Dynamics of adaptation in experimental yeast populations exposed to gradual and abrupt change in heavy metal concentration
Gorter, Florien ; Aarts, Mark ; Zwaan, B.J. ; Visser, J.A.G.M. de - \ 2015
adaptation - ecology - evolutionary - microbial - Environmental variability - evolution - fitness - genetics - population - dynamics - fungi - heavy metals - pleiotropy
Directional environmental change is a ubiquitous phenomenon that may have profound effects on all living organisms. However, it is unclear how different rates of such change affect the dynamics and outcome of evolution. We studied this question using experimental evolution of heavy metal tolerance in the baker´s yeast Saccharomyces cerevisiae. To this end, we grew replicate lines of yeast for 500 generations in the presence of (i) a constant high concentration of cadmium, nickel or zinc, or (ii) a gradually increasing concentration of these metals. We found that gradual environmental change leads to a delay in fitness increase compared to abrupt change, but not necessarily to a different fitness of evolutionary endpoints. For the non-essential metal cadmium this delay is due to reduced fitness differences between genotypes at low metal concentrations, consistent with directional selection to minimize intracellular concentrations of this metal. In contrast, for the essential metals nickel and zinc different genotypes are selected at different concentrations, consistent with stabilizing selection to maintain constant intracellular concentrations of these metals. These findings indicate diverse fitness consequences of evolved tolerance mechanisms for essential and non-essential metals, and imply that the rate of environmental change and the nature of the stressor are crucial determinants of evolutionary dynamics.
Environmental influence on the genetic correlations between life-history traits in Caenorhabditis elegans
Gutteling, E.W. ; Doroszuk, A. ; Riksen, J.A.G. ; Prokop, Z. ; Reszka, J. ; Kammenga, J.E. - \ 2007
Heredity 98 (2007)4. - ISSN 0018-067X - p. 206 - 213.
phenotypic plasticity - offspring size - trade-offs - egg-size - arabidopsis-thaliana - seed beetle - c-elegans - wild-type - loci - pleiotropy
Empirical evidence is mounting to suggesting that genetic correlations between life-history traits are environment specific. However, detailed knowledge about the loci underlying genetic correlations in different environments is scant. Here, we studied the influence of temperature (12°C and 24°C) on the genetic correlations between egg size, egg number and body mass in the nematode Caenorhabditis elegans. We used a quantitative trait loci (QTL) approach based on a genetic map with evenly spaced single nucleotide polymorphism markers in an N2 CB4856 recombinant inbred panel. Significant genetic correlations between various traits were found at both temperatures. We detected pleiotropic or closely linked QTL, which supported the negative correlation between egg size and egg number at 12°C, the positive correlation across temperatures for body mass, and the positive correlation between body mass and egg size at 12°C. The results indicate that specific loci control the covariation in these life-history traits and the locus control is prone to environmental conditions
Mapping phenotypic plasticity and genotype-environment interactions affecting life-history traits in Caenorhabditis elegans
Gutteling, E.W. ; Riksen, J.A.G. ; Bakker, J. ; Kammenga, J.E. - \ 2007
Heredity 98 (2007)1. - ISSN 0018-067X - p. 28 - 37.
arabidopsis-thaliana - drosophila-melanogaster - natural environments - genetic correlation - loci - pleiotropy - selection - fitness - span - polyphenism
Phenotypic plasticity and genotype-environment interactions (GEI) play an important role in the evolution of life histories. Knowledge of the molecular genetic basis of plasticity and GEI provides insight into the underlying mechanisms of life-history changes in different environments. We used a genomewide single-nucleotide polymorphism map in a recombinant N2 × CB4856 inbred panel of the nematode Caenorhabditis elegans to study the genetic control of phenotypic plasticity to temperature in four fitness-related traits, that is, age at maturity, fertility, egg size and growth rate. We mapped quantitative trait loci (QTL) for the respective traits at 12 and 24°C, as well as their plasticities. We found genetic variation and GEI for age at maturity, fertility, egg size and growth rate. GEI in fertility and egg size was attributed to changes in rank order of reaction norms. In case of age at maturity and growth rate, GEI was caused mainly by differences in the among-line variance. In total, 11 QTLs were detected, five QTL at 12°C and six QTL at 24°C, which were associated with life-history traits. Five QTL associated with age at maturity, fertility and growth rate showed QTL × environment interaction. These colocalized with plasticity QTL for the respective traits suggesting allelic sensitivity to temperature. Further fine mapping, complementation analyses and gene silencing are planned to identify candidate genes underlying phenotypic plasticity for age at maturity, fertility and growth
Genomic variation in dairy cattle - Identification and use
Schrooten, C. - \ 2004
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Henk Bovenhuis. - [S.l.] : S.n. - ISBN 9789085040095
melkvee - genomen - genetische variatie - kwantitatieve kenmerken - loci - melkproductie - bouw (dier) - pleiotropie - genetische verbetering - genetische merkers - dierveredeling - moleculaire genetica - dairy cattle - genomes - genetic variation - quantitative traits - loci - milk production - conformation - pleiotropy - genetic improvement - genetic markers - animal breeding - molecular genetics
The development of molecular techniques has offered possibilities to identify quantitative trait loci (QTL). Studies in dairy cattle have mainly focused on milk production traits. This thesis first gives an overview of the main identified QTL for milk production traits. Subsequently, a study to detect QTL affecting 27 conformation traits and functional traits was performed. A granddaughter design consisting of 20 Holstein-Friesian grandsires and 833 sons was analyzed by multi-marker regression. This across-family analysis suggested the presence of 61 QTL. Ten of these QTL exceeded the genomewise significance threshold. These were mainly QTL affecting body size traits and udder conformation traits.
When QTL-information is used to select for a certain trait, genetic progress in other traits may be influenced as well, due to pleiotropic effects of QTL, or due to closely linked QTL. A method was developed to identify regions affecting multiple traits. The method is based on the covariance between marker contrasts from single-trait regression analysis for different traits. Application of this method to data on fifteen traits (milk production, udder conformation, udder health and fertility traits) in our granddaughter design resulted in 59 multiple trait quantitative trait regions (MQR). Most MQR were found on BTA 6, 13, 14, 19, 22, 23 and 25.
QTL-information can be used in breeding schemes (marker-assisted selection, MAS) to increase the rate of genetic improvement. A number of multi-stage dairy cattle breeding schemes was evaluated, studying the impact of increased preselection using QTL-information. Response in multi-stage MAS-schemes was 4.5% to 31.3% higher than response in corresponding schemes without QTL-information. In some of the MAS-schemes with a reduced number of progeny tested bulls, genetic progress was identical to or higher than genetic progress in the original schemes. The results indicate opportunities to improve current breeding schemes. The gains depend on the amount of genomic variation explained by QTL.
Currently available pedigrees and methods offer excellent opportunities to identify more QTL, thus increasing the fraction of the genomic variation explained by QTL. New initiatives, like sequencing the bovine genome, will further facilitate the identification of genomic variation, and its use.
Biosafety of metabolic engineering in plants : literature study in the framework of Policy Program 347, Biological Safety of Transgenic Plants commissioned by the Dutch Ministry of Agriculture, Nature Management and Fisheries
Nap, J.L. ; Gilissen, L.J.W.J. - \ 2002
Wageningen : Plant Research International - 46
secundaire metabolieten - metabolisme - planten - engineering - bioveiligheid - toxicologie - ecologie - pleiotropie - transgene planten - secondary metabolites - metabolism - plants - engineering - biosafety - toxicology - ecology - pleiotropy - transgenic plants
|Gene transfer and expression in plants
Mlynarova, L. ; Nap, J.P.H. - \ 1997
Bratislava : VEDA (Monograph, Slovak Academy of Sciences ) - ISBN 9788022404990 - 127
genetische modificatie - recombinant dna - plantkunde - genexpressie - pleiotropie - genetic engineering - botany - gene expression - pleiotropy
Characterization of genes expressed during mesoderm formation and anteroposterior patterning in carp (Cyprinus carpio)
Stevens, C.J.M. - \ 1997
Agricultural University. Promotor(en): L.P.M. Timmermans; H.W.J. Stroband; G. te Kronnie. - S.l. : Stevens - ISBN 9789054857549 - 130
cyprinidae - karper - moleculaire genetica - genexpressie - pleiotropie - methodologie - technieken - moleculaire biologie - cyprinidae - carp - molecular genetics - gene expression - pleiotropy - methodology - techniques - molecular biology
The formation of germ layers during gastrulation and the specification and patterning of the body axes are important events in the development of the embryo. The investigations described in this thesis aimed to isolate and characterize the distribution of transcripts of genes, in particular novel genes, that are expressed during the formation of mesoderm and the patterning of the anteroposterior axis in the carp (Cyprinus carpio), a cyprinid teleost fish. Such studies may contribute to a better insight in the molecular mechanisms underlying the above processes, in two respects. Firstly, the characterization of a gene's expression pattern is one of the first steps towards the elucidation of its function. Especially the characterization of novel genes may provide a key to new insights in development. Furthermore, in studies of development, it is of importance to have markers that identify specific cell types, for example early mesodermal precursor cells. The isolation of genes that are specifically expressed in certain cell types provides such markers.
Two molecular approaches were chosen for gene isolation. Firstly, we specifically searched for homeobox genes, which encode transcription factors with important regulatory functions during development. A particular class of homeobox genes, the Hox genes, provides cells along the anteroposterior axis with positional information and the expression patterns of members of this class are excellent markers of position on this axis. Our second approach was a subtractive hybridization strategy. It was applied to isolate genes that are differentially expressed between the oocyte and the early segmentation stage, a period during which mesoderm is induced.
For the identification of homeobox-containing sequences in a carp early segmentation stage cDNA library, we used a probe that was composed of a mixture of homeobox fragments, produced by PCR. The PCR primers were designed against the most conserved regions of the homeobox. This approach yielded a number of different genes of which two are described in this thesis. The gene cdx1(Chapter 2) is a member of the caudal class of homeobox genes and is expressed in ventrolateral cells of the embryo prior to gastrulation. During gastrulation, transcripts of this gene accumulate in the posterior half of the embryo. The functions of caudal class genes of Drosophila, mouse and Xenopus indicate that genes of this class mediate the specification of posterior positional values in the embryo. Because of their characteristic distribution, cdx1 transcripts are useful markers of (ventro) posterior position in the embryo and have been used as such in the studies of mesoderm formation and anteroposterior patterning in blastoderm explants, performed in our laboratory.
A second gene isolated in the search for homeobox genes was the Hoxb-1 gene, described in Chapter 3. This gene belongs to the class of Hox genes, whose members are organized into clusters in the genome of many species. With expression reaching into the hindbrain, the Hoxb-1 gene is one of the most anteriorly expressed Hox genes. Its most prominent expression, especially during segmentation, is found in rhombomere 4. In the late segmentation stage embryo, Hoxb-1 expression is a valuable marker of this rhombomere and the neural crest cells at that level of the hindbrain.
Chapter 4 describes the cloning of genes on the basis of their differential gene expression between the oocyte and the early segmentation stage, using a subtractive hybridization strategy. Fifteen genes, identified from the oocyte stage cDNA library, are expressed in early development, when mesoderm induction occurs, and their expression disappears before the beginning of segmentation. From the early segmentation stage cDNA library, 26 genes were selected whose expression was activated during segmentation but not yet in early development, coinciding with the differentiation of the mesoderm and the patterning of the anteroposterior axis. In total 27 genes appeared to code for novel proteins and are therefore candidates for further studies and may provide a better insight into molecular mechanisms underlying developmental processes. Also in light of the large scale mutagenesis screens of zebrafish that have recently been undertaken in a number of laboratories and for which the affected genes yet need to be molecularly identified, it is important that the search for novel genes continues for only few candidate sequences are available so far. The subtractive hybridization strategy described in this thesis appears a worthwhile technique to obtain such candidate sequences.
Further investigations of these novel genes were restricted to a detailed characterization of the expression of one gene: cth1.Chapter 5 gives a description of the distribution of the mRNA transcripts of this gene during cleavage, blastula and gastrula stages. Whereas maternal cth1 mRNA is ubiquitously distributed in the blastomeres, the embryonically transcribed cth1 mRNA is expressed after the late blastula stage, in cells at the blastoderm margin which have mesodermal and endodermal fates. The cth1 transcripts disappear around midgastrulation, coinciding with the commitment of cells to the mesodermal (or endodermal) fate. In the cth1 protein, motifs containing three cysteines and one histidine (C3H) are present that are most likely zinc fingers, structures involved in the regulation of expression of target genes. The cth1 mRNA expression pattern and the gene's homology to the TIS11 family, a family of primary response genes whose expression is activated after treatment with for example growth factors, suggest a function for the cth1 gene of maintenance of the cellular potential in cells with mesodermal (and endodermal) fates, and in cells of cleavage stages. By inhibiting the expression of certain target genes, cth1 could prevent or delay the selection of certain differentiation pathways, such as for example the commitment to a mesodermal fate before midgastrulation.
Proteins containing C3H motifs are expressed in a number of species.For example in C.elegans, the PIE-1 protein is required to keep germlineblastomeres totipotent during early development, most likely by suppressing the transcription in these blastomeres. In Chapter 6 the literature on the C3H class of proteins is shortly reviewed and the hypothesis is proposed that they may be widely involved in preserving cellular potency in specification events during development.
In Chapter 7 the results presented in previous chapters are discussed, with emphasis on the proposed role for cth1 and how its activity could affect the fate of the cells expressing this gene.
Expression and silencing of cowpea mosaic virus transgenes
Sijen, T. - \ 1997
Agricultural University. Promotor(en): A. van Kammen; J. Wellink. - S.l. : Sijen - ISBN 9789054857235 - 133
koebonenmozaïekvirus - genexpressie - pleiotropie - genetische modificatie - recombinant dna - vigna - vignabonen - cowpea mosaic virus - gene expression - pleiotropy - genetic engineering - recombinant dna - vigna - cowpeas
Plant viruses are interesting pathogens because they can not exist without their hosts and exploit the plant machinery for their multiplication. Fundamental knowledge on viral processes is of great importance to understand, prevent and control virus infections which can cause drastic losses in crops. In this thesis, cowpea mosaic virus (CPMV) was studied. This virus consists of two, icosahedral particles that each carry a distinct single stranded RNA molecule of positive polarity. Several years of research have revealed much information on the genomic organisation, the strategy of gene expression and the multiplication processes of CPMV, which are described in Chapter 1, but also many aspects remain to be elucidated.
To study individual viral processes, like replication, encapsidation or cell to cell movement, transgenic plants can be generated that express individual viral genes like the replicase, coat protein or movement protein gene. A prerequisite in this approach is the presence of an efficient and reliable plant regeneration and transformation system. (CPMV) 5 natural host is the tropical grain legume cowpea, Vigna unguiculata, a plant species that is recalcitrant at regeneration. Although in experiments described in Chapter 2 fertile plants could be regenerated from nodal thin cell layer segments, the explants were not competent for Agrobacterium-mediated transformation. Possibly in further studies, these nodal explants could prove suited for another transformation method.
Therefore, tobacco, which is also a host for CPMV and highly competent for regeneration and transformation, was preferred as the species to generate transgenic plants carrying CPMV specific genes. Especially the CPMV movement proteins (MP) genes appealed to us for overexpression studies. CPMV cell to cell movement is enabled by the CPMV MPs that act to modify plasmodesmata. They are assumed to channel plasmodesmata with MP-containing tubular structures and through or with these tubules virus particles are transported to adjacent cells. To obtain more information on the plasmodesmatal modifications brought about by the MPs, transgenic tobacco plants were generated that carried the MP gene under the control of either a constitutive or an inducible 35S promoter. However, in none of these plants the MPs were expressed to detectable levels (Chapter 3). Using the potato virus X (PVX)-based expression vector, accumulation of CPMV MPs was observed in the form of tubular structures extending from the surface of infected protoplasts into the medium. These PVX-derivatives look promising for providing effective tools in future studies on the effects of the CPMV MPs in plants.
Studies on MP functioning could involve complementation experiments with a CPMV mutant that is defective in cell to cell movement. In experiments described in Chapter 4 is was analysed by a molecular approach whether the CPMV mutant N123, that was first described in 1976, could be used to this effect. As the basis of the N123 specific phenotype was found not only to rest in the movement protein gene but also in one of the two coat protein genes, this mutant seemed not very suitable for complementation studies. Presumably a recently developed CPMV mutant in which the MP gene has been replaced by the fluorescent marker protein GFP (green fluorescent protein), will be a more appropriate tool.
Transgenic Nicotiana benthamiana plants that were expressing either the CPMV MP or the replicase gene under the control of a constitutive promoter, were found to exhibit a resistant phenotype when inoculated with CPMV (Chapter 5). Protoplast studies revealed that the resistance occurred as full immunity and was maintained in the cell. Resistance was specific to viruses highly homologous to CPMV, and in addition it was found to be specifically directed against the replication of the CPMV segment of which the transgene was derived (Chapter 5). Pathogen derived resistance can be mediated either by the protein encoded by the transgene or by the transcribed mRNA. Protein -mediated resistance generally offers moderate protection against a broad range of viruses, while RNA-mediated resistance results in immunity at the cellular level. Resistance obtained in transgenic plants transformed with defective genes confirmed that an RNA-based mechanism was underlying the highly specific transgenic resistance against CPMV (Chapter 6).
Specifically in the resistant lines, the transgene mRNA steady state levels were low compared to the relative transgene nuclear transcription rates (Chapter 6). This indicated that resistance occurs from a specific, cytoplasmic RNA turnover mechanism. This process can be regarded as a post- transcriptional gene-silencing process, that is primarily induced on the transgene mRNAs but to which also incoming, homologous CPMV genomes fall victim. In addition, heterologous RNA molecules, like PVX genomes, that contain the sequences corresponding to the transgene, are eliminated (Chapter 6). By inserting sequences homologous to only parts of the transgene in the genome of PVX and studying the fate of these recombinant genomes, it was shown that the degradation process is primarily targeted to a defined region of the transgene mRNA, the 3' region. Further analyses revealed that degradation can occur at various sites within this 3' region and that not a specific sequence or structure is of predominant importance. We observed that small inserts, like of only 60 nucleotides, can tag recombinant PVX molecules for the elimination process, albeit with reduced efficiency, which suggested that the RNA turnover process carries quantitative features.
On the intruiging question why post-transcriptional gene-silencing is induced in only some of the transgenic lines, we revealed (Chapter 6) that the organisation of integrated transgene sequences has an important role. Transformation with a transgene containing a directly repeated MP gene, increased the frequency at which resistant lines arise to 60%, compared to 20% of resistant lines that occur upon transformation with a transgene with a single MP gene. Thus, the resistance process seems influenced by qualitative features of the integrated transgenes. Also, it was observed that resistance concurred with extensive methylation at the transcribed transgene sequences (Chapter 6), which could indicate an essential role of methylation at transcribed sequences in obtaining RNA-mediated pathogen derived resistance.
From these observations and from data described in literature, a model for RNA-mediated virus resistance was made and presented in Chapter 6. In Chapter 7, the post-transcriptional gene-silencing phenomenon is discussed in more details and in addition an approach is presented by which the process could be exploited to efficiently engineer virus resistance or study plant gene expression.
The amylose - free potato mutant as a model plant to study gene expression and gene silencing
Flipse, E. - \ 1995
Agricultural University. Promotor(en): E. Jacobsen; R.G.F. Visser. - S.l. : Flipse - ISBN 9789054854678 - 132
solanum tuberosum - aardappelen - genetische modificatie - recombinant dna - genexpressie - pleiotropie - solanum tuberosum - potatoes - genetic engineering - recombinant dna - gene expression - pleiotropy
In this thesis, gene-expression and gene silencing were examined for Granule Bound Starch Synthase (GBSS) which catalyses the formation of amylose and Branching Enzyme (BE) which catalyses the formation of amylopectin. The (GBSS) deficient, with iodine, red staining amylose-free (amf) potato mutant was used in order to facilitate the experiments; GBSS-gene expression and BE-gene silencing resulted in blue staining starch.
A dosage effect of the wild-type GBSS-allele on GBSS-activity and amylose content was found in a tetraploid dosage population. The presence of amylose had a distinct influence on the physico-chemical properties of the starch.
Insertion of the wild-type GBSS-gene in the amf -mutant resulted in fully and partially complemented plants. The minimum number of independently segregating active GBSS-inserts was estimated by genetic analysis after microspore staining. The complemented phenotype was normally transmitted to the F1 for the fully complemented, but not for the partially complemented plants. For one plant the partial complementation was correlated with the presence of a block of five inserts. This block was also capable of inhibiting the endogenous GBSS-gene in a wild- type. This indicates that partial complementation can be caused by co-suppression, besides low transgene expression. Co-suppression was also obtained when the full size (GBSS) sequence or (GBSS) cDNA were introduced in a wild-type potato.
Introduction of the distal 1.5 kb cDNA coding for BE in both sense and antisense orientation resulted in several transgenic plants with a small blue core in these starch granules. This could indicate the presence of loosely branched amylopectin. in the core of the starch granules. The expression of the endogenous BE-gene was largely or fully inhibited as judged by the absence of BE mRNA and protein. This did not result in a measurable effect on the branching degree, but resulted in altered physico-chemical properties of the starch compared to amf -starch.
It is concluded that this study shows that the amf -mutant is successfully used as a model plant to examine different aspects of gene expression.
Early nodulin gene expression and the action of nod factors in Vicia sativa
Vijn, I. - \ 1995
Agricultural University. Promotor(en): A. van Kammen; T. Bisseling. - S.l. : Vijn - ISBN 9789054853794 - 101
wortelknolletjes - knobbelvorming - fabaceae - genexpressie - pleiotropie - root nodules - nodulation - fabaceae - gene expression - pleiotropy
Bacteria of the genera Rhizobium, Bradyrhizobium or Azorhizobium secrete lipo-oligosaccharide signal molecules, which play a pivotal role in the induction of early steps of root nodule formation on legumes. In these nodules the bacteria are hosted and a proper environment is provided for the bacteria to fix atmospheric nitrogen into ammonia, making the plant for its growth independent of nitrogen compounds in the soil.
The goal of the research described in this thesis is to provide insight in the mechanism by which the Nod factors secreted by Rhizobium bacteria initiate root nodule formation. Such kind of study requires a plant-bacteria system in which both the morphological and molecular changes induced by the Nod factor can be examined. Working on the Nod factors secreted by Rhizobium leguminosarum bv. viciae the choice of the host plant is limited to the genera Pisum, Lathyrus, Lens and Vicia. We decided that Viciasativa (vetch) would be the most useful plant for our studies, since the plant is small and various morphological changes, like root hair deformation and the formation of nodule primordia, that are induced by the Nod factors can easily be observed (Chapter 1).
Nod factors can induce the expression of early nodulin genes. These genes are expressed during different developmental stages of root nodule formation and the expression of these genes can therefore be used as molecular markers of root nodule development and Nod factor induced processes. To study Nod factor induced plant responses in V.sativa on the molecular level, the homologues of the early nodulin genes of pea (Pisum sativum) were isolated from V. sativa and their expression pattern was studied by in situ hybridization during root nodule development (Chapter 2).
To obtain information about the pathway from Nod factor to early nodulin gene expression, the mechanisms controlling ENOD12 expression were studied. With a promoter analysis in transgenic V.hirsuta root nodules we have identified that the 200 bp immediately upstream of the transcription start are sufficient to induce nodule specific and Nod factor induced expression (Chapter 3). For the isolation of transcription factors involved in controlling ENOD12 expression an expression library was screened and a preliminary characterization of cDNA clones encoding polypeptides that bind to the PsENOD12 promoter is described in Chapter 4.
In Chapter 5 early nodulin gene expression during Nod factor induced morphological changes, like root hair deformation and nodule primordia induction, was studied. Furthermore we examined whether RNA and protein synthesis are required for root hair deformation and for the activation of the early nodulin genes. These studies provided new insights about the mode of action of Nod factors.
In Chapter 6 is discussed to what extent V. sativa is a suitable host plant to study the mode of action of Nod factors and in which way the studies reported in this thesis have contributed to elucidate the mechanism by which Nod factors induce a diversity of plant responses.
A molecular analysis of L-arabinan degradation in Aspergillus niger and Aspergillus nidulans
Flipphi, M.J.A. - \ 1995
Agricultural University. Promotor(en): A.J.J. van Ooyen; J. Visser. - S.l. : Flipphi - ISBN 9789054853923 - 165
aspergillus - celwanden - koolhydraten - cellulose - celmembranen - fermentatie - voedselbiotechnologie - glycosidasen - polysacchariden - genexpressie - pleiotropie - moleculaire genetica - aspergillus - cell walls - carbohydrates - cellulose - cell membranes - fermentation - food biotechnology - glycosidases - polysaccharides - gene expression - pleiotropy - molecular genetics
This thesis describes a molecular study of the genetics ofL-arabinan degradation in Aspergillus niger and Aspergillus nidulans. These saprophytic hyphal fungi produce an extracellular hydrolytic enzyme system to depolymerize the plant cell wall polysaccharideL-arabinan. Chapter 1 surveys the occurrence, properties and applications ofL-arabinanolytic enzymes (arabinases). The A.niger system, which constitutes an endolytic endo-1,5-α-L-arabinase (ABN A) and two distinct α-L-arabinofuranosidases (ABF A and ABF B), has been a frequent subject of investigation in the past and represents the best characterizedL-arabinanolytic system to date. These three enzymes are all glycosylated. Current knowledge on the induction of fungal arabinase expression is summarized in this Chapter. Furthermore, the structure of the polysaccharide substrate and its function in the plant cell wall matrix are introduced.
In Chapters 2 to 5, the cloning and characterization of the structural genes coding for the three glycosyl hydrolases from the A. nigerL-arabinan-degrading complex are described. A. niger abf A and abf B ar e the first eukaryotic ABF-encoding genes to be isolated and sequenced, abn A is the first ABN-encoding gene published. Chapter 2 reports on the isolation of the abf A gene encoding ABF A, the minor extracellular ABF. This gene could be cloned by utilizing ABF Aspecific cDNA as the probe. This cDNA was immunochemically identified from a cDNA library generated fromL-arabitol-induced myceliurn of an A. nigerD-xylulose kinase mutant. This mutant is unable to grow onL-arabitol and features enhanced expression of all three arabinases when transferred to medium containing this pentitol as sole carbon source. In Chapter 3 , the cloning of the ABN A-encoding gene (abn A) is described. This gene was isolated following the same strategy as with abf A, although a second cDNA library had to be generated first. The induction process was immunochemically monitored in order to establish the proper induction conditions for the new library. The abn A gene and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and the cDN A. The N-terminal amino acid sequences of ABN A and a CNBr-derived peptide were determined. Several transcription initiation sites and one polyadenylation site could be identified. The structural region codes for a protein of 321 amino acids and is interrupted by three introns. Extracellular ABN A consists of 302 amino acid residues with a deduced molecular weight of 32.5 kDa and a theoretical pl of 3.5. For the protein, an apparent pl of 3.0 and an apparent molecular weight of 43 kDa, determined upon SDS-PAGE, were previously reported. Chapter 4 documents the isolation and characterization of the abf B gene, coding for the major extracellular ABF. The determination of N-terminal amino acid sequences from ABF B and CNBr-generated peptides allowed the design of deoxyoligonucleotide mixtures which enabled the cloning of abf B. When utilized as primers in a polymerase chain reaction (PCR), ABF B-specific amplification products emerged, one of which was used to probe the gene. The abf B gene and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and of ABF B- specific cDNA isolated from the library described in Chapter 3. Several transcription initiation sites and one polyadenylation site could be identified. The structural region is a single open reading frame and codes for a protein of 499 amino acids. The mature enzyme consists of 481 amino acid residues with a deduced molecular weight of 50.7 kDa and a theoretical pl of 3.8. An apparent pl of 3.5 and an apparent molecular weight of 67 kDa, determined upon SDS-PAGE, were previously reported. The abf B gene product was suggested to be identical to the ABF purified and characterized by Kaji and Tagawa (Biochim Biophys Acta 207 : 456-464 (1970)). Considering the non-amino acid content of the latter protein, a molecular weight of 64 kDa could be deduced for ABF B. In Chapter 5 , the abf A gene and its gene product were characterized by DNA sequence analyses of the genomic DNA and of the cDNA for which the isolation was described in Chapter 2. The N-terminal amino acid sequences of ABF A and a CNBr-derived peptide were determined. One transcription initiation site and two polyadenylation sites could be identified. The structural region is interrupted by seven introns and codes for a protein of 628 amino acids. Mature ABF A consists of 603 amino acid residues with a deduced molecular weight of 65.4 kDa and a theoretical pl of 3.7. For this ABF, an apparent pi of 3.3 and an apparent molecular weight of 83 kDa, determined upon SDS-PAGE, were previously documented.
Although the three enzymes are all active against (1->5)-α-glycosidic bonds betweenL-arabinofuranosides, ABF A, ABF B and ABN A are genetically unrelated. ABF A was found to be N -glycosylated whereas ABF B and ABN A were not - these enzymes are only O -glycosylated. For each gene, arabinaseoverproducing strains were generated by introducing multiple gene copies in A.niger or in A.nidulans uridine auxotrophic strains through co-transformation. Transformants were isolated upon primary selection for uridine prototrophy. Subsequent overproduction of the genes introduced was demonstrated in these recombinant strains upon growth on sugar beet pulp, both immunochemically and by assaying enzyme activity. abf A was shown to be expressed in the heterologous host A.nidulans, despite the absence of an abf A gene equivalent in this organism. High-copy number A.niger abf B transformants featured impaired secretion of other extracellular proteins upon growth on sugar beet pulp. ABN A overproduction was found to be limited to approximately five times the wild-type level in A.niger abn A transformants, but not in A.nidulans transformants. Such a limitation was not observed in case of the ABFs.
In Chapters 5 and 6, the regulation ofL-arabinan degradation is addressed. The structural genes seem to be regulated mainly at the transcriptional level. Additional copies of either A13F-encoding gene in A.niger were shown to result in a reduction, but not in total silencing of the expression of the wild-type ABN Aencoding gene upon induction with either sugar beet pulp orL-arabitol ( Chapter 5 ). The reduction of the expression level of abn A correlated with the abf gene dosage. The repression effected by extra abf B gene copies was more stringent and more persistent than that elicited by additional abf A copies. Although observed with both inducers, these phenomena were more outspoken and more persistent on sugar beet pulp. Similar, but more moderate effects were observed towards the expression of the other abf gene in multiple copy abf A- and abf B-transformants. It was proposed that the abf genes titrate two distinct gene activators both involved in coordination of arabinase gene expression. However, the three genes were shown to respond differently upon a mycelial transfer toL-arabitol-containing medium, indicating that gene-specific factors are also involved. Four distinct sequence motifs were found in common in the promoter regions of the three genes. One of these elements is identical to the A.nidulans CREA-motif, which has been shown to mediate carbon catabolite repression on several A.nidulans enzyme systems. Arabinase expression in A.niger is known to be repressed in the presence ofD-glucose. Two other motifs are highly homologous to cAMP-responsive elements described in other organisms. For the fourth motif no functional analogues could be found, but the element was found to be present in several other fungal genes which are not involved inL-arabinan degradation at all. It is therefore likely that none of these common elements confer system-specific regulation.
The presumed involvement ofL-arabitol in the induction process of fungal arabinases was further emphasized by the induction characteristics of an A. nidulans mutant unable to grow on the end-product ofL-arabinan degradation,L-arabinose, nor onL-arabitol ( Chapter 6).L-Arabitol is an intermediate ofL-arabinose catabolism in Aspergilli. This mutant was shown to lack NAD +-dependentL-arabitol dehydrogenase activity resulting inL-arabitol accumulation, both intracellularly and in the culture medium, wheneverL-arabinose is present. Upon submerged growth on various carbon sources in the presence ofL-arabinose, the mutant featured enhanced expression of the enzymes involved in extracellularL-arabinan degradation, and of those of the intracellularL-arabinose catabolism. The co-substrates on which the mutant secreted large amounts of arabitol simultaneously exhibited high arabinase expression and featured reduced growth.L-Arabitol secretion and enzyme production were also observed on a mixed carbon source ofD-glucose andL-arabinose, resulting in normal growth. Hence, in the presence ofL- arabinose, the carbon catabolite repression conferred byD-glucose in the wild-type, is overruled in the mutant.
In Chapter 7 , ABN A is shown to have remote sequence similarity with four bacterial xylanolytic glycosyl hydrolases (three β-D-xylosidases and an endo-1,4-β-D-xylanase), three of which feature activity against para -nitrophenyl-α-L-arabinofuranoside. This synthetic compound is commonly utilized to assay potential ABF activity, whereas it is known to be an inhibitor of the fourth enzyme. The homology became evident only after multi pie-sequence alignments and hydrophobic cluster analysis. It was proposed that these enzymes share a binding site for a terminal non-reducing α-linkedL-arabinofuranosyl residue and that they all belong to glycosyl hydrolase family 43. Implications from these suggestions were discussed. The ABFs could not be assigned to an established glycosyl hydrolase family.
Based on theL-arabinolytic system of the brown-rot fungus Monilinia fructigena, the sequence similarity found amongst ABF A and bacterial pullulan-degrading enzymes, and ABF expression levels under carbon starvation conditions and onD-glucose as the carbon source, distinct functions inL-arabinan and plant cell-wall degradation were proposed for ABF A and ABF B. ABF A would be essentially cell-wall associated and act to degradeL-arabinan fragments generated by ABN A. ABF B activity would be important for the primary release of small amounts ofL-arabinose which initiate induction of various endolytic systems to degrade plant cell walls, and thus function in substrate sensing. In line with these considerations, the involvement of other, not yet identified glycosyl hydrolases inL-arabinan degradation by A.niger was suggested.
Induction and repression of arabinase gene expression are further discussed in Chapter 7 . The results of the studies in A.niger (Chapter 5) and A.nidulans (Chapter 6) were interpreted in a mutual context. The identity of the lowmolecular-weight compound directly responsible for induction of arabinase gene expression, was addressed. BothL-arabinose andL-arabitol are likely candidates to fulfil such a role. However, it was not possible to weigh the actual inductive capacities ofL-arabinose andL-arabitol due to their in vivo convertibility and the carbon catabolite repression elicited by the pentose. Competition for such a compound provides an alternative explanation for the phenomena observed in Chapter 5. The involvement of the transcriptional repressor CREA in arabinase gene expression is not limited to the direct repression of structural and regulatory genes of theL-arabinan-degrading system. It also plays a role in inducer exclusion and end-product repression, two processes shown to be eminently involved in the regulation ofL-arabinan degradation in wild-type A.nidulans. Fungal growth rate was suggested to be related to derepression of theL-arabinan-degrading system. The possible involvement of cAMP in arabinase gene expression, as suggested by the presence of potential cis -acting cAMP-responsive elements in the structural genes, was considered. Various ways by which cAMP might modulate arabinase synthesis were surveyed.
Molecular aspects of avirulence and pathogenicity of the tomato pathogen Cladosporium fulvum
Ackerveken, G.F.J.M. van den - \ 1993
Agricultural University. Promotor(en): P.J.G.M. de Wit. - S.l. : Van den Ackerveken - ISBN 9789054851486 - 135
tomaten - passalora fulva - plantenziekteverwekkende schimmels - solanum lycopersicum - moleculaire biologie - genexpressie - pleiotropie - tomatoes - passalora fulva - plant pathogenic fungi - solanum lycopersicum - molecular biology - gene expression - pleiotropy
The molecular understanding of host-pathogen interactions and particularly of specificity forms the basis for studying plant resistance. Understanding why a certain plant species or cultivar is susceptible and why other species or cultivars are resistant is of great importance in order to design new strategies for future crop protection by molecular plant breeding.
In this thesis molecular aspects of avirulence and pathogenicity of the tomato pathogen Cladosporium fulvum are described. The interaction C. fulvum - tomato is an excellent model system to study fungus - plant specificity as the communication between pathogen and plant is confined to the apoplast (intercellular space). The ability to obtain intercellular fluid from C. fulvum -infected tomato leaves enabled the isolation and characterization of plant and fungal compounds that might play an important role in pathogenesis and/or the induction of resistance. The purification and characterization of a race-specific peptide elicitor provides the basis for most of the experiments described in this thesis. This peptide was thought to be produced only by races of C. fulvum avirulent on tomato genotypes carrying the resistance gene Cf9 , on which the elicitor induced necrosis.
Molecular aspects of avirulence of C. fulvum were first studied by the isolation and characterization of the cDNA encoding the AVR9 race-specific peptide elicitor (Chapter 2). The peptide was shown to be indeed produced by C. fulvum . Races virulent on tomato genotype Cf9 , lack the avr9 gene and do not produce the peptide elicitor thereby evading recognition by tomato genotypes carrying the corresponding resistance gene Cf9. To prove that the avr9 gene is a genuine avirulence gene, races virulent on tomato genotype Cf9 , were transformed with the cloned avr9 gene (Chapter 3). The cultivar-specificity of the transformants was changed from virulent to avirulent on tomato genotype Cf9. The avr9 gene can therefore be considered to be a genuine avirulence gene, the first fungal avirulence gene cloned. Additional proof for the role of the avirulence gene avr9 in specificity was provided by the disruption of avr9 in two races avirulent on tomato genotype Cf9 , by gene replacement, resulting in transformants virulent on tomato genotype Cf9 , (Chapter 4).
The avr9 gene encodes a 63 amino acids precursor protein. Removal of a signal peptide results in an extracellular peptide of 40 amino acids. Proteases of C. fulvum are involved in further processing this extracellular peptide by removal of N-terminal amino acids resulting in peptides of 32, 33 and 34 amino acids. Plant factors are responsible for further processing, resulting in the stable peptide elicitor of 28 amino acids (Chapter 5).
The avirulence gene avr9 is highly expressed in C. fulvum while growing inside the tomato leaf. The expression of avr9 is induced in C. fulvum grown in vitro under conditions of nitrogen limitation. The high expression of avr9 in C. fulvum growing inside the tomato leaf might be caused by nitrogen limiting conditions in the apoplast (Chapter 6).
Pathogenicity of C. fulvum was studied at the molecular level by the isolation of two genes encoding extracellular proteins (ECPs). The ecp1 and ecp2 genes were isolated via the amino acid sequence of ECP1, and polyclonal antibodies raised against ECP2, respectively (Chapter 7). The expression of the ecp genes is highly induced in planta as compared to the in vitro situation. The availability of the cloned ecp genes now enables us to study the role and importance of these genes during pathogenesis by reporter gene analysis and gene disruption.
Two models describing the C. fulvum -tomato interaction are presented, dealing with basic compatibility and race-specific incompatibility, respectively (Chapter 8). The improved understanding of pathogen recognition can be exploited in future research to elucidate the role of putative receptors in the resistant plant involved in perception of elicitors and induction of active plant defence.
Differential gene expression in Phytophthora infestans during pathogenesis on potato = [Differentiele genexpressie in Phytophthora infestans tijdens de pathogenese op aardappel]
Pieterse, C.M.J. - \ 1993
Agricultural University. Promotor(en): P.J.G.M. de Wit; F.P.M. Govers. - S.l. : Pieterse - ISBN 9789054851585 - 151
plantenziekteverwekkende schimmels - solanum tuberosum - aardappelen - phytophthora infestans - genexpressie - pleiotropie - plant pathogenic fungi - solanum tuberosum - potatoes - phytophthora infestans - gene expression - pleiotropy
The plant pathogenic fungus Phytophthora infestans (Mont.) de Bary is the causal agent of potato late blight, the most important and devastating fungal disease of potato. The molecular and cellular processes involved in pathogenicity of this hemibiotrophic fungus are poorly understood. The aim of the research described in this thesis was to gain more insight in these processes. A novel and unbiased approach was explored to isolate P . infestans genes which might have a function in pathogenesis. The approach was based on the assumption that the expression of pathogenicity genes of P . infestans , i.e. genes which are essential for the establishment and maintenance of basic compatibility or for the increase of disease severity, is specifically induced or significantly increased during pathogenesis. The strategy implies the isolation of in planta induced ( ipi ) genes by differential screening of a genomic library of P.infestans , subsequent characterization of their gene products, and elucidation of their function in pathogenesis.
By differential screening, nine distinct in planta induced genes were isolated, i.e. ipiA , ipiB , ipiC , ipiD , ipiJ1 , ipiJ2 , ipiN , ipiO and ipiQ . Expression studies revealed that the ipiB and ipiO genes have a transient expression pattern during pathogenesis with the highest levels in early stages of the interaction. The seven other ip i-genes show a continuous 5-10 fold increase in expression level during growth of the fungus in the host.
The P.infestans genes ipiA , ipiB , ipiC and ipiO were further characterized. Two of these appeared to encode ubiquitous proteins: ipiA , which was renamed ubi3R , encodes ubiquitin and ipiC , renamed calA , codes for calmodulin. Both proteins are highly conserved in eukaryotic organisms and have been shown to play important roles in basic cellular processes such as selective degradation of abnormal proteins (ubiquitin) and signal transduction (calmodulin). This is the first time that an increased expression of ubiquitin and calmodulin genes is found in association with pathogenicity of a plant pathogenic fungus. However, the significance of increased ubiquitin and calmodulin levels in P.infestans during pathogenesis remains to be resolved. The ipiB gene belongs to a gene family consisting of at least three highly homologous genes, ipiB1 , ipiB2 and ipiB3 , which are clustered on the genome in a head-to-tail arrangement. The ipiB genes encode novel glycine-rich proteins (GRPs) of 301 (IPI-B1), 343 (IPI-B2), and 347 (IPI-B3) amino acids (aa) respectively. The primary structure of the IPI-B proteins is composed of a N-terminal signal sequence followed by a large, highly repetitive glycine-rich domain. The glycine-rich domain is predominantly composed of two repeats with the core sequences A/V-G-A-G-L-Y-G-R and G-A-G-Y/V-G-G, respectively. The characteristic primary structure is also found in plant GRPs of which some have been shown to be associated with the plant cell wall. The P. infestans genome contains two ipiO genes, ipiO1 and ipiO2 , which are closely linked and arranged in an inverted orientation. The ipiO genes encode two nearly identical 152 aa proteins which have no homology with any known sequence. As the IPI-B proteins, the IPI-O proteins contain a N-terminal signal sequence suggesting that they are excreted. In addition, the IPI-O proteins have a putative N-glycosylation site and a typical Arg-Gly-Asp (RGD) tripeptide motif. The RGD tripeptide is conserved in several mammalian extracellular adhesion proteins in which it functions as a cell attachment motif. Whether the IPI-O proteins have adhesion properties is unknown.
The four in planta induced P . infestans genes ubi3R , calA , ipiB and ipiO , are among the first of the limited group of oomycetous genes to be isolated and sequenced. As in most known oomycetous genes, the coding sequence of these genes is not interrupted by introns. Within 100 nucleotides upstream of their ATG start codon, a conserved motif matching the consensus sequence GCTCATTYYNCA(A/T)TTT was identified. Comparison of the 5' nontranscribed regions of eight distinct oomycetous genes revealed that this sequence motif surrounds the transcription start point of the majority of these genes, suggesting that oomycetous genes have a GCTCATTYYNCA(A/T)TTT sequence preference for transcription initiation.
The expression of the ipiB and ipiO genes was studied in more detail. The ipiB genes as well as the ipiO genes are expressed in germinating cysts prior to host penetration. During pathogenesis both genes are transiently expressed. The highest mRNA levels are found in early stages of infection. Also during initial stages of interactions of P.infestans with a racespecific resistant host and the nonhost Solanum nigrum L., the expression of the ipiB and ipiO genes is induced. During growth of the fungus in vitro, nutrient deprivation appeared to be a strong stimulus for the induction of ipiB and ipiO gene expression. For several other pathogenesis related genes characterized in fungal plant pathogens it has been shown that nutrient starvation induces their expression. Whether starvation conditions mimic the nutrient condition encountered upon infection and, as such, function as a general stimulus for the activation of pathogenicity genes remains to be established.
Manipulation of gene expression in P.infestans is an important tool to study gene function. The possibility to manipulate gene expression in P. infestans by anti-sense RNA was tested. First, the promoters of five oomycetous genes, among which the P.infestans ubi3R gene, were fused to the reporter β-glucuronidase ( GUS ) gene of E. coli and the activity of the promoters was determined in transient expression assays as well as in stable transformants. The hsp70 and ham34 genes of Bremia lactucae appeared to have the highest activity. When the GUS gene in the anti-sense orientation fused to the ham34 promoter, is introduced in transgenic P.infestans containing sense GUS constructs, the accumulation of GUS is effectively inhibited. This indicates that the anti-sense technique is suitable to study gene function in P.infestans.
Nodulin gene expression in the developing pea root nodule
Govers, F. - \ 1987
Agricultural University. Promotor(en): A. van Kammen; T. Bisseling. - Wageningen : Govers - 126
genexpressie - moleculaire biologie - knobbelvorming - erwten - pisum sativum - pleiotropie - wortelknolletjes - gene expression - molecular biology - nodulation - peas - pisum sativum - pleiotropy - root nodules
Infection of leguminous plants with bacteria of the genus Rhizobium results in a symbiotic interaction which brings about the development of an entirely new organ on the plant, the root nodule. Within this organ about half of the plant cells are inhabited by bacteroids, the endosymblotic form of the bacterial partner. The bacteroids reduce atmospheric nitrogen (N 2 ) to ammonia (NH 3 ) which the plant uses as a nutrient. The host plant, on the other hand, provides the rhizobia with carbon compounds as an energy source for the nitrogen fixation process. Development of a nitrogen fixing root nodule is a true co-operation between two genomes. In chapter II the plant and bacterial genes involved in this symbiotic interaction are reviewed.
The research reported in this thesis deals with the identification of plant genes which are specifically expressed during the development of the symbiosis, the so-called nodulin genes and, in addition, bears upon the question which bacterial genes are involved in regulating the expression of nodulin genes.
By comparing the RNA content of pea root nodules induced by Rhizobiumleguminosarum and uninfected pea roots by means of invitro translation, we have been able to demonstrate the expression of twenty one nodulin genes (chapter III). Out of a nodule cDNA library seven pea nodulin cDNA clones were selected (chapter IV). The appearance of nodulin mRNAs during pea root nodule development, studied by analyses of invitro translation products and Northern blots, showed that nodulin genes are differentially expressed. Thus, nodulins could be classified into two groups: class I or early nodulins and class II nodulins which are expressed later in nodule development (chapter III and IV).
To determine the origin and the nature of signals responsible for the induction of nodulin genes, we mimicked the microaerobic conditions supposed to exist in root nodules and showed that in pea roots microaerobiosis by itself is not sufficient to trigger the induction of nodulin gene expression (chapter V). Also, in pea tumors formed by Agrobacteriumtumefaciens no expression of nodulin genes was detected (chapter VI). Therefore, it seems likely that nodulin gene expression is induced by specific signals from Rhizobium and we have searched for the Rhizobium genes responsible for such signals.
Analysis of nodulin gene expression in non-effective nodules, formed by Rhizobium mutants and engineered RhizobiumandAgrobacterium strains, showed that Rhizobium genes required for nodulation ( nod genes) are involved in the induction of at least one early nodulin gene (chapter VI), whereas Rhizobium genes essential for the nitrogen fixation process ( nif and fix genes) are not required for the induction of nodulin gene expression (chapter III IV and VI). In noneffective nodules the final accumulation level of class II nodulin mRNAs is reduced, indicating that the gene products of the Rhizobiumnif and fix genes, directly or indirectly via the products of the nitrogen fixation process, regulate the level of nodulin gene expression.
Finally, in chapter VII the implications of the reported results are discussed in the perspective of the possible functions of nodulins in the developing root nodule and the way nodulin gene expression may be induced and regulated.