for disease control
Engineering metabolic highways in Lactococci and other lactic acid bacteria
Vos, W.M. de; Hugenholtz, J. - \ 2004
Trends in Biotechnology 22 (2004)2. - ISSN 0167-7799 - p. 72 - 79.
controlled gene-expression - complete genome sequence - exopolysaccharide production - lactate-dehydrogenase - sugar catabolism - streptococcus-thermophilus - molecular characterization - functional-analysis - diacetyl production - zymomonas-mobilis
Lactic acid bacteria (LAB) are widely used in industrial food fermentations and are receiving increased attention for use as cell factories for the production of food and pharmaceutical products. Glycolytic conversion of sugars into lactic acid is the main metabolic highway in these Gram-positive bacteria and Lactococcus lactis has become the model organism because of its small genome, genetic accessibility and simple metabolism. Here we discuss the metabolic engineering of L. lactis and the value of metabolic models compared with other LAB, with a particular focus on the food-grade production of metabolites involved in flavour, texture and health.
Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells
Pijlman, G.P. ; Vermeesch, A.M.G. ; Vlak, J.M. - \ 2003
Journal of Invertebrate Pathology 84 (2003)3. - ISSN 0022-2011 - p. 214 - 219.
nuclear polyhedrosis-virus - exigua multicapsid nucleopolyhedrovirus - autographa-californica - spodoptera-exigua - functional-analysis - defective genomes - deletion mutants - serial passage - in-vivo - identification
Successive Viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did not show the accumulation of such concatemers. When introduced into SeUCR1 and ScIZD2109 cells, the non-hr ori concatemers generated in Se301 cells were maintained but did not increase. This suggests that the non-hr ori confers a strong selective advantage in Se301 cells, but not or to a lesser extent in the other cell lines. The cell line-specific accumulation of non-hr ori concatemers might be due to a higher intrinsic recombination frequency in Se301 cells and may reflect tissue related differences involving some host cell factor(s). Since non-hr ori concatemers in Se301 cells were more abundant in intracellular than in extracellular viral DNA preparations, episomal replication and the requirement of a minimal DNA size for packaging into micleocapsids is hypothesized. (C) 2003 Elsevier Inc. All rights reserved.
Increased exopolysaccharide production in Lactococcuc lactis due to increased levels of expression of the NIZO B40 eps gene cluster
Boels, I.C. ; Kranenburg, R. van; Kanning, M.W. ; Chong, B.F. ; Vos, W.M. de; Kleerebezem, M. - \ 2003
Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 5029 - 5031.
rheological properties - functional-analysis - sugar nucleotide - biosynthesis - glycolysis
Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented food products. This is the first report demonstrating that homologous overexpression of a complete eps gene cluster in Lactococcus lactis leads to increased EPS production levels. A ninefold-elevated EPS plasmid copy number led to an almost threefold increase in the eps expression level, resulting in an almost fourfold increase in the NIZO B40 EPS production level. It was previously reported that increased EPS precursor levels did not influence NIZO B40 EPS production levels. However, the present results indicate that the maximal NIZO B40 EPS production level is limited by the activity level of the expression products of the eps gene cluster rather than by the level of EPS precursors.
Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells
Pijlman, G.P. ; Schijndel, J. van; Vlak, J.M. - \ 2003
Journal of General Virology 84 (2003). - ISSN 0022-1317 - p. 2669 - 2678.
nuclear polyhedrosis-virus - non-hr origin - exigua multicapsid nucleopolyhedrovirus - bacterial artificial chromosome - dna-replication - in-vivo - functional-analysis - defective genomes - escherichia-coli - identification
Repeated baculovirus infections in cultured insect cells lead to the generation of defective interfering viruses (DIs), which accumulate at the expense of the intact helper virus and compromise heterologous protein expression. In particular, Autographa californica multicapsid nucleopolyhedovirus (AcMNPV) DIs are enriched in an origin of viral DNA replication (ori) not associated with the homologous regions (hrs). This non-hr ori is located within the coding sequence of the non-essential p94 gene. We investigated the effect of a deletion of the AcMNPV non-hr ori on the heterologous protein expression levels following serial passage in Sf21 insect cells. Using homologous ET recombination in E coli, deletions within the p94 gene were made in a bacterial artificial chromosome (BAC) containing the entire AcMNPV genome (bacmid). All bacmids were equipped with an expression cassette containing the green fluorescent protein gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). For the parental (intact) bacmid only, a strong accumulation of DIs with reiterated non-hr oris was observed. This was not observed for the mutants, indicating that removal of the non-hr ori enhanced the genetic stability of the viral genome upon passaging. However, for all passaged viruses it was found that the entire BAC vector including the expression cassette was spontaneously deleted from the viral genome, leading to a rapid decrease in GFP and CSFV-E2 production. The rationale for the (intrinsic) genetic instability of the BAC vector in insect cells and the implications with respect to large-scale production of proteins with bacmid-derived baculoviruses are discussed.
|Generation and analysis of a repeat DNA fragment of SeMNPV by serial undiluted passage in Se301 insect cells
Yang, K. ; Pijlman, G.P. ; Yu, M. ; Vlak, J.M. ; Pang, Y. - \ 2002
Acta Biochimica et Biophysica Sinica 34 (2002)5. - ISSN 1672-9145 - p. 608 - 614.
nuclear polyhedrosis-virus - exigua multicapsid nucleopolyhedrovirus - non-hr origin - spodoptera-exigua - in-vivo - functional-analysis - defective genomes - baculovirus dna - replication - identification
Two major clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from insect hosts have been previously identified. In order to gain insight into DNA replication of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV, Groups II), the essential cis-acting DNA segments were studied. A strain, named Se-4, was plaque-purified from SeMNPV isolate US1. PCR, ELT-PCR and REN showed that Se-4 was genetically relatively homogeneous and retained the full-length of a hypervariable region which is usually prone to deletion from SeMNPV genome. To study the stability of this isolate in vitro, Se-4 was serially passaged in the Se301 cell line up to 10 times without dilution. Intracellular viral DNA extracted from every passage was analyzed by REN. A novel 3.5 kb PstI fragment was observed in passage 7 and the relative intensities of the bands increased with subsequent passages. In passage 10, the molar ratio of the fragment was much higher than those of any other viral DNA fragments. This fragment was thus expected to contain an important cis-acting element for SeMNPV DNA replication. The fragment was cloned and sequenced and it was found that it overlapped 3 525 bp with the published SeMNPV genome sequence (GenBank AF169823), from 81 014 nt to 84 538 nt. The region contained the SeMNPV non-hr origin (ori) of DNA replication and some ORFs including partial vlf-1, partial p26, Se84 as well asSe83, Se85, Se86, which are unique to SeMNPV. As compared to Autographa californica MNPV (Groups I), which also generated hypermolar DNA fragments containing the non-hr ori by serial undiluted passage in IPLB-SF-21 cell culture, our results provided in vitro evidence that the non-hr ori may also play an important role in the viral infection cycle of Groups II NPVs
Metabolic engineering of lactic acid bacteria for the production of nutraceuticals
Hugenholtz, J. ; Sybesma, W. ; Groot, M.N. ; Wisselink, W. ; Ladero, V. ; Burgess, K. ; Sinderen, D. van; Piard, J.C. ; Eggink, G. ; Smid, E.J. ; Savoy, G. ; Sesma, F. ; Jansen, T. ; Hols, P. ; Kleerebezem, M. - \ 2002
Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 82 (2002)1-4. - ISSN 0003-6072 - p. 217 - 235.
controlled gene-expression - lactococcus-lactis - exopolysaccharide biosynthesis - streptococcus-thermophilus - lactobacillus-plantarum - glycosyltransferase genes - nucleotide-sequence - diacetyl production - functional-analysis - nmr-spectroscopy
Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed