The D-galacturonic acid catabolic pathway in Botrytis cinerea
Zhang, L. ; Thiewes, H. ; Kan, J.A.L. van - \ 2011
Fungal Genetics and Biology 48 (2011)10. - ISSN 1087-1845 - p. 990 - 997.
mold hypocrea-jecorina - aspergillus-nidulans - filamentous fungi - identification - pathogenesis - expression - virulence - aldolase - mutants - enzymes
d-galacturonic acid is the most abundant component of pectin, one of the major polysaccharide constituents of plant cell walls. Galacturonic acid potentially is an important carbon source for microorganisms living on (decaying) plant material. A catabolic pathway was proposed in filamentous fungi, comprising three enzymatic steps, involving d-galacturonate reductase, l-galactonate dehydratase, and 2-keto-3-deoxy-l-galactonate aldolase. We describe the functional, biochemical and genetic characterization of the entire d-galacturonate-specific catabolic pathway in the plant pathogenic fungus Botrytis cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-l-galactonate aldolase gene (Bclga1). Their expression levels were highly induced in cultures containing GalA, pectate, or pectin as the sole carbon source. The four proteins were expressed in Escherichia coli and their enzymatic activity was characterized. Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on d-galacturonic acid, pectate, or pectin as the sole carbon source. In Aspergillus nidulans and A. niger, the first catabolic conversion only involves the Bcgar2 ortholog, while in Hypocrea jecorina, it only involves the Bcgar1 ortholog. In B. cinerea, however, BcGAR1 and BcGAR2 jointly contribute to the first step of the catabolic pathway, albeit to different extent. The virulence of all B. cinerea mutants in the d-galacturonic acid catabolic pathway on tomato leaves, apple fruit and bell peppers was unaltered.
Comparison of two GM maize varieties with a near-isogenic non-GM variety using transcriptomics, proteomics and metabolomics
Barros, E. ; Lezar, S. ; Anttonen, M.J. ; Dijk, J.P. van; Rohlig, R.M. ; Kok, E.J. ; Engel, K.H. - \ 2010
Plant Biotechnology Journal 8 (2010)4. - ISSN 1467-7644 - p. 436 - 451.
gene-expression - h-1-nmr spectroscopy - safety assessment - food - tool - nmr - hybridization - microarrays - mutants - plants
P>The aim of this study was to evaluate the use of four nontargeted analytical methodologies in the detection of unintended effects that could be derived during genetic manipulation of crops. Three profiling technologies were used to compare the transcriptome, proteome and metabolome of two transgenic maize lines with the respective control line. By comparing the profiles of the two transgenic lines grown in the same location over three growing seasons, we could determine the extent of environmental variation, while the comparison with the control maize line allowed the investigation of effects caused by a difference in genotype. The effect of growing conditions as an additional environmental effect was also evaluated by comparing the Bt-maize line with the control line from plants grown in three different locations in one growing season. The environment was shown to play an important effect in the protein, gene expression and metabolite levels of the maize samples tested where 5 proteins, 65 genes and 15 metabolites were found to be differentially expressed. A distinct separation between the three growing seasons was also found for all the samples grown in one location. Together, these environmental factors caused more variation in the different transcript/protein/metabolite profiles than the different genotypes.
Involvement of the mannose phosphotransferase system of Lactobacillus plantarum WCFS1 in peroxide stress tolerance
Stevens, M.J.A. ; Molenaar, D. ; Jong, A. de; Vos, W.M. de; Kleerebezem, M. - \ 2010
Applied and Environmental Microbiology 76 (2010)11. - ISSN 0099-2240 - p. 3748 - 3752.
lactic-acid bacteria - lactate utilization - oxygen - mutants - glucose
A Lactobacillus plantarum strain with a deletion in the gene rpoN, encoding the alternative sigma factor 54 (sigma(54)), displayed a 100-fold-higher sensitivity to peroxide than its parental strain. This feature could be due to sigma(54)-dependent regulation of genes involved in the peroxide stress response. However, transcriptome analyses of the wild type and the mutant strain during peroxide exposure did not support such a role for sigma(54). Subsequent experiments revealed that the impaired expression of the mannose phosphotransferase system (PTS) operon in the rpoN mutant caused the observed increased peroxide sensitivity
Functional study on GTP hydrolysis by the GTP-binding protein from Sulfolobus solfataricus, a member of the HflX family
Huang, B. ; Wu, H. ; Hao, N. ; Blombach, F. ; Oost, J. van der; Li, X. ; Zhang, X.C. ; Rao, Z. - \ 2010
Journal of Biochemistry 148 (2010)1. - ISSN 0021-924X - p. 103 - 113.
ras p21 - activation - mutants - gtpases - loop - substitutions - purification - resolution - software - complex
GTPase domains from members of the HflX protein family have their catalytic glutamine residue of the DxxGQ motif substituted by phenylalanine, while they are still able to hydrolyse GTP. This appears to challenge the traditional view of GTP hydrolysis mechanism of Ras-like GTPases. SsGBP from the hyperthermophilic archaeon Sulfolobus solfataricus provided the first crystal structure of the HflX family. Here, we report structure-based mutagenesis analyses on SsGBP. Six-point mutations were individually introduced in the Ras-like GTPase domain including regions of P-loop, switches I and II. Intrinsic GTPase activities and thermal stabilities of these variants together with the wild-type full-length SsGBP and its isolated GTPase domain were analysed. Both functional and structural analyses of G235P and G235S mutants, which showed total and partial loss of the GTP hydrolyzing activity, respectively, support our hypothesis that the role of aligning a nucleophilic water molecule by the Ras Gln60 residue is replaced by the backbone amide group of Gly235 in SsGBP. Together with functional studies of other mutants, we conclude that the classical view of GTP hydrolysis mechanism likely remains the same in the HflX family with a twist in the entity of the nucleophilic alignment
A nodule-specific protein secretory pathway required for nitrogen-fixing symbiosis
Wang, D. ; Griffitts, J. ; Starker, C. ; Fedorova, E. ; Limpens, E.H.M. ; Ivanov, S.E. ; Bisseling, T. ; Long, S. - \ 2010
Science 327 (2010)5969. - ISSN 0036-8075 - p. 1126 - 1129.
signal peptidase activity - medicago-truncatula - root-nodules - endoplasmic-reticulum - gene-expression - membrane - fixation - mutants - define - plant
The nitrogen-fixing symbiosis between Sinorhizobium meliloti and its leguminous host plant Medicago truncatula occurs in a specialized root organ called the nodule. Bacteria that are released into plant cells are surrounded by a unique plant membrane compartment termed a symbiosome. We found that in the symbiosis-defective dnf1 mutant of M. truncatula, bacteroid and symbiosome development are blocked. We identified the DNF1 gene as encoding a subunit of a signal peptidase complex that is highly expressed in nodules. By analyzing data from whole-genome expression analysis, we propose that correct symbiosome development in M. truncatula requires the orderly secretion of protein constituents through coordinated up-regulation of a nodule-specific pathway exemplified by DNF1
Jubileum voor de zandraket
Sikkema, A. - \ 2010
Resource: weekblad voor Wageningen UR 4 (2010)8. - ISSN 1874-3625 - p. 18 - 20.
arabidopsis thaliana - mutanten - genomen - genetische bronnen - genetische modellen - plantenfysiologie - wetenschappelijk onderzoek - arabidopsis thaliana - mutants - genomes - genetic resources - genetic models - plant physiology - scientific research
Je kunt ’m in Nederland op elke straathoek tegenkomen tussen de trottoirtegels: de zandraket of Arabidopsis thaliana. Dit jaar viert dit ‘onkruid’ zijn zilveren jubileum als modelplant van de plantenwetenschappers. Onderzoek aan dit plantje heeft geleid tot detailkennis van vrijwel alle moleculaire processen in planten.
Germinator: A software package for high-throughput scoring and curve fitting of Arabidopsis seed germination
Joosen, R.V.L. ; Kodde, J. ; Willems, L.A.J. ; Ligterink, W. ; Plas, L.H.W. van der; Hilhorst, H.W.M. - \ 2010
The Plant Journal 62 (2010)1. - ISSN 0960-7412 - p. 148 - 159.
quantitative trait loci - inbred line population - image-analysis - thaliana - tolerance - dormancy - mutants - stress - biosynthesis - expression
Over the past few decades seed physiology research has contributed to many important scientific discoveries and has provided valuable tools for the production of high quality seeds. An important instrument for this type of research is the accurate quantification of germination; however gathering cumulative germination data is a very laborious task which is often prohibitive to the execution of large experiments. In this paper we present the Germinator package: a simple, highly cost efficient and flexible procedure for high-throughput automatic scoring and evaluation of germination that can be implemented without the use of complex robotics. The Germinator package contains three modules; 1) design of experimental setup with various options to replicate and randomize samples; 2) automatic scoring of germination based on the color contrast between the protruding radicle and seed coat on a single image; 3) curve fitting of cumulative germination data and the extraction, recap and visualization of the various germination parameters. The curve fitting module enables analysis of general cumulative germination data and can be used for all plant species. We show that the automatic scoring system works for Arabidopsis thaliana and Brassica spp. seeds, but is likely to be applicable to other species, as well. In this paper we show the accuracy, reproducibility and flexibility of the Germinator package. We have successfully applied it to evaluate natural variation for salt tolerance in a large population of Recombinant Inbred Lines (RIL) and were able to identify several QTL for salt tolerance. Germinator is a low-cost package that allows the monitoring of several thousands of germination tests, several times a day by a single person.
Genome-wide screen for Listeria monocytogenes genes important for growth at high temperatures
Veen, S. van der; Abee, T. ; Vos, W.M. de; Wells-Bennik, M.H.J. - \ 2009
FEMS Microbiology Letters 295 (2009)2. - ISSN 0378-1097 - p. 195 - 203.
staphylococcus-aureus - bacillus-subtilis - stress tolerance - escherichia-coli - cell-division - heat-shock - expression - virulence - identification - mutants
Listeria monocytogenes is a Gram-positive food-borne pathogen that is able to grow over a wide temperature range. Although the class I and class III heat-shock genes are known to play an important role in heat shock, information on genes that are essential for growth at high temperatures is scarce. To determine which genes are important for growth at high temperatures (42.5-43 degrees C), we performed a random insertion screening in L. monocytogenes, rendering 28 temperature-sensitive mutants. These mutants showed insertions in genes that play a role in transcription regulation, cell-wall biosynthesis, cell division, translation, transport, sensing, and specific stress responses like the SOS response and the class III heat-shock response. Some of these mutants showed altered morphological characteristics such as cell elongation, reduced cell length, or sickle shapes. Furthermore, the majority of the mutants showed increased heat inactivation after exposure to 55 degrees C compared with the wild-type strain. The role of the specific genes in relation to growth at high temperatures is discussed
Genome-wide investigation into roles of Arabidosis receptor-like proteins in pathogen defense
Ellendorff, U. - \ 2009
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Bart Thomma. - [S.l. : S.n. - ISBN 9789085853206 - 141
planten - arabidopsis thaliana - verdedigingsmechanismen - plantenziekteverwekkers - planteiwitten - pathogenesis-gerelateerde eiwitten - genoomanalyse - mutanten - genexpressie - plant-microbe interacties - gene silencing - plants - arabidopsis thaliana - defence mechanisms - plant pathogens - plant proteins - pathogenesis-related proteins - genome analysis - mutants - gene expression - plant-microbe interactions - gene silencing
Receptor-like proteins (RLPs) are receptors on the surface of plant cells that are important for the activation of disease resistance. Furthermore, some RLPs are important for plant development. The Arabidopsis genome contains 57 genes encoding RLPs. A genome wide collection of RLP gene knock-out mutants was assembled and functionally analyzed for defects in defense and development. This resulted in the identification of an RLP that plays a role in hormone perception, and two RLPs that play a role in non-host resistance, the phenomenon that a plant species is typically resistant to pathogens of other plant species.
RNA silencing is a regulatory mechanism by which the expression of genes is downregulated or entirely suppressed. In this thesis, it is demonstrated for the first time that this mechanism is important for defense of Arabidopsis against a fungal pathogen; the vascular wilt fungus Verticillium. This is an extremely important pathogen of over 200 plant species including economically important crops.
|Corina, een vroege mutant van Conference
Meijer, H. ; Dieren, M.C.A. van - \ 2009
De Fruitteelt 99 (2009)4. - ISSN 0016-2302 - p. 13 - 13.
fruitteelt - peren - rassen (planten) - mutanten - fruit growing - pears - varieties - mutants
Corina, rasnaam Saels, is een stabiele, vroege mutant van het perenras Conference en werd gevonden door de gebroeders Saels in België. Samen met de Belgische boomkwekerij René Nicolaï hebben zij een concept voor marktintroductie en afzet ontwikkeld
Genes involved in carotene synthesis and mating in Blakeslea trispora
Kuzina, V. ; Ramirez-Medina, H. ; Visser, H. ; Ooyen, A.J.J. van; Cerda-Olmedo, E. ; Berg, J.A. van den - \ 2008
Current Genetics 54 (2008)3. - ISSN 0172-8083 - p. 143 - 152.
beta-carotene - cdna-aflp - saccharomyces-cerevisiae - expression data - biosynthesis - phycomyces - light - mucorales - lycopene - mutants
Mating of Blakeslea trispora and other molds of the order Mucorales requires the interaction of mycelia of opposite sex, (+) and (-), leading to the development of specialized structures and to an enhanced accumulation of beta-carotene. Industry obtains beta-carotene by co-cultivating appropriate strains of Blakeslea (mated cultures). Gene transcription in single and mated cultures was assayed by cDNA-AFLP, a technique to observe the differential expression of subsets of mRNA fragments. Overexpression in mated cultures is about ten times more frequent than underexpression. We obtained and sequenced fragments of 97 candidate genes that appeared to be overexpressed during mating and confirmed four of them by reverse transcription and real-time PCR. Comparisons with gene sequences from other organisms suggest functions in carotene biosynthesis (4 genes), energy metabolism (8), cell wall synthesis (1), transfer of acetyl groups (1), and regulatory processes (10). Sodium acetate inhibited sexual overexpression in about two-thirds of the candidate genes and acted as a signal with broad effects on the metabolism and the morphology of mated cultures. Our work offers new materials for the study of carotene biosynthesis and its regulation and for the improvement of carotene production with Mucorales.
Development of a quantitative real-time PCR for determination of genotype frequencies for studies in baculovirus population biology
Zwart, M.P. ; Oers, M.M. van; Cory, J.S. ; Lent, J.W.M. van; Werf, W. van der; Vlak, J.M. - \ 2008
Journal of Virological Methods 148 (2008)1-2. - ISSN 0166-0934 - p. 146 - 154.
nuclear polyhedrosis-virus - polymerase-chain-reaction - trichoplusia-ni - sybr-green - mosaic-virus - wild-type - rt-pcr - nucleopolyhedrovirus - mutants - larvae
Two bacmid-derived Autographa californica Multiple-capsid Nucleopolyhedrovirus genotypes ¿ that differ only in a short tag sequence for differential PCR recognition ¿ were generated. By electron microscopy, these genotypes were found to have identical polyhedra morphology. Mixtures of quantified polyhedra were made and used to validate a SYBR Green I-based quantitative real-time PCR (qPCR) to determine genotype frequencies in mixed genotype populations. The PCR could accurately quantify genotype ratios over a range of 8 orders of magnitude. Only a small correction of the genotype ratio was necessary to obtain a valid result. Low levels of aspecific background (a fluorescent signal when the template corresponding with the primer set used is not present) were measured in these validation experiments and in a typical laboratory setup. A small fitness difference between the genotypes generated was observed in a median lethal dose bioassay. The bacmid-derived virus genotypes generated and the qPCR assays are valuable tools for studying the population biology of baculoviruses.
Niet-waardplant resistentie tegen Phytophthora infestans in modelplant Arabidopsis thaliana
Vossen, E.A.G. van der; Arkel, G. van - \ 2008
phytophthora infestans - arabidopsis - mutanten - arabidopsis thaliana - genetische modificatie - plaagresistentie - vatbaarheid - phytophthora infestans - arabidopsis - mutants - arabidopsis thaliana - genetic engineering - pest resistance - susceptibility
In een verzameling van ca. 5.000 Arabidopsis deletielijnen wordt gezocht naar mutanten die een zeldzame vatbaarheid vertonen voor P. infestans
Experimental ecology and evolution of microbial diversity : the role of spatial structure
Habets, M.G.J.L. - \ 2008
Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Arjan de Visser. - S.l. : s.n. - ISBN 9789085048619 - 102
micro-organismen - diversiteit - biodiversiteit - evolutie - ecologie - adaptatie - heterogeniteit - mutanten - microbiële diversiteit - microorganisms - diversity - biodiversity - evolution - ecology - adaptation - heterogeneity - mutants - microbial diversity
In the light of the competitive exclusion principle, which states that complete competitors cannot coexist, many explanations have been sought to explain the high diversity found in nature. The most common explanation is the niche differentiation hypothesis: coexistence is obtained through differentiation of species in ecological niches. Spatial structure is thought to be a factor capable of providing opportunities for niche differentiation. We have focused on four aspects of spatial structure enabling genetic diversity to emerge and /or to be maintained.
First of all, population fragmentation, resulting from growth in spatially structured habitats, can increase diversity, because the resulting smaller subpopulations, due to their smaller population size, are more likely to adaptively diverge. By allowing small and large populations of E. coli to evolve for 500 generations in two different nutrient environments, we test this hypothesis. The results demonstrate higher variance in fitness among small populations, and consequently more heterogeneous adaptive trajectories for small populations, some of which surprisingly lead to higher fitness peaks than reached by even the best adapted large population.
In a short-term invasion experiment between a superior E. coli competitor and its inferior ancestor, we demonstrate that populations residing in structured environments experience slower invasion dynamics of beneficial mutations than well-mixed populations due to limited dispersal, and therefore local competition. Moreover, our results demonstrate a deceleration of invasion with increasing size of the invading subpopulation. This is caused by a decrease of inter specific competition relative to intra specific competition. Since inferior competitors are present in the community for a longer period of time, they can recombine with other persisting lineages or obtain new mutations, some of which might be beneficial. It is therefore possible that polymorphisms arise which would not have had the opportunity to emerge in a well-mixed environment. Even though both population fragmentation and slower competitive dynamics can increase the emergence of diversity, they do not provide a means for their maintenance.
Environmental heterogeneity on the other hand can cause maintenance of diversity. Environmental heterogeneity can be introduced by spatial structure, e.g. by providing gradients in biotic and abiotic factors, thereby increasing the number of niches. By allowing E. coli populations to evolve for 900 generations in either a well-mixed environment or two structured environments (with or without dispersal), we demonstrate stable coexistence of diversity in structured populations without dispersal. This can be attributed to negative frequency-dependent fitness interactions among niche specialists that either inhabit existing niches provided by the heterogeneous environment or new niches constructed by organisms inhabiting the environment.
In addition to examining aspects of spatial structure that provide means for populations to diversify, we examine a specific consequence of slower dynamics and environmental heterogeneity: the probability of mutators to hitchhike to fixation. Understanding the emergence of mutators is not only scientifically important, but also relevant for human health, since high frequencies of mutators have been found in bacterial populations and drug resistant mutants arise more often in mutator populations. E. coli mutator populations were introduced at different starting frequencies in a well-mixed environment and two structured environments differing in their dispersal rate. Contrary to expectations, we find an advantage in the rate of invasion for mutators in well-mixed environments. Faster competitive dynamics may allow a rapid increase of population size and hence a greater supply of mutations for subsequent adaptation. Due to a delay in mutator extinction in structured environments at low frequencies, mutators may gain from fluctuating conditions.
Global analysis of gene expression in flower buds of Ms-cd1 Brassica oleracea conferring male sterility by using an Arabidopsis microarray
Kang, Jungen ; Zhang, Guoyu ; Bonnema, A.B. ; Fang, Zhiyuan ; Wang, Xiaowu - \ 2008
Plant Molecular Biology 66 (2008)1-2. - ISSN 0167-4412 - p. 177 - 192.
male gametophyte development - lathyrus-odoratus l - pollen-tube growth - mother cell-wall - pectin methylesterase - anther development - thaliana - identification - mutants - transcriptome
The dominant male sterility gene Ms-cd1 is identified in Brassica oleracea. Electron microscopical observations revealed that abortion of pollen development starts after tetrad formation. This important male sterility phenotype is characterized by lack of degradation of the primary pollen mother cell (PMC) wall and delayed degradation of callose surrounding the tetrads and thus arrest of microspore release. Gene expression of the male sterile and fertile buds was analyzed by heterologous hybridization of Brassica oleracea cRNA onto an Arabidopsis whole genome oligonucleotide microarray. A total of 277 suppressed genes including 40 kinase-, 32 cell wall modification and 29 transport related genes were found to be significantly down regulated >3-fold in the male sterile mutant. The vast majority of the differentially expressed transcripts are found to present late pollen stage specific genes. Kinase genes, cell wall modification genes and ion transport genes were greatly over-represented when compared to their percentage of all flower bud expressed genes and represent 36.5% of the genes suppressed by Ms-cd1. Our results also suggest that Ms-cd1 may blocks an anther developmental pathway with a small number of genes suppressed in tapetum cells which prevent the degradation of callose and PMC wall, which further leads to the suppression of a large number of genes involved in signaling pathways, cell wall modification and ion transport in pollen grains.
Vossen, E.A.G. van der; Loonen, A.E.H.M. - \ 2007
solanum - phytophthora - ziekteresistentie - mutanten - genexpressie - solanum - phytophthora - disease resistance - mutants - gene expression
In dit project wordt onderzocht in hoeverre R-genen in de plant verschillende werkingsmechanismen hebben
Tagging target genes of the mat1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A)
Keszthelyi, A. ; Jeney, A. ; Kerenyi, Z. ; Mendes, O. ; Waalwijk, C. ; Hornok, L. - \ 2007
Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 91 (2007)4. - ISSN 0003-6072 - p. 373 - 391.
pheromone precursor genes - mating-type gene - neurospora-crassa - sordaria-macrospora - sexual development - species complex - regulated genes - mutants - zeae - complementation
Mating type in filamentous ascomycetes is controlled by idiomorphic alleles, named MAT1-1 and MAT1-2, which contain 1-3 genes. Of these genes MAT1-1-1 and MAT1-2-1 encode putative transcription factors and are thus considered to be the major regulators of sexual communication and mating. Fungi with no known sexual stage may also have fully functional mating type genes and therefore it was plausible to hypothesize that the MAT products may also regulate other types of genes not involved directly in the mating process. To identify putative target genes of these transcription factors in Fusarium verticillioides, ¿MAT1-2-1 knock out mutants were produced and transcript profiles of mutant and wild type were compared by means of differential cDNA hybridization. Clones, either up- or down-regulated in the ¿MAT1-2-1 mutant were sequenced and a total of 248 sequences were blasted against the NCBI database as well as the Gibberella zeae and Gibberella moniliformis genomes. Fifty-five percent of the clones were down-regulated in the mutant, indicating that the MAT1-2-1 product positively affected these tagged sequences. On the other hand, 45% were found to be up-regulated in the mutant, suggesting that the MAT1-2-1 product also exerted a negative regulatory function on this set of genes. Sequences involved in protein synthesis and metabolism occurred more frequently among the clones up-regulated in the mutant, whereas genes belonging to cell signalling and communication were especially frequently tagged among the sequences down-regulated in the mutant
Medicago LYK3, an entry receptor in rhizobial nodulation factor signaling
Smit, P. ; Limpens, E.H.M. ; Geurts, R. ; Fedorova, E. ; Dolgikh, E. ; Gough, C. ; Bisseling, T. - \ 2007
Plant Physiology 145 (2007). - ISSN 0032-0889 - p. 183 - 191.
protein-kinase - lotus-japonicus - calcium spiking - truncatula - mutants - gene - domain - perception - infection - responses
Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.
An evolvable oestrogen receptor activity sensor: development of a modular system for integrating multiple genes into the yeast genome
Fox, J.E. ; Bridgham, J.T. ; Bovee, T.F.H. ; Thornton, J.W. - \ 2007
Yeast 24 (2007)5. - ISSN 0749-503X - p. 379 - 390.
green fluorescent protein - saccharomyces-cerevisiae - antiestrogenic activities - directed evolution - in-vitro - populations - adaptation - efficient - mutants - assay
To study a gene interaction network, we developed a gene-targeting strategy that allows efficient and stable genomic integration of multiple genetic constructs at distinct target loci in the yeast genome. This gene-targeting strategy uses a modular plasmid with a recyclable selectable marker and a multiple cloning site into which the gene of interest is cloned, flanked by two long regions of homology to the target genomic locus that are generated using adaptamer primers. We used this strategy to integrate into a single yeast strain components of the oestrogen receptor (ER) signalling network, comprising the human ER and three reporter genes driven by oestrogen response elements (EREs). The engineered strain contains multiple reporters of ligand-dependent receptor signalling, providing sensitive, reproducible, rapid, low-cost quantitative assays of ER activity in order to screen potential receptor agonists. Further, because two of the ERE-driven reporter genes are required for growth in deficient media, the strain's growth rate - and therefore its fitness - depends on ligand-induced ER activity. This evolvable oestrogen receptor activity sensor (EERAS) can therefore provide the foundation of a long-term experimental evolution strategy to elucidate ER structure-function relations and ligand-receptor evolution.
Control of FWA gene silencing in Arabidopsis thaliana by SINE-related direct repeats
Kinoshita, Y. ; Saze, H. ; Kinoshita, T. ; Miura, A. ; Soppe, W.J. ; Koornneef, M. ; Kakutani, T. - \ 2007
The Plant Journal 49 (2007)1. - ISSN 0960-7412 - p. 38 - 45.
novo dna methylation - medea polycomb gene - epigenetic control - cytosine methylation - tandem repeats - small rnas - transposons - maintenance - mutants - locus
A unique feature of late-flowering fwa epigenetic mutations is that the phenotype is caused by ectopic expression of the homeobox gene FWA. During normal development the FWA gene is expressed specifically in the endosperm in an imprinted manner. Ectopic FWA expression and disruption of imprinting can be induced in mutants of a CG methyltransferase MET1 (methyltransferase 1) or a chromatin-remodeling gene DDM1 (decrease in DNA methylation 1), suggesting that the proper FWA expression depends on cytosine methylation. However, critical methylated residues controlling FWA silencing are not pinpointed. Nor is it understood how the FWA gene is initially methylated and silenced in wild-type plants. Here we mapped sequences critical for FWA silencing by application of RdDM (RNA-directed DNA methylation) to a ddm1-induced stable fwa epiallele. Transcription of double-stranded RNA corresponding to the tandem direct repeats around the FWA transcription start site induced de novo DNA methylation, transcriptional suppression and phenotypic reversion. The induced changes were heritable even without the transgene, which correlates with inheritance of CG methylation in the direct repeats. The newly silenced FWA allele was transcribed in an endosperm-specific and imprinted manner, as is the case for the wild-type FWA gene. The results indicate that methylation of the direct repeats, which presumably originated from a short interspersed nuclear element (SINE), is sufficient to induce proper epigenetic control of the FWA gene