Recognition of Verticillium effector Ave1 by tomato immune receptor Ve1 mediates Verticillium resistance in diverse plant species
Song, Yin - \ 2017
Wageningen University. Promotor(en): B.P.H.J. Thomma; P.J.G.M. de Wit. - Wageningen : Wageningen University - ISBN 9789463437950 - 231
disease resistance - defence mechanisms - immunity - plant-microbe interactions - plant pathogens - verticillium dahliae - verticillium - tomatoes - solanum lycopersicum - receptors - genes - tobacco - nicotiana glutinosa - potatoes - solanum tuberosum - solanum torvum - humulus lupulus - cotton - gossypium hirsutum - transgenic plants - arabidopsis thaliana - ziekteresistentie - verdedigingsmechanismen - immuniteit - plant-microbe interacties - plantenziekteverwekkers - verticillium dahliae - verticillium - tomaten - solanum lycopersicum - receptoren - genen - tabak - nicotiana glutinosa - aardappelen - solanum tuberosum - solanum torvum - humulus lupulus - katoen - gossypium hirsutum - transgene planten - arabidopsis thaliana
Plant-pathogenic microbes secrete effector molecules to establish disease on their hosts, whereas plants in turn employ immune receptors to try and intercept such effectors in order to prevent pathogen colonization. Based on structure and subcellular location, immune receptors fall into two major classes; cell surface-localized receptors that comprise receptor kinases (RKs) and receptor-like proteins (RLPs) that monitor the extracellular space, and cytoplasm-localized nucleotide-binding domain leucine-rich repeat receptors (NLRs) that survey the intracellular environment. Race-specific resistance to Verticillium wilt in tomato (Solanum lycopersicum) is governed by the tomato extracellular leucine-rich repeat (eLRR)-containing RLP-type cell surface receptor Ve1 upon recognition of the effector protein Ave1 that is secreted by race 1 strains of the soil-borne vascular wilt Verticillium dahliae. Homologues of V. dahliae Ave1 (VdAve1) are found in plants and in a number of plant pathogenic microbes, and some of these VdAve1 homologues are recognized by tomato Ve1. The research presented in this thesis aims to characterize the role of the tomato cell surface-localized immune receptor Ve1, and its homologues in other diverse plant species, in Verticillium wilt resistance.
Potatoes, pathogens and pests : effects of genetic modifi cation for plant resistance on non-target arthropods
Lazebnik, Jenny - \ 2017
Wageningen University. Promotor(en): J.J.A. Loon; M. Dicke. - Wageningen : Wageningen University - ISBN 9789463431620 - 151
solanum tuberosum - potatoes - oomycetes - phytophthora infestans - genetic engineering - transgenic plants - disease resistance - risk assessment - nontarget organisms - arthropods - insect pests - herbivores - trophic levels - ecological risk assessment - greenhouse experiments - field experimentation - solanum tuberosum - aardappelen - oömyceten - phytophthora infestans - genetische modificatie - transgene planten - ziekteresistentie - risicoschatting - niet-doelorganismen - geleedpotigen - insectenplagen - herbivoren - trofische graden - ecologische risicoschatting - kasproeven - experimenteel veldonderzoek
Currently, fungicides are necessary to protect potato crops against late blight, Phytophthora infestans, one of the world’s most damaging crop pathogens. The introgression of plant resistance genes from wild potato species targeted specifically to the late blight pathogen into susceptible potato varieties may alleviate the environmental impact of chemical control. Genetically modified plants are subject to an environmental risk assessment, and this includes testing for risks to the non-target arthropod community associated with the crop. The thesis begins with a review about the main plant defense responses and their role in influencing sequential interactions between herbivores and plant pathogens. The experimental chapters each focus on different aspects of the interaction between potato plants (both resistant and susceptible), the target pathogen (P. infestans) and several non-target insects. With each chapter, the scope widens: from the molecular gene expression in potato leaves in response to sequential attacks, to field scale biodiversity analyses. At the molecular level, one of the main findings was that the genomic position of the Rpi-vnt1 insertion conferring resistance to P. infestans influenced potato gene expression measured in leaves, when interacting with the non-target insect pests Myzus persicae (Green peach aphid) and Leptinotarsa decemlineata (Colorado potato beetle). Insect performance differed between the resistant GM and susceptible non-GM comparator. In the following chapter, the differences in insect performance were tested across a range of conventionally bred cultivars varying in resistance to P. infestans. Differences in M. persicae performance between several cultivars greatly outweighed the differences previously detected between the GM and non-GM comparator. These results are crucial in shaping the way risk is assessed in the context of GM crops, and these results are supported in our experiments assessing effects on biodiversity with pitfall traps in the field. The third trophic level was also addressed by comparing the performance of the parasitoid Aphidius colemani reared on GM and non-GM fed aphids, both with an without exposure to P. infestans. Differences in parasitoid performance were only found on the susceptible cultivar when inoculated with P. infestans. In the last experimental chapter the risk assessment is taken to the field comparing pitfall trap catches over two years and in two countries. Different methods for statistical analysis of biodiversity data were compared to arrive at recommendations for such analysis in the framework of environmental risk assessments. Drawing on these lessons, the discussion ends with ideas for the development of protocols for environmental risk assessments in the light of expected scientific progress in agricultural biotechnology.
Starch meets biotechnology : in planta modification of starch composition and functionalities
Xu, Xuan - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade. - Wageningen : Wageningen University - ISBN 9789462579200 - 169
starch - potato starch - potatoes - solanum tuberosum - plant biotechnology - biotechnology - genetic engineering - transgenic plants - modified starches - phosphate - arabidopsis thaliana - plant breeding - zetmeel - aardappelzetmeel - aardappelen - solanum tuberosum - plantenbiotechnologie - biotechnologie - genetische modificatie - transgene planten - gemodificeerd zetmeel - fosfaat - arabidopsis thaliana - plantenveredeling
Storage starch is an energy reservoir for plants and the major source of calories in the human diet. Starch is used in a broad range of industrial applications, as a cheap, abundant, renewable and biodegradable biopolymer. However, starch needs to be modified before it can fulfill the required properties for specific industrial applications. Genetic modification of starch, as a green technology with environmental and economic advantages, has attracted increasingly attention. Many achievements obtained from earlier studies have demonstrated the feasibility and potential of using this approach to produce starches with novel properties (Chapter 2).
The main objective of this research was to produce novel starches with enhanced functionalities through genetic modification, while gaining a better understanding of storage starch biosynthesis. A focus on potato was warranted as it represents a superior model system for storage starch biosynthesis studies and for the production of starches with novel properties. To this end, a number of enzymes from various sources have been expressed in potato tubers to modify starch phosphate content and polysaccharide structure, since these two characteristics have long been recognized as key features in starch properties.
To modify starch phosphate content and explore starch (de)phosphorylation, a human phosphatase enzyme named laforin, and modifications of it, were introduced into potato (Chapter 3). Interestingly, modified starches exhibited a significantly higher phosphate content rather than the expected lower phosphate content. Transcriptome analysis showed that the increase in phosphate content was a result of upregulation of starch phosphorylating genes, which revealed a compensatory response to the loss of phosphate content in potato starch. Furthermore, the increase of phosphate content in potato starch was reached to a threshold level. This was in line with the observations in the modified starches from overexpressed- Glucan water dikinase (GWD1) transgenic plants (Chapter 4). Furthermore, overexpression of two starch dikinases from Arabidopsis thaliana, glucan water dikinase 2 and 3 (AtGWD2 and AtGWD3), did not result in a significant increase in phosphate content of potato starch (Chapter 5). Taken together, these results indicated that phosphate content of potato starch is under strict control.
Morphological analysis of starch granules containing different levels of phosphate content confirmed the indispensible role of phosphate content in the normal formation of starch granules, since cracked granules were observed in the starches containing low phosphate content, while irregular bumpy shaped granules were observed in the tubers from plants containing high phosphate content. Interestingly, further analyses on the expression level of genes involved in starch metabolism and sugar-starch conversion suggested that starch phosphorylation might affect starch synthesis by controlling the carbon flux into starch while simultaneously modulating starch-synthesizing genes. Further studies are needed to confirm this finding (Chapter 4).
To produce starches with novel structures, an (engineered) 4, 6-α-glucanotransferase (GTFB) from Lactobacillus reuteri 121 was introduced into potato tubers (Chapter 6). The resulting starches showed severe changes in granule morphology, but not in starch fine structure. Transcriptome analysis revealed the existence of a self-repair mechanism to restore the regular packing of double helices in starch granules, which possibly resulted in the removal of novel glucose chains potentially introduced by the (engineered) GTFB.
This research successfully generated starches with various functionalities, including altered gelatinization characteristics (Chapter 3 and 4), improved freeze-thaw stability (Chapter 4) and higher digestibility (Chapter 6). The exploitation of relationships between starch characteristics and starch properties revealed that starch properties represent the outcome of the combined effect of many factors and are highly dependent on the genetic background in which the modification has been performed.
In conclusion, the research described in this thesis demonstrates the great potential of genetic modification in producing starches with novel properties. Meanwhile, these results revealed the presence of complex and exquisite molecular regulation mechanisms for starch biosynthesis in potato. In future research, these regulations need to be taken into account for the relational design of starch in planta. Certainly, a better understanding of the process of starch metabolism in storage organs would be a great step forward towards tailoring starch in an economically important crop such as potato.
Targeted and non-targeted effects in cell wall polysaccharides from transgenetically modified potato tubers
Huang, J.H. - \ 2016
Wageningen University. Promotor(en): Harry Gruppen; Henk Schols. - Wageningen : Wageningen University - ISBN 9789462576292 - 126
potatoes - cell walls - polysaccharides - transgenic plants - pectins - tubers - xyloglucans - genetically engineered foods - galactans - characteristics - nontarget effects - effects - aardappelen - celwanden - polysacchariden - transgene planten - pectinen - knollen - xyloglucanen - genetisch gemanipuleerde voedingsmiddelen - galactanen - karakteristieken - onbedoelde effecten - effecten
The plant cell wall is a chemically complex network composed mainly of polysaccharides. Cell wall polysaccharides surround and protect plant cells and are responsible for the stability and rigidity of plant tissue. Pectin is a major component of primary cell wall and the middle lamella of plants. However, pectin biosynthesis in planta and the mechanisms underlying the influence of structural differences arising from a modified biosynthesis machinery on functional properties remain poorly understood. In our research, the changes in the chemical structures of cell wall polysaccharides after transgenic modification of potato tuber polysaccharides were examined. The cell wall material from potato wild-type varieties, from known and from new potato transgenic lines targeting changes of the homogalacturonan or rhamnogalacturonan I backbone were isolated and characterized. The modified cell wall polysaccharides were examined by determining their individual monosaccharide levels on fresh weight base and their cell wall characteristic parameters, and levels of acetylation and methyl esterification of cell wall pectin. Data for both targeted and non-targeted structures of cell wall polysaccharides from wild-type and transgenic potatoes were obtained. A shorter galactan side chain was found from the buffer soluble pectin and calcium bound pectin of β-galactosidase (β-Gal) transgenic lines. All pectin fractions from rhamnogalacturonan lyase (RGL) transgenic lines had less galactan chains attached to their rhamnogalacturonan I backbones. Two uridine diphosphate-glucose 4-epimerase (UGE) transgenic lines, UGE 45 and UGE 51, had diverse effects on length of the galactan side chain. The xyloglucans from the RGL and UGE transgenic lines retained its XXGG building blocks but differed in the proportion of repeating units compared to the respective wild-type varieties. In contrast, the β-Gal transgenic lines predominantly consisted of XXXG-type xyloglucan in the 4 M alkali extract, but showed XXGG-type building blocks in 1 M alkali extract. In addition, a quick-screening method was validated and used to analyze 31 transgenic lines and their respective wild-type potato varieties. An overall comparison of pectin backbone, pectin side chains, acetylation and methyl-esterification of pectin, pectin content and (hemi)cellulose content of cell wall polysaccharides from these transgenic lines provided a better insight in the frequency, level and combination of both targeted and non-targeted structural changes compared to that of their respective wild-type varieties. The same evaluation method was used to correlate cell wall composition in wild-type and selected transgenic lines and their established gene expression with the texture of corresponding cooked potato cubes. Changed physical properties for the genetically modified tubers could be connected to specific cell wall characteristics. Tubers from transgenic lines containing cell wall pectin with short galactan side chains were less firm after cold processing compared to wild-type tubers. The enhanced understanding of transgenic modifications of potato tubers resulting into significant targeted and non-targeted modifications in cell wall polysaccharides will lead to a better selection of potato lines with tailored cell wall characteristics and desired properties of the tubers during processing.
Potato cell walls are composed of pectin, hemicellulose and cellulose. Cell wall polysaccharides are responsible for the stability, rigidity and flexibility of plant tissue. Pectin, a major component of primary plant cell walls, primarily consists of homogalacturonan (HG) and rhamnogalacturonan I (RG-I). To understand the structure–function relationships of potato cell wall pectin, this study aimed to identify the characteristics of both pectin and other polysaccharides as present in cell wall material (CWM) and of individual polysaccharide populations from wild-type potato varieties and their respective transgenic potato lines.
Chapter 1 gives a general introduction to the fine chemical structures of potato cell wall polysaccharides, the main models of cell wall architecture and the cell wall-degrading enzymes, which include pectinases, hemicellulases and cellulases. In addition, transgenic modification of the cell wall through the heterologous expression of various enzymes from fungal or plant origin that could modify potato cell wall polysaccharides in planta is addressed. Transgenic modifications of potato cell wall polysaccharides that targeted pectin structures and cellulose levels are summarised. However, due to unsuccessful starch removal during CWM isolation and incomplete analysis of CWM yield and composition, characteristics regarding the different cell wall polysaccharides from previously-studied transgenic potato lines are hardly available.
CWMs were extracted from the Karnico (wild-type) potato and its transgenic lines that expressed either β-galactosidase or rhamnogalacturonan lyase (Chapter 2). Improved starch removal procedures proved to be successful. Pectic polysaccharides were fractionated from CWMs of wild-type potato and its transgenic lines β-Gal-14 and RGL-18. Most β-Gal-14 pectin populations had less galactose (Gal) than wild-type, indicating that the transgenic line had shorter galactan side chains, although the side chain length differed for individual pectin populations. The ratio of HG:RG-I was introduced to evaluate the pectin backbone structure. High HG:RG-I ratios were consistently found in RGL-18 pectic polysaccharide populations. A low level of RG-I segments in combination with lower Gal contents indicated the removal of the galactan-rich RG-I segments in all pectin populations of RGL transgenic lines. In addition, RGL-18 transgenic modification increased the methyl-esterification and lowered the acetylation of pectins present in hot buffer extracts, when compared to wild-type. No effect on pectin esterification was found for β-Gal transgenic lines. Side effects of the mutation generated unexpected changes in the various pectin populations.
The xyloglucan structure was extensively modified after transgenic modification of the pectin structure. Two xyloglucan extracts were obtained from the Karnico and its β-Gal-14 and RGL-18 transgenic lines (Chapter 3). The extracts of the Karnico and RGL-18 lines were mainly comprised of the XXGG-type xyloglucan as represented by XXGG and XSGG as predominant repeating units. In contrast, the XXXG-type xyloglucan was primarily present in the β-Gal-14 4 M alkali extract built up by LLUG repeats, although XXGG type of xyloglucan was present in the 1M alkali extract. Both the RGL and β-Gal transgenic lines had different proportions of xyloglucan building blocks (XSGG/XXGG ratios) than wild-type. After transgenic modification of pectin backbone or pectin side chains, the xyloglucan structures has been biosynthetically modified by plant itself.
Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyses the conversion of UDP-glucose into UDP-galactose, which hypothetically should lead to more galactose being built into the cell wall polysaccharides. CWMs from the Kardal (wild-type) potato and its UGE45-1 and UGE51-16 transgenic lines were isolated, fractionated and characterised (Chapter 4). Both the UGE45 and UGE51 genes encoded for UGE enzymes, but the corresponding transgenic lines exhibited different modifications of the galactan side chains and of other cell wall structures. The Gal content of CWM from the UGE45-1 transgenic line was 38% higher than that of the wild-type and resulted in longer pectin side chains. The Gal content present in CWM from UGE51-16 was 17% lower than that of wild-type, which resulted in a slightly shorter galactan side chains for most pectin populations. Both UGE transgenic lines showed a decreased acetylation and an increased methyl-esterification of the cell wall pectin. Side effects were found in the xyloglucan structures of the transgenes as reflected by different proportions of XSGG/XXGG repeating units in comparison to wild-type. Pectin side chain biosynthesis had not only a varying level of galactan side chain modification, but also influenced the structure and possibly the interaction of other cell wall polysaccharides.
In Chapter 5, a new screening strategy is introduced to evaluate higher numbers of transgenic potato tubers via CWM yield and sugar composition. A total of four wild-type potato varieties and 31 transgenic lines were evaluated to determine the effects on targeted structures including RG-I or HG pectin backbone elements, galactan or arabinogalactan side chains, acetyl groups of pectin and cellulose levels. Modification of the pectin backbone or pectin side chains in the transgenic lines has either a simultaneous increase or simultaneous decrease of HG:RG-I ratio, side chain length and methyl-esterification of pectin. The pectin esterification transgenic line exhibited only limited side effects. The cellulose level targeted lines had also high HG:RG-I ratios, longer galactan chains and similar pectin content compared to the wild-type, indicative for a less branched pectin backbone with longer side chains. From the monosaccharide composition data, various pectin and cell wall characteristics parameters are suggested as powerful indicators of cell wall polysaccharide structure.
In Chapter 6, the achievements of this research are summarised and discussed in the context of potato cell wall architecture. The strategy and outcome of a quick screening method for multiple transgenic lines and an in-depth analysis of individual pectin and xyloglucan populations for the evaluation of potato CWMs is discussed. Furthermore, the texture of steam-cooked potatoes and the stability of potato cubes after freeze-thaw cycles are correlated with gene expression and cell wall composition in wild-type and selected transgenetically modified potato tubers. CWMs from transgenetically modified potatoes showed different physical properties during processing. In isolated CWMs, acetylation of cell wall pectin, molar Gal levels and starch content were the main parameters that could be related to the texture or firmness of tubers. Tubers from transgenic lines that resulted in shorter pectin side chains felt apart more easily after several freeze-thaw cycles than wild-type tubers and tubers with an increased length of pectin side chains. The modification of both targeted as well as non-targeted structures have now been shown to occur in many different potato transgenic lines, but precise mechanisms and consequences for the cell wall architecture remain unclear. Research performed so far, as well as research needed for getting a better understanding of plant cell wall architecture, is discussed.
Economic impact of the Commission's 'opt-out' proposal on the use of approved GM crops : quick assessment of the medium-term economic consequences
Hoste, R. ; Wagenberg, C.P.A. van; Wijnands, J.H.M. - \ 2015
LEI Wageningen UR (Report / LEI Wageningen UR 2015-097) - ISBN 9789086157259 - 51
transgenic plants - crops - genetic engineering - soyabeans - economic impact - agricultural sector - food industry - feed industry - european union - france - germany - poland - hungary - transgene planten - gewassen - genetische modificatie - sojabonen - economische impact - landbouwsector - voedselindustrie - veevoederindustrie - europese unie - frankrijk - duitsland - polen - hongarije
The European Commission proposed the opportunity for individual EU Member States to restrict or prohibit the use of GMOs in food or feed on their territory (a national ‘opt-out’). The economic impact on individual sectors of the feed and food chain (the vegetable oil and meal industry, trade, animal feed industry, livestock sector) of a possible opt-out policy for soy by individual Member States has been assessed by LEI Wageningen UR.
A single scenario was defined in which the four countries France, Germany, Poland and Hungary choose an ‘opt-out’ policy for soy. Consequences of this switch to non-GM soy and substitutes were assessed both quantitatively and qualitatively for feed prices, for production costs for animal production, for crushing industry and for trade, with a focus on the medium term
Governing agricultural sustainability : Global lessons from GM crops
Macnaghten, Philip ; Carro-Ripalda, Susana - \ 2015
Taylor and Francis Inc. - ISBN 9781138891777 - 244
transgenic plants - sustainable agriculture - transgene planten - duurzame landbouw
Although GM crops are seen by their advocates as a key component of the future of world agriculture and as part of the solution for world poverty and hunger, their uptake has not been smooth nor universal: they have been marred by controversy and all too commonly their regulation has been challenged as inadequate, even biased. This book aims to understand these dynamics, examining the impacts of GM crops in diverse contexts and their potentials to contribute to sustainable agricultural futures. Part 1 draws on research from three global 'rising powers' - Brazil, India and Mexico - exploring the views of scientists, farmers and publics. Using a diverse array of ethnographic and qualitative methodologies, the book examines the dynamics that have underpinned the controversy in three diverse geo-political contexts, the manner in which dominant institutional framings have been closely aligned with the interests of powerful elites, and the multiple ways in which these have been resisted through local, symbolic and material practices. Part 2 comprises a series of short comment pieces from 11 leading social and natural scientists responding to the question of how to develop a policy framework for the responsible innovation of sustainable, culturally appropriate and socially just agricultural GM technologies. This innovative book offers new insights for researchers and postgraduates in Science and technology studies, Agro-ecology and Environmental Studies, Development studies, Anthropology, Human Geography, Sociology, Political Science, Public Administration, Latin American studies, and Asian studies.
Effecten van een verbod op het gebruik van genetisch gemodificeerde soja als veevoedergrondstof. Quick scan van de gevolgen voor Nederland
Wagenberg, C.P.A. van; Hoste, R. - \ 2015
Wageningen : LEI Wageningen UR (LEI Report 2015-109) - ISBN 9789086157143 - 26
transgenic plants - crops - genetic engineering - soyabeans - fodder - economic impact - netherlands - transgene planten - gewassen - genetische modificatie - sojabonen - veevoeder - economische impact - nederland
If the Netherlands, alongside Germany, France, Poland, and Hungary, decides to ban genetically modified (GM) soy in animal feed, the use of soy products in animal feed in these five countries will have to decrease by 40 to 50% to ensure that the EU demand for non-GM soy does not exceed the supply on the world market. The extra costs to Dutch livestock farmers over a period of 3 to 5 years as a result of the more expensive non-GM soy and alternative protein sources are estimated at between €60 and €100 million a year, with approximately 80% being borne by poultry farmers. Livestock numbers and productivity will then be maintained. A partial shift in trade flows from animal feed ingredients can be expected from import in the west of the EU - for example, through the port of Rotterdam - to intra-EU flows from production areas within the EU to consumers and via the waterway axis from regions east of the EU, such as Ukraine. Less soy will enter the EU via the Netherlands. This deficit can be offset by the increased demand for alternative protein sources, which will be partly imported from overseas. The effects on Dutch ports, the transport sector, and employment will depend on the nature of the trade flow shifts.
Voorstel voor een co-existentie monitoringsprogramma t.b.v. het naast elkaar bestaan van genetisch gemodificeerde (GG) en niet-GG teelten in toekomstige praktijksituaties. 3. Suikerbiet
Wiel, C.C.M. van de; Kok, E.J. ; Scholtens, I.M.J. ; Smulders, M.J.M. ; Lotz, L.A.P. - \ 2015
Wageningen UR - 36
akkerbouw - veldgewassen - suikerbieten - vermenging - kruisbestuiving - genetische modificatie - uitkruisen - transgene planten - nederland - arable farming - field crops - sugarbeet - mixing - cross pollination - genetic engineering - outcrossing - transgenic plants - netherlands
Beschrijving van een voorstel voor een concreet co-existentiemonitoringprogramma (CMP) voor suikerbiet dat is aangepast aan de specifieke gewaseigenschappen van suikerbiet. De gemaakte keuzen t.b.v. een pragmatische invulling van het voorgestelde CMP worden in de opvolgende hoofdstukken toegelicht op basis van de huidige stand van zaken in het wetenschappelijk onderzoek aan (trans)genverspreiding in suikerbiet. Er is nog geen ervaring met een CMP in suikerbiet, noch is er een (Europese) standaard voor. Voor het bereiken van een zo pragmatisch mogelijke aanpak wordt zoveel mogelijk aangesloten bij al bestaande evaluatiepraktijken in de suikerbietenteelt
Voorstel voor een co-existentie monitoringsprogramma t.b.v. het naast elkaar bestaan van genetisch gemodificeerde (GG) en niet-GG teelten in toekomstige praktijksituaties. 2. Aardappel
Wiel, C.C.M. van de; Kok, E.J. ; Scholtens, I.M.J. ; Smulders, M.J.M. ; Lotz, L.A.P. - \ 2015
Wageningen UR - 36
akkerbouw - aardappelen - veldgewassen - vermenging - kruisbestuiving - genetische modificatie - uitkruisen - transgene planten - nederland - arable farming - potatoes - field crops - mixing - cross pollination - genetic engineering - outcrossing - transgenic plants - netherlands
Het rapport is als volgt opgezet. Eerst wordt een voorstel voor een concreet coexistentiemonitoringprogramma (CMP) voor aardappel beschreven dat is aangepast aan de specifieke gewaseigenschappen van aardappel. De gemaakte keuzen t.b.v. een pragmatische invulling van het voorgestelde CMP worden in de opvolgende hoofdstukken toegelicht op basis van de huidige stand van zaken in het wetenschappelijk onderzoek aan (trans)genverspreiding in aardappel. Er is nog geen ervaring met een CMP in aardappel, noch is er een (Europese) standaard voor. Voor het bereiken van een zo pragmatisch mogelijke aanpak wordt zoveel mogelijk aangesloten bij al bestaande evaluatiepraktijken in de aardappelteelt. Dat betekent dat zoveel mogelijk aangesloten is bij de bestaande controlepraktijk zoals die uitgevoerd wordt door de NAK. Dat laat onverlet dat co-existentiemonitoring niet tot de staande praktijk van de NAK gerekend kan worden en dat nu ook nog niet vastligt dat de NAK deze monitoring inderdaad gaat doen. Praktische uitvoering hangt af van besluitvorming over de uiteindelijke invulling van de co-existentiemonitoring zodra GG aardappelteelt geïntroduceerd zou gaan worden.
Voorstel voor een co-existentie monitoringsprogramma t.b.v. het naast elkaar bestaan van genetisch gemodificeerde (GG) en niet GG-teelten in toekomstige praktijksituaties. 1. Maïs
Wiel, C.C.M. van de; Kok, E.J. ; Scholtens, I.M.J. ; Dolstra, O. ; Smulders, M.J.M. ; Lotz, L.A.P. - \ 2015
Wageningen UR - 33
akkerbouw - veldgewassen - maïs - genetische modificatie - vermengen - uitkruisen - transgene planten - nederland - arable farming - field crops - maize - genetic engineering - blending - outcrossing - transgenic plants - netherlands
In het rapport wordt een voorstel voor een concreet co-existentiemonitoringprogramma (CMP) voor maïs beschreven dat is aangepast aan de specifieke gewaseigenschappen van maïs. De gemaakte keuzen t.b.v. een pragmatische invulling van het voorgestelde CMP worden in de opvolgende hoofdstukken toegelicht op basis van de huidige stand van zaken in het wetenschappelijk onderzoek aan (trans)genverspreiding in maïs. Er is nog beperkte ervaring met een CMP in maïs, bijvoorbeeld in Portugal, Tsjechië en Slowakije waar Bt MON810 maïs op beperkte schaal verbouwd wordt. Er is ook geen (Europese) standaard voor een CMP, maar er is wel voor maïs als eerste een Best Practice Document door het European Co-existence Bureau (ECoB) van het JRC uitgebracht (Rizov & Rodríguez-Cerezo 2014).
Ecological and experimental constraints for field trials to study potential effects of transgenic Bt-crops on non-target insects and spiders
Booij, C.J.H. - \ 2014
Bilthoven : Cogem (Report / Plant Research International, Business Unit Biointeractions and Plant Health 592) - 58
transgene planten - schade - risicoschatting - onbedoelde effecten - veldproeven - nadelige gevolgen - niet-doelorganismen - akkerbouw - gewasbescherming - transgenic plants - damage - risk assessment - nontarget effects - field tests - adverse effects - nontarget organisms - arable farming - plant protection
Veldproeven zijn een belangrijk onderdeel van de milieurisicobeoordeling bij de toelatingsprocedure voor teelt van genetisch gemodificeerde gewassen , met name om eventuele nadelige effecten op zogenaamde niet-doelwitorganismen te onderkennen.
Prof. Justus Wesseler over overheidsingrijpen bij nieuwe biotechnologische ontwikkelingen
Wesseler, J.H.H. - \ 2014
genetische modificatie - transgene planten - transgene organismen - biotechnologie - schade - rijst - india - economische aspecten - genetic engineering - transgenic plants - transgenic organisms - biotechnology - damage - rice - economic aspects
Overheidsregels rond biotechnologie en genetisch gemodificeerde gewassen leiden vaak tot substantieel hoge investeringskosten. Die hebben weer een lager niveau van productontwikkeling tot gevolg en een concentratie in de industrie, een herschikking van onderzoeksprioriteiten en een verschuiving van onderzoek en ontwikkeling naar landen met minder stringente regelgeving. Die trend leidde zelfs tot schade aan duurzame ontwikkeling uit oogpunt van milieu en volksgezondheid.
Expression of natural human b1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans
Hesselink, T. ; Rouwendal, G.J.A. ; Henquet, M.G.L. ; Florack, D.E.A. ; Helsper, J.P.F.G. ; Bosch, H.J. - \ 2014
Transgenic Research 23 (2014)5. - ISSN 0962-8819 - p. 717 - 728.
golgi-apparatus - murine beta-1,4-galactosyltransferase - beta 1,4-galactosyltransferase - transgenic plants - gene - cells - localization - antibodies - oligosaccharides - glycoproteins
b1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human b1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of biantennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat a2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.
Understanding the role of L-type lectin receptor kinases in Phytophthora resistance
Wang, Y. - \ 2014
Wageningen University. Promotor(en): Francine Govers, co-promotor(en): W. Shan; Klaas Bouwmeester. - Wageningen : Wageningen University - ISBN 9789462571327 - 214
phytophthora - phytophthora capsici - oömycota - plantenziekteverwekkende schimmels - plant-microbe interacties - arabidopsis - transgene planten - genexpressie - receptoren - kinasen - genen - ziekteresistentie - immuniteit - phytophthora - phytophthora capsici - oomycota - plant pathogenic fungi - plant-microbe interactions - arabidopsis - transgenic plants - gene expression - receptors - kinases - genes - disease resistance - immunity
Phytophthora pathogens are notorious for causing severe damage to many agriculturally and ornamentally important plants. Effective plant resistance depends largely on the capacity to perceive pathogens and to activate rapid defence. Cytoplasmic resistance (R) proteins are well-known for activation of plant immunity upon recognition of matching effectors secreted by Phytophthora. However, Phytophthora pathogens are notoriously difficult to control due to their rapid adaptation to evade R protein-mediated recognition. Hence, exploring novel resistance components is instrumental for developing durable resistance. Receptor-like kinases (RLKs) function as important sentinels in sensing exogenous and endogenous stimuli to initiate plant defence. One RLK that was previously identified as a novel Phytophthora resistance component is the Arabidopsis L-type lectin receptor kinase LecRK-I.9. This RLK belongs to a multigene family consisting of 45 members in Arabidopsis but whether or not the other members function in Phytophthora resistance was thus far unknown. The research described in this thesis was aimed at unravelling the role of LecRKs in plant immunity, in particular to Phytophthora pathogens.
Chapter I describes various Phytophthora diseases and the current understanding of the mechanisms underlying plant innate immunity with emphasis on disease resistance to Phytophthora pathogens.
In Chapter II, we describe the development of a new Arabidopsis-Phytophthora pathosystem. We demonstrated that Phytophthora capsici is capable to infect Arabidopsis. Inoculation assays and cytological analysis revealed variations among Arabidopsis accessions in response to different P. capsici isolates. Moreover, infection assays on Arabidopsis mutants with specific defects in defence showed that salicylic acid signaling, camalexin and indole glucosinolates biosynthesis pathways are required for P. capsici resistance (Chapter IIa). The importance of these pathways in Arabidopsis resistance was supported by the finding that the corresponding marker genes are induced upon infection by P. capsici (Chapter IIb). This model pathosystem can be used as an additional tool to pinpoint essential components of Phytophthora resistance.
We then exploited Arabidopsis-Phytophthora pathosystems to uncover the role of LecRKs in Phytophthora resistance. In Chapter III we describe a systematic phenotypic characterization of a large set of Arabidopsis LecRK T-DNA insertion lines. The T-DNA insertion lines were assembled and assayed for their response towards different Phytophthora pathogens. This revealed that next to LecRK-I.9, several other LecRKs function in Phytophthora resistance. We have also analysed whether the LecRKs are involved in response to other biotic and abiotic stimuli. Several T-DNA insertion lines showed altered responses to bacterial or fungal pathogens, but none of the lines showed visible developmental changes under normal conditions or upon abiotic stress treatment. Combining these phenotypic data with LecRK expression profiles obtained from publicly available datasets revealed that LecRKs that are hardly induced or even suppressed upon infection, might still have a function in pathogen resistance. Computed co-expression analysis revealed that LecRKs with similar function display diverse expression patterns.
Arabidopsis LecRK clade IX comprises two members. T-DNA insertion mutants of both LecRK-IX.1 and LecRK-IX.2 showed gain of susceptibility to non-adapted Phytophthora isolates and therefore the role of these two LecRKs in Phytophthora resistance was further investigated. In Chapter IV we describe that overexpression of either LecRK-IX.1 or LecRK-IX.2 in Arabidopsis resulted in increased resistance to Phytophthora, but also induced plant cell death. A mutation in the kinase domain abolished the ability of LecRK-IX.1 and LecRK-IX.2 to induce Phytophthora resistance as did deletion of the lectin domain. Cell death induction however, only required the kinase, not the lectin domain. Since transient expression of both LecRKs in Nicotiana benthamiana also resulted in increased Phytophthora resistance and induction of cell death, we used N. benthamiana to explore downstream components required for LecRK-IX.1- and LecRK-IX.2-mediated Phytophthora resistance and cell death. Virus-induced gene silencing of candidate signaling genes revealed that NbSIPK1/NPT4 is essential for LecRK-IX.1-mediated cell death but not for Phytophthora resistance. Collectively, these results illustrate that the Phytophthora resistance mediated by LecRK-IX.1 and LecRK-IX.2 is independent of the cell death phenotype. By co-immunoprecipitation we identified putative interacting proteins, one of which was an ATP-binding cassette (ABC) transporter. A homolog in Arabidopsis, the ABC transporter ABCG40, was found to interact in planta with both LecRK-IX.1 and LecRK-IX.2. Similar to the LecRK mutants, Arabidopsis ABCG40 mutants showed compromised Phytophthora resistance, indicating that ABCG40 has a function in Phytophthora resistance.
In Chapter V, we describe the generation of stable transgenic N. benthamiana plants expressing Arabidopsis LecRK-I.9 or LecRK-IX.1. Multiple transgenic lines were obtained varying in transgene copy number and transgene expression level. Ectopic expression of LecRK-I.9 resulted in reduced plant sizes and aberrant leaf morphology. In addition, expression of LecRK-IX.1 induced plant cell death. Transgenic N. benthamiana lines expressing either LecRK-I.9 or LecRK-IX.1 showed increased resistance towards P. capsici or Phytophthora infestans. This demonstrated that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their role in Phytophthora resistance upon interfamily transfer.
Based on the results obtained on Arabidopsis LecRKs, we speculated that LecRKs in other plant species could play a similar role in Phytophthora resistance. In Chapter VI, we focus on LecRKs in two Solanaceous plants, i.e. N. benthamiana and tomato. By exploring genome databases, we identified 38 and 22 LecRKs in N. benthamiana and tomato, respectively. Phylogenetic analysis revealed that both N. benthamiana and tomato lack LecRKs homologous to Arabidopsis LecRKs of clades I, II, III and V, but contain a Solanaceous-specific clade of LecRKs. Functional analysis of various Solanaceous LecRKs using virus-induced gene silencing followed by infection assays revealed that homologs of Arabidopsis LecRK-IX.1 and LecRK-IX.2 in N. benthamiana and tomato are implicated in Phytophthora resistance. These results indicate that the role of clade IX LecRKs in Phytophthora resistance is conserved across plant species.
In Chapter VII, the experimental data presented in this thesis are summarized and discussed in a broader context. We present an overview of the current understanding of LecRKs in plant immunity and discuss how LecRKs can be exploited to improve plant resistance.
Safety aspects of genetically modified crops with abiotic stress tolerance
Liang, C. ; Prins, T.W. ; Wiel, C.C.M. van de; Kok, E.J. - \ 2014
Trends in Food Science and Technology 40 (2014)1. - ISSN 0924-2244 - p. 115 - 122.
risk-assessment - salt tolerance - transcription factors - salinity tolerance - transgenic plants - drought tolerance - gene flow - vegetable crops - gm crops - ipt gene
Abiotic stress, such as drought, salinity, and temperature extremes, significantly reduce crop yields. Hence, development of abiotic stress-tolerant crops by modern biotechnology may contribute to global food security. Prior to introducing genetically modified crops with abiotic stress tolerance to the market, a food and environmental safety assessment is generally required. Although worldwide harmonised comparative approach is currently provided, risk assessors still face challenges to assess genetically modified crops with abiotic stress-tolerance. Here, we discuss current developments of abiotic stress tolerance as well as issues concerning food and environmental safety assessment of these crops, including current approaches, challenges and future directions.
R gene stacking by trans- and cisgenesis to achieve durable late blight resistance in potato
Zhu, S. - \ 2014
Wageningen University. Promotor(en): Evert Jacobsen; Richard Visser, co-promotor(en): Jack Vossen. - Wageningen : Wageningen University - ISBN 9789461735706 - 164
solanum tuberosum - aardappelen - phytophthora infestans - oömycota - plantenziekteverwekkende schimmels - ziekteresistentie - genen - cisgenese - transgene planten - plantenveredeling - genetische modificatie - solanum tuberosum - potatoes - phytophthora infestans - oomycota - plant pathogenic fungi - disease resistance - genes - cisgenesis - transgenic plants - plant breeding - genetic engineering
Among the many diseases of potato (Solanum tuberosum L.), which is the third food crop in the world after wheat and rice, late blight caused by the oomycete pathogen Phytophthora infestans, is one of the most serious diseases. In the last century, major resistance (R) genes were introgressed mainly from the wild species Solanum demissum into the cultivated potato Solanum tuberosum. However, introgression of late blight resistance genes by interspecific crosses followed by backcrosses, proved to be associated with linkage drag problems. The desired R gene is then closely linked with one or more unfavorable genes. Moreover, the obtained resistance in the varieties could be easily overcome by fast evolving virulence among P.infestans isolates. The introduction of the A2 mating type from Mexico to Europe resulted in genetically more diverse and complex P.infestans offspring, since initially only the A1 mating type existed. Therefore, new strategies for breeding varieties with durable and broad spectrum resistance needed to be developed.
Previous research indicated that varieties containing single major R genes did not show durable resistance. Therefore, the potato breeding and research community abandoned the introgression of major R genes and started breeding for horizontal resistance by combining multiple partial resistance genes. This quantitative resistance breeding approach was also not successful because the levels of resistance were too low, breeding was too complicated and the spectrum was not as broad as anticipated. Nowadays, the introgression of major R genes regained interest and two ways of resistance breeding can be distinguished: 1. molecular marker assisted resistance breeding or 2. genetic modification (GM) of existing varieties with cloned major R genes.
In this thesis, the time-saving GM approach has been investigated to achieve durable resistance against potato late blight in existing varieties by stacking of major R genes via transgenesis and cisgenesis (Chapters 2, 3, 4). These R genes are so called cisgenes and are unmodified copies of genes from the same or crossable species, harboring their own promoter and terminator sequences.
The main difference between cisgenesis and transgenesis is the resulting (end) product. The end products for the latter case are transformants, which contain transgenes, that can come from a very different species, such as the selection marker gene nptII coding for antibiotic resistance from bacteria. However, the end products of cisgenesis, called cisformants, only harbor cisgenes (which are natural genes from the same or crossable species). These cisformants are selected by PCR for the presence of R gene(s) and for the absence of vector backbone sequences. In our study, functionality of the individual R genes, in trans- and cisformants containing stacked R genes, was determined by detached leaf assays (DLA) using avirulent isolates and by agro-infiltration with Avr genes matching every single R gene. Their foliar resistance was also tested in the field, and their resistance in tubers was tested in the lab.
In order to ensure durability, an accurate and robust system must be available to monitor virulence in P.infestans populations. Differential sets with plants containing single R genes are important and developed in many crops in order to facilitate both resistance breeding and genetic research on pathogen populations in different locations worldwide. The existing conventional differential potato set of Mastenbroek was updated and a start was made to develop a GM differential set with cloned R genes in individual transformants of cv Desiree (Chapter 5).
In Chapter 2, R genes with broad and complementary resistance spectrum were selected as a first step for R gene stacking. Selection for these R genes was performed using DLA with 44 selected late blight isolates. Out of four R genes (Rpi-sto1, Rpi-vnt1.1, Rpi-blb3, and R3a), three were selected for stacking experiments, Rpi-sto1 from S. stoloniferum, Rpi-vnt1.1 from S. venturii and Rpi-blb3 from S. bulbocastanum. Cv Desiree transformants containing these three single R genes conferred resistance to 40, 43 and 37 out of 44 isolates, respectively. The R3a containing transformant conferred resistance to only five out of 44 isolates. These three broad spectrum R genes were then combined in one binary vector pBINPLUS containing nptII as kanamycin resistance marker. Transformants containing nptII and the three R genes showed foliar resistance in DLA against two isolates PIC99189 (avrsto1, Avrvnt1, avrblb3) and EC1 (Avrsto1, avrvnt1, Avrblb3). Furthermore, the functions of these three individualR genes were confirmed using the cross reacting Avr genes from the pathogen, since no isolates were available to distinguish the function of each R gene individually due to the broad resistance spectrum. The resistance spectrum of transformants containing the three R genes Rpi-sto1, Rpi-vnt1.1 and Rpi-blb3 showed after DLA the expected sum of resistance spectrum from all three individual R genes and no indications for epistatic effects were observed (Chapter 2). These triple R genes containing transformants showed also full resistance in the field after inoculation with IPO-C (Avrsto1, Avrvnt1, avrblb3) both in 2011 and 2012. Furthermore, these three R genes were inherited to the next generation as a cluster and retained their functionality after crossing. Generally, resistance in tubers of these plants showed also the summed spectrum of all individual R genes in both generations, as was the case in the foliar resistance test. It was remarkable that transgenic Desiree plants, harboring Rpi-sto1 or Rpi-blb3,showed increased resistance in tubers, while their functional homologs Rpi-blb1 and R2, did not show resistance in tubers of conventionally bred materials. The integration of T-DNA borders and vector backbone sequences was also investigated. Around 45% of the triple R gene containing transformants harbored one or two T-DNA copies, without the integration of T-DNA borders and vector backbone (Chapter 3).
The introduction of multiple R genes was also applied to produce cisformants, plants containing only cisgenes. Three approaches were taken: 1) two cisgenes were introduced through one marker free transformation vector, 2) two cisgenes were introduced through two separate marker free vectors by co-transformation, 3) co-transformation of two vectors, one only containing nptII, and the other one is a marker free transformation vector harboring three cisgenes. This co-transformation was followed by sexual crossing to remove selection marker nptII. All three approaches were successful in the production of cisformants. The first approach produced a high percentage (73%) of cisformants but, in contrast to transgenic plants, the percentage of plants showing full resistance in DLA was relatively low (42%). The second approach produced only 4% of cisformants with stacked R genes, due to the high incidence of vector backbone sequence integration from two vectors used for co-transformation. All transformants obtained by the third approach showed full late blight resistance, which was very efficient compared to the first two approaches. This must be due to the use of the nptII selection marker. After crossing, the integration of both T-DNAs appeared to be unlinked in all tested transformants. Therefore, cisformants with active R genes could be obtained. The resistance level in tubers of cisformants was more frequently sufficient in plants with integration of two or more T-DNA copies, as it was also observed in the triple R gene transformants (Chapter 3). Not only the R genes from cisformants obtained using the third approach but also the cisformants from the first approach showed clustered inheritance in a crossing population, while the R genes segregated independently in the crossing population from a cisformant obtained using the second approach (Chapter 4).
The potato late blight differential set is used to characterize the virulence of P.infestans isolates, consisting of eleven plants which are expected to represent eleven different late blight R genes. Most differential plants were found to be susceptible to current late blight isolates, with the exception of the MaR8 and MaR9 plants. It had already been described that additional R genes were present in some members of this differential set. In Chapter 5, all eleven differential plants were tested for a hypersensitive reaction towards seven Avr genes. Only in three differential plants (MaR1, MaR2 and MaR4) no additional R genes were found, while for example MaR3,MaR8 andMaR9 contained multiple R genes. The conventional differential set was extended with F1 and BC1 segregants harboring a reduced number of these R genes and potentially containing only one R gene (R3a, R3b, R8 or R9, respectively) and with plants containing recently cloned R genes (Rpi-blb3, Rpi-sto1, Rpi-blb1, Rpi-pta1, Rpi-blb2, Rpi-vnt1.1 and Rpi-chc1). A disadvantage of the (extended) conventional differential set is that their genetic background is different which is complicating the use of this set. Moreover, for none of the extended differential plants it can be ruled out that different additional R genes are present. Therefore, a GM differential set consisting of ten transformants of cv Desiree, each harboring a single R gene was compiled. This GM differential set is more reliable for characterization of P.infestans isolates and for the functional test of individual R genes, due to the isogenic background. As a proof of concept, the conventional and the GM differential sets were compared using recently collected isolates from Dutch fields in detached leaf assays. It was found that plants containing Rpi-blb3, Rpi-blb1, Rpi-chc1, R8, R9, Rpi-vnt1.1 and Rpi-blb2 showed a broader resistance spectrum as compared to R1, R3a, R3b andR4. Furthermore, the application of the GM differential set to monitor virulence towards the different R genes in local late blight populations using trap fields was investigated. The extended conventional and the GM differential sets are on continuously growing lists, which can be in the future updated with better performing, genetically more isogenic plants harboring novel R genes, or when new R genes are transformed into cv Desiree.
In the general discussion (chapter 6), related topics from different experimental chapters are discussed simultaneously, some additional experimental data are provided and a broader view on the research area is given.
In summary, five main conclusions can be drawn from this work: 1. broad spectrum resistance in leaf and tuber with stable inheritance can be achieved by gene stacking via transgenesis and cisgenesis; 2. The frequency of cisformants with sufficient resistance at foliage and tuber level is lower than in transformants; 3. Avr genes are highly needed to test for functionality of all stacked R genes in trans- or cisformants; 4. the GM differential set can be used to accurately characterize P.infestans isolates and to assess the employability of certain R genes in particular geographic locations; and 5. genetic transformation is a unique way to improve existing susceptible potato varieties such as the cvs Bintje and Russet Burbank which are grown at relatively large areas worldwide.
Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody
Häkkinen, S.T. ; Raven, N. ; Henquet, M.G.L. ; Laukkanen, M.L. ; Anderlei, T. ; Pitkänen, J.P. ; Twyman, R.M. ; Bosch, H.J. ; Oksman-Caldentey, K.M. ; Schillberg, S. ; Ritala, A. - \ 2014
Biotechnology and Bioengineering 111 (2014)2. - ISSN 0006-3592 - p. 336 - 346.
enzyme replacement therapy - cell-suspension-culture - monoclonal-antibody - recombinant proteins - hyoscyamus-muticus - transgenic plants - tissue-cultures - nitrogen - optimization - glucocerebrosidase
Recombinant pharmaceutical proteins expressed in hairy root cultures can be secreted into the medium to improve product homogeneity and to facilitate purification, although this may result in significant degradation if the protein is inherently unstable or particularly susceptible to proteases. To address these challenges, we used a design of experiments approach to develop an optimized induction protocol for the cultivation of tobacco hairy roots secreting the full-size monoclonal antibody M12. The antibody yield was enhanced 30-fold by the addition of 14¿g/L KNO3, 19¿mg/L 1-naphthaleneacetic acid and 1.5¿g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formation and morphological changes in the inner cortex and pericycle cells, indicating that the improved productivity was at least partially based on the enhanced efficiency of antibody secretion. We found that 57% of the antibody was secreted, yielding 5.9¿mg of product per liter of induction medium. Both the secreted and intracellular forms of the antibody could be isolated by protein A affinity chromatography and their functionality was confirmed using vitronectin-binding assays. Glycan analysis revealed three major plant complex-type glycans on both forms of the antibody, although the secreted form was more homogeneous due to the predominance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment. Biotechnol. Bioeng. 2013;9999: 1–11.
Fructan biosynthesis in crop plants : the molecular regulation of fructan biosynthesis in chicory (Cichorium intybus L.)
Arkel, J. van - \ 2013
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Ingrid van der Meer. - S.l. : s.n. - ISBN 9789461736635 - 158
cichorium intybus - gewassen - zea mays - solanum tuberosum - transgene planten - koolhydraten - fructanen - biosynthese - inuline - polymerisatie - cichorium intybus - crops - zea mays - solanum tuberosum - transgenic plants - carbohydrates - fructans - biosynthesis - inulin - polymerization
Fructan is a polymer of fructose produced by plants and microorganisms. Within the plant kingdom about 45.000 species accumulate fructan as storage carbohydrate in addition to, or instead of, starch. Fructan accumulating species are mainly found in temperate and sub-tropical regions with seasonal or sporadic rainfall. During the last decades, the use of fructan in the (food) industry has rapidly evolved, because of its health promoting characteristics and interesting functional properties.Chicory (Cichorium intybus L.) is a biennial taproot-bearing crop plant that is grown for the production of inulin on an industrial scale. Inulin, a ß(2,1) linked linear fructan with a terminal glucose residue, is stored in the chicory taproots. The degree of polymerisation (DP) determines the application of the inulin and hence the value of the crop. This leads us to the central question of this thesis:
What regulates the fructan yield and the degree of polymerisation, and how can we modify this?
The DP is highly dependent on the field conditions and harvest time, and therefore the first step in answering this question was tostudy the regulation of fructan (inulin) metabolism throughout the growing season. This is described in Chapter 2. Metabolic aspects of inulin production and degradation in chicory were monitored in the field and under controlled conditions. We determined the concentrations of soluble carbohydrates, the inulin mean degree of polymerisation (mDP), inulin yield, gene expression and activity of enzymes involved in inulin metabolism in the taproots. Inulin biosynthesis - catalysed by sucrose: sucrose 1-fructosyltransferase (EC 126.96.36.199) (1-SST) and fructan: fructan 1-fructosyltransferase (EC 188.8.131.52) (1-FFT) - started at the onset of taproot development. Inulin yield increased with time following a sigmoid curve reaching a maximum in November. The maximum inulin mDP of 15 was reached in September and then gradually decreased. Based on the changes observed in the pattern of inulin accumulation, we defined three phases in the growing season and analysed product formation, enzyme activity and gene expression in these defined periods. The results were validated by performing experiments under controlled conditions in climate rooms. Our results show that the decrease in 1-SST is not regulated by day length and temperature. From mid-September onwards the mDP decreased gradually although inulin yield still increased. This is most probably the result from back-transfer activity of 1-FFT and fructan exohydrolase activity (EC 184.108.40.206) (1-FEH). In plants 1-FEH catalyses the breakdown of fructan in order to release the stored carbohydrates necessary in periods of stress, like cold or drought periodsor flowering. This information was used to design two strategies to obtain the desired, increased inulin DP and yield. Overexpression of 1-SSTwas performed to increase the mDP and to keep the sucrose concentration low, to prevent 1-FFT from depolymerizing inulin. The result was a higher mDP during the growing season. Unfortunately, no effect on the mDP was seen at the end of the growing season, most probably due to activity of FEH. Secondly, anFEH I antisense fragment was introduced into chicory in order to block depolymerisation at the end of the growing season. This resulted in a reduction in FEH Iexpression upon cold induction, but had only minor effects on the mDP. The degradation of inulin was most probably caused by the remaining 1-FEH activity. Overall this study showed that inulin metabolism in chicory is tightly regulated, but also revealed options to further steer inulin metabolism in chicory.
The next step in answering the central question was to study the regulation of the genes involved in fructan biosynthesis. In Chapter 3this was studied at three different levels. Firstly, fructan gene expression and carbohydrate concentrations were studied in axial sections of mature chicory root, revealing the highest expression levels and carbohydrate levels in the phloem. Correlations were found between the gene expression patterns of 1-SST, 1-FFT and the carbohydrate levels, suggesting a possible involvement of sugars in the regulation of 1-SSTand 1-FFTgene expression. Secondly, the induction of 1-SSTand 1-FFTexpression was studied in excised chicory leaves. Expression of both 1-SSTand 1-FFTwas induced upon sucrose and glucose feeding, suggesting that both genes are at least partly regulated in the same way. Upon fructose feeding, the induction of fructan biosynthesis was less pronounced than with sucrose. The expression of 1-SSTwas induced by fructose but this resulted in only low amounts of 1-kestose. The expression of 1-FFTwas not induced upon fructose feeding.Thirdly, to further unravel the mechanism of induction, the promoters of 1-SSTand 1-FFTfrom chicory were isolated and characterized through in silicoand in planta(only 1-FFT) analysis. Computational analysis of fructosyltransferase (FT) promoters revealed elements that are common in fructan biosynthesis-promoters among different species and also occur in Arabidopsis promoter sequences. One of these elements is predominantly present in genes involved in sugar metabolism and transport. This element did also contain a core sequence involved in MYB transcription factor binding important for fructan biosynthesis activation in wheat, as was published recently. An 1100bp 1-FFTpromoter fragment was shown to be functional in transgenic chicory and in the non-fructan accumulating plants species, Arabidopsis and potato. Application of carbohydrates resulted in the expression of the reporter gene GUS comparable to 1-FFTexpression upon carbohydrate feeding in chicory. This study provides information on the regulation of inulin biosynthesis, suggestions for studies on transcription factors, and provides a promoter for steering the expression of fructan biosynthetic genes in transgenic plants. An alternative way for the production of inulin with the desired DP and yield, circumventing the problems in chicory rather than trying to solve them, is the introduction of the fructan biosynthetic pathway in non-fructan metabolizing and catabolizing plant species.
Towards this end we have expressed the inulin synthesizing enzymes, 1-SST and 1-FFT from Jerusalem artichoke, in maize and potato, as described inChapter 4. Transgenic maize plants produced inulin type fructan (at 3.2 milligram per gram kernel) and kernel development was not affected. Potato tubers expressing 1-SSTaccumulated 1.8 milligram inulin per gram tuber while tubers with a combined expression of 1-SSTand 1-FFTaccumulated 2.6 milligram inulin per gram tuber. Inulin accumulation in maize kernels was modulated by kernel development, first peaking in young seeds and then decreasing again through degradation during late kernel development. In potato, inulin mDP was relatively stable throughout tuber development and little evidence of degradation was observed. The accumulation of 1-kestose in transgenic maize correlated positively with kernel sucrose concentration and introduction of the fructan biosynthetic pathway in a high-sucrose maize background increased inulin accumulation to 41 milligram per gram kernel kernel. This study shows the importance of sugar availability and the absence of degradation mechanisms in platform crops for tailor-made fructan production.
Further evaluation of the production of tailor-made inulin and putative platform crops is discussed in Chapter5.Here we come to the conclusion that the mDP, the distribution and yield depend on the origin of the fructan biosynthesis genes and the availability of sucrose in the host. The combination of genes from different origins could result in new types and different lengths of fructan molecules resulting in (new) specific properties of fructan. Limitations for the production of tailor-made fructan in chicory are not seen in putative new platform crops, such as sugar beet, sugarcane and rice.
The work described in this thesis on fructan biosynthesis in chicory and in new platform crops has resulted in new insights that will lead new applied and fundamental research in this field.
N-Glycosylation of Plant-produced Recombinant Proteins
Bosch, H.J. ; Castilho, A. ; Loos, A. ; Schots, A. ; Steinkeller, H. - \ 2013
Current Pharmaceutical Design 19 (2013)31. - ISSN 1381-6128 - p. 5503 - 5512.
reactive carbohydrate determinants - human monoclonal-antibody - sialic acid transporter - arabidopsis-thaliana - transgenic plants - storage vacuoles - endoplasmic-reticulum - high-level - beta-1,4-n-acetylglucosaminyltransferase iii - human beta-1,4-galactosyltr
Plants are gaining increasingly acceptance as a production platform for recombinant proteins. One reason for this is their ability to carry out posttranslational protein modifications in a similar if not identical way as mammalian cells. The capability of plants to carry out human-like complex glycosylation is well known. Moreover, the targeted manipulation of the plant N-glycosylation pathway allows the production of proteins carrying largely homogeneous, human-type oligosaccharides. These outstanding results have placed plants in a favourable position compared to other eukaryotic expression systems. This review provides a comprehensive summary of the N-glycosylation of plant-produced recombinant proteins, the possible impact of plant-specific N-glycans on the human immune system, and recent advances in engineering the plant N-glycosylation pathway towards the synthesis of (complex) human-type glycan structures, highlighting challenges and achievements in the application of these powerful technologies
Unveiling and deploying durability of late blight resistance in potato : from natural stacking to cisgenic stacking
Kwang-Ryong Jo, - \ 2013
Wageningen University. Promotor(en): Richard Visser; Evert Jacobsen, co-promotor(en): Jack Vossen. - S.l. : s.n. - ISBN 9789085855798 - 168
solanum tuberosum - aardappelen - ziekteresistentie - phytophthora infestans - plantenziekteverwekkende schimmels - verdedigingsmechanismen - genkartering - transgene planten - solanum tuberosum - potatoes - disease resistance - phytophthora infestans - plant pathogenic fungi - defence mechanisms - gene mapping - transgenic plants
The potato, which receives an increased attention as a food crop, has long been in threats from the oomycete Phytophthora infestans, the causal agent of late blight. This disease still remains the most important constraint in potato producing regions of the world. It might cause the complete destruction of the foliage and tubers of potato if meteorological conditions are conducive to the onset and spread of late blight epidemics. Although fungicides applications provide sufficient levels of late blight control, they impose high input costs to the farmer, are detrimental to human and environment and increase the capacity of the pathogen to develop resistance to the active ingredients of fungicides applied. The increased genetic diversity in P. infestanspopulations due to sexual recombination between two mating types in many parts of the world and the emergence of fungicide resistant strains poses the necessity to develop potatoes that possess high levels of durable resistance as an alternative to the use of fungicides. Clones MaR8 and MaR9 from the Mastenbroek differential set, used to assess virulence towards Rgenes, have been known for their strong resistance to P. infestans. This also holds for cultivar Sarpo Mira which has retained resistance in the field over several years without fungicide applications. Uncovering genetic basis of such, partly naturally-formed, late blight resistance is the prerequisite for the implementation of durable resistance in a breeding scheme. In this study, MaR8, MaR9and cv. Sarpo Mira were used as plant materials for unveiling durability of late blight resistance in potato. First, F1 mapping populations from crosses between these resistant materials with susceptible parents were assessed for late blight resistance in field trials and in detached leaf assays (DLA) after inoculation with an incompatible P. infestans isolate IPO-C. A 1:1 segregation of resistance and susceptibility was observed in the MaR8derived-F1 population in field trials, but not in detached leaf assays. NBS profiling and Rgene cluster directed profiling (CDP), followed by marker landing in the newly sequenced potato genome, referred to as “anchored scaffold approach”, led to the mapping of R8at a new locus on chromosome IX rather than on chromosome XI, the previously suggested chromosomal position (Chapter 2). The Rgene mediated resistance reaction in potato is a consequence of an (in)direct interaction between the pathogen Avrand host Rgene product that leads to a hypersensitive cell death (HR). We screened a wide collection of RXLR effectors of P. infestansfor eliciting cell death in the differential potato MaR8 by agroinfiltration (Chapter 3). R8-specific cell death to one effector PITG_07558, termed AVR8, co-segregated with the R8-mediated resistance to P. infestansisolate IPO-C in a F1 population. From the notion that Avr8is identical to effector AvrSmira2that was previously found to associate with field resistance in cultivar Sarpo Mira, we performed genetic mapping studies in a Sarpo Mira-based F1 population and indeed Rpi-Smira2localized in the R8locus. To investigate the geographical and phylogenetic origin of R8in the Solanumgene pool, we conducted functional screens for AVR8 responsiveness in 98 wild genotypes (72 accessions of 40 species) of Solanumsection Petota. We identified twelve AVR8 responding Solanum accessions originating both from Central and South America. Interestingly, our study involving late blight resistance from the differential plant MaR9described that it is near the R8 locus on chromosome IX (Chapter 4). An integrated approach combining 1. a Rgene ”de-stacking” approach using Rgene specific marker analysis and effector responses, 2. the whole plant climate cell assay, and 3. CDP profiling enabled a clear picture for the presence of two closely linked genes, termed R9aand R9b. It was shown that R9alocates in a Tm-22 cluster of NB-LRR genes and, most likely will be a member of the Tm-22 Rgene family (Chapter 4). The identified fully co-segregating Tm-2 likeCDP markers were used to select the R9agene-containing BAC clone, demonstrating the possibility of BAC landing by marker saturation in the targeted chromosomal regions (Chapter 5). For cloning R9agene, a bacterial artificial chromosome (BAC) library derived from the differential plant MaR9, was screened with co-segregating Rgene CDP markers whereby two overlapping BAC clones carrying CDP markers were obtained. Sequence annotation of the complete insert of these BAC clones identified the presence of two complete Rgene analogs (RGA9.1 andRGA9.2) of the NB-LRR class in one BAC clone. Two RGAs, including their natural regulatory transcriptional elements, were subcloned by long-range PCR into a binary vector for plant transformation. After transformation, it was found that RGA9.1was able to complement the susceptible phenotype in cultivar Desiree. RGA9.1, now designated R9a,encodes a CC-NB-LRR protein of the Tm2 family, where the LRR consensus is only loosely fitted. Agroinfiltration-based effector screens for identifying the Avrgenes matching the R9agene was performed, leading to the discovery of Avrblb2 homologs which trigger R9amediated hypersensitivity in Nicotiana benthamiana (Chapter 5).Resistance profiling with 54 P. infestans isolates showed that MaR9 and S.xedinense accessions had similar resistance spectra as the Rpi-blb2containing cultivar Bionica. Transformation of potato with resistance genes and antibiotic resistance markers encounters consumers’ criticism. These criticisms are considerably less if only resistance genes from crossable species, and no antibiotic resistance selection marker is used. Genes deriving from crossable species are referred to as cisgenes. For the production of cisgenic potatoes with a broader resistance spectrum and potential durability, Agrobacterium-mediated marker free transformation and PCR selection of transformants was performed. This way four potato cultivars (Atlantic, Bintje, Potae9 and Doip1) were successfully transformed with a construct containing two cisgenic Rpigenes (Rpi-vnt1from Solanum venturiiand Rpi-sto1from Solanum stoloniferum) (Chapter 6). Resistance assays in untransformed varieties with five P. infestansisolates showed that cvs. Potae9 and Doip1 were already resistant to certain isolates. Single Rpigene containing transgenic plants for all 4 varieties were obtained and used as references. Marker free transformation with a construct containing two Rpi genes (cisgenesis) was compared to kanamycin assisted selection (transgenesis) in terms of regeneration and transformation frequency, vector backbone integration, and T-DNA copy number. In addition, the different time tracks to harvest regenerated shoots for the selection of PCR positive regenerants for one or both Rpi-genes were studied. Through further analyses involving phenotypic evaluations in the greenhouse, agroinfiltration of avirulence (Avr) genes and detached leaf assays, totally eight cisgenic plants were selected. Two cisgenic plants of cv. Altantic and four of cv. Bintje, were selected that showed broad spectrum late blight resistance due to the activity of both Rpigenes. Based on characterization of two cisgenic transformants of cv. Potae9, it was demonstrated that the existing late blight resistance spectrum has been broadened by adding the two Rpigenes. Finally, results from this study are discussed in terms of genetic and molecular mechanism of durability and cisgenic deployment to address the challenges of the durable resistant potato variety development (Chapter 7). We pursue possible options for durability in the nature of the Rgenes or their cognate Avrgenes. The comparative analysis of several features of available R-AVR pairs shows that major components for producing durability are the copy number variation in the P. infestansgenome and abundance of the Avrgene in different isolates. As a counterpart of such an Avrgene, potato Rgenes that display broad spectrum resistance and often have abundant functional homologs among various wild Solanumspecies could be optional for Rgene combinations providing durability. Multiple years’ on-site-monitoring of resistance spectrum in natural Rgene stacks demonstrates that stacking of several broad spectrum Rpigenes or even “defeated” Rgenes could sum up to high levels of resistance potentially capable to provide durability to commercial potato cultivars. Our data about acquirement of complementary resistance spectrum by cisgenic introduction of two broad spectrum resistance genes into cultivars support a first step into that direction.