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.