Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Recognition of Verticillium effector Ave1 by tomato immune receptor Ve1 mediates Verticillium resistance in diverse plant species
Song, Yin - \ 2017
University. Promotor(en): Bart Thomma; Pierre 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 - tomaten - receptoren - genen - tabak - aardappelen - katoen - transgene planten

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.

(+)-Valencene production in Nicotiana benthamiana is increased by down-regulation of competing pathways
Cankar, K. ; Jongedijk, E.J. ; Klompmaker, M. ; Majdic, T. ; Mumm, R. ; Bouwmeester, H.J. ; Bosch, H.J. ; Beekwilder, M.J. - \ 2015
Biotechnology Journal 10 (2015)1. - ISSN 1860-6768 - p. 180 - 189.
plant transformation - biosynthetic-pathway - terpenoid metabolism - squalene synthase - tobacco - expression - artemisinin - arabidopsis - reductase - precursors
Plant sesquiterpenes, such as (+)-valencene, artemisinin, and farnesene are valuable chemicals for use as aromatics, pharmaceuticals, and biofuels. Plant-based production systems for terpenoids critically depend on the availability of farnesyl diphosphate (FPP). Currently, these systems show insufficient yields, due to the competition for FPP of newly introduced pathways with endogenous ones. In this study, for the first time an RNAi strategy aiming at silencing of endogenous pathways for increased (+)-valencene production was employed. Firstly, a transient production system for (+)-valencene in Nicotiana benthamiana was set up using agroinfiltration. Secondly, silencing of the endogenous 5-epi-aristolochene synthase (EAS) and squalene synthase (SQS) that compete for the FPP pool was deployed. This resulted in a N. benthamiana plant that produces (+)-valencene as a prevalent volatile with a 2.8-fold increased yield. Finally, the size of the FPP pool was increased by overexpression of enzymes that are rate-limiting in FPP biosynthesis. Combined with silencing of EAS and SQS, no further increase of (+)-valencene production was observed, but emission of farnesol. Formation of farnesol, which is a breakdown product of FPP, indicates that overproducing sesquiterpenes is no longer limited by FPP availability in the cytosol. This study shows that metabolic engineering of plants can effectively be used for increased production of desired products in plants. Keywords: 5-Epi-aristolochene synthase · Metabolic engineering · RNAi · Squalene synthase
in tissue culture of lilium explants may become heavily contaminated by the standard initiation procedure
Askari Rabori, N. ; Wang, Y.G. ; Klerk, G.J.M. de - \ 2014
Propagation of ornamental plants 14 (2014)2. - ISSN 1311-9109 - p. 49 - 56.
sodium-hypochlorite - bud regeneration - cell-cultures - plant-tissue - in-vitro - sterilization - tobacco
In tissue culture of Lilium, the standard initiation procedure brought about substantial contamination in two ways. (1) When scales were detached from the mother bulb, microorganisms could enter via the wound. This source of contamination was strongly enhanced by the negative hydrostatic pressure within the scales by which nonsterile fluid was sucked up at detachment. Contamination decreased strongly when the scales were detached from bulbs submerged in 0.03% NaClO. Evidence is presented that this type of contamination was endogenous, i.e., localized in the interior of the explant. (2) During the rinsing of scales after surface-sterilization, the rinsing water became contaminated with microorganisms associated with the scales that had not been killed during surface-sterilization. This caused cross-contamination. This type of additional contamination was controlled by rinsing in 0.03% NaClO instead of 'sterile' water. In our conditions, these initiation-related sources of contamination led to ca. 20% and ca. 25% contamination, respectively, of otherwise uninfected scales.
Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves
Outchkourov, N.S. ; Carollo, C.A. ; Gomez Roldan, M.V. ; Vos, C.H. de; Bosch, H.J. ; Hall, R.D. ; Beekwilder, M.J. - \ 2014
Frontiers in Plant Science 5 (2014). - ISSN 1664-462X - 9 p.
r2r3-myb gene family - mass-spectrometry - tomato fruit - arabidopsis-thaliana - biosynthesis - tobacco - accumulation - metabolomics - plants - health
Coloration of plant organs such as fruit, leaves and flowers through anthocyanin production is governed by a combination of MYB and bHLH type transcription factors (TFs). In this study we introduced Rosea1 (ROS1, a MYB type) and Delila (DEL, a bHLH type), into Nicotiana benthamiana leaves by agroinfiltration. ROS1 and DEL form a pair of well-characterized TFs from Snapdragon (Antirrhinum majus), which specifically induce anthocyanin accumulation when expressed in tomato fruit. In N. benthamiana, robust induction of a single anthocyanin, delphinidin-3-rutinoside (D3R) was observed after expression of both ROS1 and DEL. Surprisingly in addition to D3R, a range of additional metabolites were also strongly and specifically up-regulated upon expression of ROS1 and DEL. Except for the D3R, these induced compounds were not derived from the flavonoid pathway. Most notable among these are nornicotine conjugates with butanoyl, hexanoyl, and octanoyl hydrophobic moieties, and phenylpropanoid-polyamine conjugates such as caffeoyl putrescine. The defensive properties of the induced molecules were addressed in bioassays using the tobacco specialist lepidopteran insect Manduca sexta. Our study showed that the effect of ROS1 and DEL expression in N. benthamiana leaves extends beyond the flavonoid pathway. Apparently the same transcription factor may regulate different secondary metabolite pathways in different plant species.
Black shank of tobacco in the former Dutch East Indies, caused by Phytophthora nicotianae
Zadoks, J.C. - \ 2014
Leiden, The Netherlands : Sidestone Press - ISBN 9789088902833 - 206
nicotiana tabacum - tabak - phytophthora nicotianae - oömycota - plantenziekten - nederlands indië - tobacco - oomycota - plant diseases - netherlands east indies
Jacob van Breda de Haan is known as the author of the name Phytophthora nicotianae n.sp., the causal agent of ‘black shank’, an important disease of tobacco. Who was he? Where did he work? What did he publish? He published in Dutch, 1896, in a Dutch colonial report series. Next question: what more on tobacco diseases was written in obscure, colonial Dutch documents? Another scientist, Thung Tjeng Hiang, better known as the first Wageningen professor of plant virology, presented two original papers in Dutch on ‘black shank’ with the word ‘epidemiologie’ in their title, 1931 and 1938. Therewith Thung was an early bird in plant disease epidemiology. The foundational paper by van Breda de Haan and two important papers by Thung are presented here in English translation. Both authors worked in the former Dutch East Indies, present Indonesia, the first on the island of Sumatera, the latter on that of Java. Both were in the service of tobacco planters; they had to solve immediate problems as fast as possible. In a pioneer situation, van Breda de Haan was confronted with a sudden seedling disease which devastated the tobacco seedlings in the seed beds and which, yes, could lead to ‘black shank’ in adult plants. Thung, working in a well-organized environment, had to prevent ‘black shank’ in the tobacco plantations. Both authors were successful in controlling disease by means of a combination of ecological intervention and chemical treatment. Whereas van Breda de Haan could only dream of genetic control, Thung could incorporate the use of a fairly resistant cultivar in his recommendations. The 1896 paper has epidemiological observations scattered throughout, without using the word epidemiology. The 1931 and 1938 papers are probably ‘firsts’ in the Dutch phytopathological literature having epidemiology in their title, one an early study in quantitative, comparative epidemiology and the other an early version of landscape epidemiology. The three papers are preceded by a sketch of tobacco cultivation in the former Dutch East Indies, describing the position of the two authors in the tobacco scene; they are followed by a long-due biography of a forgotten plant pathologist, Jacob van Breda de Haan.
The Tomato spotted wilt virus cell-to-cell movement protein (NSM) triggers a hypersensitive response in Sw-5 containing resistant tomato lines and Nicotiana benthamiana transformed with the functional Sw-5b resistance gene copy.
Hallwass, M. ; Silva de Oliveira, A. ; Dianese, E.C. ; Lohuis, D. ; Boiteux, L.S. ; Inoue-Nagata, A.K. ; Resende, R.O. de; Kormelink, R.J.M. - \ 2014
Molecular Plant Pathology 15 (2014)9. - ISSN 1464-6722 - p. 871 - 880.
mosaic-virus - lycopersicon-esculentum - nonstructural protein - capsicum-chinense - coat protein - plant-cells - rna segment - tswv - tospovirus - tobacco
Although the Sw-5 gene cluster has been cloned, and Sw-5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum SR1 plants transformed with a copy of the Sw-5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N.benthamiana transformed with Sw-5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non-structural TSWV proteins tested, the TSWV cell-to-cell movement protein (NSM) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non-replicative pEAQ-HT expression vector system. HR was induced in Sw-5b-transgenic N.benthamiana as well as in resistant near-isogenic tomato lines after agroinfiltration with a functional cell-to-cell movement protein (NSM) from a resistance-inducing (RI) TSWV strain (BR-01), but not with NSM from a Sw-5 resistance-breaking (RB) strain (GRAU). This is the first biological demonstration that Sw-5-mediated resistance is triggered by the TSWV NSM cell-to-cell movement protein.
Silencing an N-Acyltransferase-Like Involved in Lignin Biosynthesis in Nicotiana attenuata Dramatically Alters Herbivory-Induced Phenolamide Metabolism
Gaquerel, E. ; Kotkar, H. ; Onkokesung, N. ; Galis, I. ; Baldwin, I.T. - \ 2013
PLoS One 8 (2013)5. - ISSN 1932-6203
arabidopsis-thaliana - jasmonic acid - insect herbivores - mediated defenses - down-regulation - pathway - tobacco - accumulation - evolution - shikimate
In a transcriptomic screen of Manduca sexta-induced N-acyltransferases in leaves of Nicotiana attenuata, we identified an N-acyltransferase gene sharing a high similarity with the tobacco lignin-biosynthetic hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) gene whose expression is controlled by MYB8, a transcription factor that regulates the production of phenylpropanoid polyamine conjugates (phenolamides, PAs). To evaluate the involvement of this HCT-like gene in lignin production as well as the resulting crosstalk with PA metabolism during insect herbivory, we transiently silenced (by VIGs) the expression of this gene and performed non-targeted (UHPLC-ESI/TOF-MS) metabolomics analyses. In agreement with a conserved function of N. attenuata HCT-like in lignin biogenesis, HCT-silenced plants developed weak, soft stems with greatly reduced lignin contents. Metabolic profiling demonstrated large shifts (up to 12% deregulation in total extracted ions in insect-attacked leaves) due to a large diversion of activated coumaric acid units into the production of developmentally and herbivory-induced coumaroyl-containing PAs (N',N''-dicoumaroylspermidine, N',N''-coumaroylputrescine, etc) and to minor increases in the most abundant free phenolics (chlorogenic and cryptochlorogenic acids), all without altering the production of well characterized herbivory-responsive caffeoyl- and feruloyl-based putrescine and spermidine PAs. These data are consistent with a strong metabolic tension, exacerbated during herbivory, over the allocation of coumaroyl-CoA units among lignin and unusual coumaroyl-containing PAs, and rule out a role for HCT-LIKE in tuning the herbivory-induced accumulation of other PAs. Additionally, these results are consistent with a role for lignification as an induced anti-herbivore defense.
MYB8 Controls Inducible Phenolamide Levels by Activating Three Novel Hydroxycinnamoyl-Coenzyme A:Polyamine Transferases in Nicotiana attenuata[W][OA]
Onkokesung, N. ; Gaquerel, E. ; Kotkar, H. ; Kaur, H. ; Baldwin, I.T. ; Galis, I. - \ 2012
Plant Physiology 158 (2012)1. - ISSN 0032-0889 - p. 389 - 407.
ultraviolet-b radiation - transcription factor - plant defense - acid-amides - convergent evolution - simulated herbivory - insect herbivores - responses - tobacco - metabolism
A large number of plants accumulate N-acylated polyamines (phenolamides [PAs]) in response to biotic and/or abiotic stress conditions. In the native tobacco (Nicotiana attenuata), the accumulation of two major PAs, caffeoylputrescine and dicaffeoylspermidine (DCS), after herbivore attack is known to be controlled by a key transcription factor, MYB8. Using a broadly targeted metabolomics approach, we show that a much larger spectrum of PAs composed of hydroxycinnamic acids and two polyamines, putrescine and spermidine, is regulated by this transcription factor. We cloned several novel MYB8-regulated genes, annotated as putative acyltransferases, and analyzed their function. One of the novel acyltransferases (AT1) is shown to encode a hydroxycinnamoyl-coenzyme A:putrescine acyltransferase responsible for caffeoylputrescine biosynthesis in tobacco. Another gene (acyltransferase DH29), specific for spermidine conjugation, mediates the initial acylation step in DCS formation. Although this enzyme was not able to perform the second acylation toward DCS biosynthesis, another acyltransferase gene, CV86, proposed to act on monoacylated spermidines, was isolated and partially characterized. The activation of MYB8 in response to herbivore attack and associated signals required the activity of LIPOXYGENASE3, a gene involved in jasmonic acid (JA) biosynthesis in N. attenuata. These new results allow us to reconstruct a complete branch in JA signaling that defends N. attenuata plants against herbivores: JA via MYB8’s transcriptional control of AT1 and DH29 genes controls the entire branch of PA biosynthesis, which allows N. attenuata to mount a chemically diverse (and likely efficient) defense shield against herbivores.
An avirulent tomato powdery mildew isolate induces localized acquired resistance to a virulent isolate in a spatiotemporal manner
Seifi, A. ; Nonomura, T. ; Matsuda, Y. ; Toyoda, H. ; Bai, Y. - \ 2012
Molecular Plant-Microbe Interactions 25 (2012)3. - ISSN 0894-0282 - p. 372 - 378.
oidium-neolycopersici - hypersensitive response - ol-genes - lycopersicon - virus - identification - pathogens - tobacco - plants
Hypersensitive response (HR) of plant cells to the attack of pathogens induces resistance to subsequent attacks by a broad spectrum of pathogens, leading to acquired resistance. In this study, we characterized the localized acquired resistance (LAR) in the epidermal cells of tomato. First, we report the discovery of a new isolate of tomato powdery mildew occurring in Japan, KTP-02, which has a different virulence spectrum compared with the previously-characterized isolate, KTP-01. Using these two isolates, we investigated LAR phenomenon in the epidermal cells of tomato plants carrying the Ol-4 resistance gene. Ol-4 encodes a nucleotide-binding site leucine-rich repeat protein that triggers HR in the epidermal cells in response to KTP-01 but not KTP-02. We mounted a single conidium of KTP-01 on a single tomato epidermal cell and then monitored the progress of HR in that cell by live microscopy. Once HR occurred in that cell, we mounted a single conidium of KTP-02 on cells adjacent to or at one-cell distance from the first challenged cells, in different time points. With a digital microscope, we consecutively tracked the progress of HR (i.e., induction of LAR) in those cells. Results showed that, in tomato plants carrying the Ol-4 gene, HR to KTP-01 results in induction of HR in the adjacent epidermal cells challenged with KTP-02. Our results show that LAR can be triggered only in adjacent cell layer and lasts 24 to 48 h after HR occurred in the first cell. We did not observe the reverse phenomenon, induced susceptibility to KTP-01 by KTP-02. Altogether, we report an advanced technique for investigating LAR phenomena, and provide data on spatiotemporal characteristics of LAR in tomato epidermal cells.
Exploring opportunities for diversification of specialized tobacco farms in the Northwest of Argentina
Chavez Clemente, M.D. - \ 2012
University. Promotor(en): Alfons Oude Lansink, co-promotor(en): Paul Berentsen. - S.l. : s.n. - ISBN 9789461734204 - 161
tabak - gespecialiseerde landbouw - gewasproductie - landbouwbedrijven - diversificatie - specialisatie - inkomen - risico - bodemdegradatie - argentinië - tobacco - specialized farming - crop production - farms - diversification - specialization - income - risk - soil degradation - argentina
In the Northwest of Argentina tobacco (Nicotiana tabacum L.) is economically and socially important. Tobacco mono-cropping, excessive tillage and inadequate irrigation management cause soil degradation. This and also tobacco production dependence on government subsidies and concern about health damage from tobacco consumption calls for research on diversification. The aim of this thesis was to explore opportunities for diversification of specialized tobacco farms in the Northwest of Argentina.
Plant neighbor detection through touching leaf tips precedes phytochrome signals
Wit, M. de; Kegge, W. ; Evers, J.B. ; Vergeer-van Eijk, M.H. ; Gankema, P. ; Voesenek, L.A.C.J. ; Pierik, R. - \ 2012
Proceedings of the National Academy of Sciences of the United States of America 109 (2012)36. - ISSN 0027-8424 - p. 14705 - 14710.
arabidopsis-thaliana - shade avoidance - petiole elongation - light quality - growth - responses - ethylene - gene - tobacco - perception
Plants in dense vegetation compete for resources, including light, and optimize their growth based on neighbor detection cues. The best studied of such behaviors is the shade-avoidance syndrome that positions leaves in optimally lit zones of a vegetation. Although proximate vegetation is known to be sensed through a reduced ratio between red and far-red light, we show here through computational modeling and manipulative experiments that leaves of the rosette species Arabidopsis thaliana first need to move upward to generate sufficient light reflection potential for subsequent occurrence and perception of a reduced red to far-red ratio. This early hyponastic leaf growth response is not induced by known neighbor detection cues under both climate chamber and natural sunlight conditions, and we identify a unique way for plants to detect future competitors through touching of leaf tips. This signal occurs before light signals and appears to be the earliest means of above-ground plant–plant signaling in horizontally growing rosette plants.
A potato pathogenesis-related protein gene, StPRp27, contributes to race-nonspecific resistance against Phytophthora infestans
Shi, X. ; Tian, Z. ; Liu, Jun ; Vossen, E.A.G. van der; Xie, Conghua - \ 2012
Molecular Biology Reports 39 (2012)2. - ISSN 0301-4851 - p. 1909 - 1916.
broad-spectrum resistance - late blight resistance - quantitative resistance - solanum-bulbocastanum - r-gene - expression - infection - tobacco - identification - population
Late blight caused by Phytophthora infestans is the most important disease of potato. Many efforts have been made to understand molecular mechanism of the durable resistance to address the challenge raised by rapid evolution of the pathogen. A pathogenesis related protein (PR) gene StPRp27 was previously isolated from the potato leaves challenged by P. infestans. The sequence analysis and expression pattern reveal that StPRp27 may be associated with resistance to P. infestans. In present research, transient expression of StPRp27 in Nicotiana benthamiana enhanced resistance to P. infestans isolates 99189 and PY23 indicating its potential contribution to the disease resistance. These findings were also confirmed by over-expression of StPRp27 in potato cv. E-potato 3, which significantly slowed down the development of the disease after inoculation with a mixture of P. infestans races. Further, silencing of StPRp27 homologous genes in N. benthamiana harboring dominant Phytophthora resistance gene Rpi-blb1 or Rpi-blb2 showed no effects on the resistance triggered by these R genes. Our results suggest that StPRp27 contributes to a race-nonspecific resistance against P. infestans by inhibiting the disease development and has a potential use in selection and breeding for durable resistance to late blight
A large proportion of esophageal cancer cases and the incidence difference between regions are attributable to lifestyle risk factors in China
Wu, M. ; Veer, P. van 't; Zhang, Z.F. ; Wang, X.S. ; Gu, X.P. ; Han, R.Q. ; Yang, J. ; Zhang, X.F. ; Liu, A.M. ; Kok, F.J. ; Kampman, E. ; Zhao, J.K. - \ 2011
Cancer Letters 308 (2011)2. - ISSN 0304-3835 - p. 189 - 196.
squamous-cell carcinoma - high-epidemic area - jiangsu province - confidence-intervals - alcohol-drinking - stomach-cancer - smoking - fractions - definition - tobacco
A population-based case-control study was conducted in a high-risk area (Dafeng) and a low-risk area (Ganyu) of Jiangsu province, China. In this analysis, the population attributable fraction (PAF) was calculated to quantify the etiology of risk factors; the relative attributable risk (RAR) was applied to explore how much of the incidence difference could be explained by variations in the distribution of risk factors. Results showed that unhealthy lifestyles accounted for a high fraction of esophageal cancer in China. Dissimilar distribution of several lifestyle factors, together with hereditary variations may be largely responsible for the incidence difference between areas.
Potato R1 resistance gene confers resistance against Phytophthora infestans in transgenic tomato plants
Faino, L. ; Carli, P. ; Testa, A. ; Cristinzio, G. ; Frusciante, L. ; Ercolano, M.R. - \ 2010
European Journal of Plant Pathology 128 (2010)2. - ISSN 0929-1873 - p. 233 - 241.
late blight resistance - real-time pcr - copy number - dna fragments - mosaic-virus - transformation - zygosity - tobacco - expression - efficient
Tomato is challenged by several pathogens which cause loss of production. One such pathogen is the oomycete Phytophthora infestans which is able to attack all the aerial parts of the plant. Although a wide range of resistance sources are available, genetic control of this disease is not yet successful. Pyramiding R-genes through genetic transformation could be a straightforward way to produce tomato and potato lines carrying durable resistance to P. infestans. In this work the R1 potato gene was transferred into tomato lines. The tomato transgenic lines were analyzed by using q-RT-PCR and progeny segregation to determine the gene copy number. To test the hypothesis that R1 represents a specifically regulated R-gene, transgenic tomato plants were inoculated with P. infestans isolate 88133 and IPO. All the plants containing the R1 gene were resistant to the late blight isolate IPO-0 and susceptible to isolate 88133. These results provide evidence for specific activation of the R1 gene during pathogen challenge. Furthermore, evidence for enhancement of PR-1 gene expression during P. infestans resistance response was obtained.
Nicotiana benthamiana as a Production Platform for Artemisinin Precursors
Herpen, T.W.J.M. van; Cankar, K. ; Nogueira, M. ; Bosch, H.J. ; Bouwmeester, H.J. ; Beekwilder, M.J. - \ 2010
PLoS One 5 (2010)12. - ISSN 1932-6203 - 11 p.
antimalarial-drug artemisinin - expression system - molecular-cloning - plants - biosynthesis - annua - reductase - tobacco - yield - acid
Background Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment. Methodology/Principal Findings Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-ß-diglucoside. This compound accumulated to 39.5 mg.kg-1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana. Conclusion/Significance This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed.
De Terugkeer van Tabak
Keulemans, M. ; Bosch, H.J. - \ 2009
Natuurwetenschap & Techniek 2009 (2009)1. - ISSN 1573-6083 - p. 49 - 53.
tabak - biotechnologie - nieuwe cultuurgewassen - tobacco - biotechnology - new crops
Tabak als schoonmaker Onthouden dat woord: ‘fytoremediatie’. De modieuze term, die steeds vaker opduikt in de vakbladen, staat voor genetisch gewijzigde planten die vervuiling opruimen. En de tabaksplant zou de tabaksplant niet zijn, als hij niet ook hier van de partij was. In Engeland experimenteren onderzoekers van de universiteit van Cambridge bijvoorbeeld met tabaksplanten die in staat zijn om restjes explosieven te verwerken tot onschadelijke stoffen. Ideaal voor oude munitiedepots en verlaten slagvelden, aldus de onderzoekers
PVYNTN elicits a diverse gene expression response in different potato genotypes in the first 12 h after inoculation
Baebler, S. ; Krecic-Stres, H. ; Rotter, A. ; Kogovsek, P. ; Cankar, K. ; Kok, E.J. ; Gruden, K. ; Kovac, M. ; Zel, J. ; Pompe-Novak, M. ; Ravnikar, M. - \ 2009
Molecular Plant Pathology 10 (2009)2. - ISSN 1464-6722 - p. 263 - 275.
mosaic-virus-infection - arabidopsis-thaliana - cysteine proteinases - real-time - y-ntn - resistance - tobacco - defense - plants - microarray
Host gene expression changes in the early response to potato virus Y-NTN interaction were compared in two differently sensitive potato cultivars: the resistant cultivar SantE and the sensitive cultivar Igor. Hybridization of potato TIGR cDNA microarrays allowed us to monitor the expression of approximately 10 000 genes simultaneously at 0.5 and 12 h post-inoculation (hpi). Microarray data, analysed by statistics and data mining, were complemented by subtraction library construction and sequence analysis to validate the findings. The expression profiles of the two cultivars were similar and faint at 0.5 hpi, but they differed substantially at 12 hpi. Although, at 0.5 hpi, cv. SantE responded by the differential expression of a greater number of genes, at 12 hpi the number was higher in cv. Igor. The majority of genes in this cultivar were down-regulated at 12 hpi, indicating a host gene shut-off. Suites of genes that exhibited altered transcript abundance in response to the virus were identified, and included genes involved in the processes of photosynthesis, perception, signalling and defence responses. The expression of the considerable number of genes associated with photosynthesis was surprisingly up-regulated as early as 0.5 hpi and down-regulated at 12 hpi in both cultivars. The expression of genes involved in perception and signalling was increased in the sensitive cultivar at 12 hpi. By contrast, a simultaneous strong defence response at the transcriptional level was evident in the resistant cultivar, as shown by the up-regulation of genes involved in brassinosteroid, polyamine and secondary metabolite biosynthesis, and of genes coding for pathogenesis-related proteins.
Requirements for ER-Arrest and Sequential Exit to the Golgi of Tomato Spotted Wilt Virus Glycoproteins
Ribeiro, D.M.O.G. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2009
Traffic 10 (2009)6. - ISSN 1398-9219 - p. 664 - 672.
plant endoplasmic-reticulum - green fluorescent protein - copii vesicle formation - brefeldin-a - transient association - soluble-proteins - export sites - cells - tobacco - maturation
The envelope glycoproteins Gn and Gc are major determinants in the assembly of Tomato spotted wilt virus (TSWV) particles at the Golgi complex. In this article, the ER-arrest of singly expressed Gc and the transport of both glycoproteins to the Golgi upon co-expression have been analyzed. While preliminary results suggest that the arrest of Gc at the ER (endoplasmic reticulum) did not appear to result from improper folding, transient expression of chimeric Gc, in which the transmembrane domain (TMD) and/or cytoplasmic tail (CT) were swapped for those from Gn, showed that the TMD of Gn was sufficient to allow ER-exit and transport to the Golgi. Expression of both glycoproteins in the presence of overexpressed Sar1p-specific guanosine nucleotide exchange factor Sec 12p, resulted in ER-retention demonstrating that the viral glycoproteins are transported to the Golgi in a COPII (coat protein II)-dependent manner. Inhibition of ER-Golgi transport by brefeldin A (BFA) had a similar effect on the localization of Gn. However, inhibition of ER (endoplasmic reticulum) to Golgi transport of co-expressed Gc and Gn by overexpression of Sec 12p or by BFA revealed distinct localization patterns, i.e. diffuse ER localization versus concentration at specific spots
Microspore embryogenesis in barley: anther pre-treatment stimulates plant defence gene expression
Jacquard, C. ; Mazeyrat-Gourbeyre, F. ; Devaux, P. ; Boutilier, K.A. - \ 2009
Planta 229 (2009)2. - ISSN 0032-0935 - p. 393 - 402.
hordeum-vulgare l. - in-vitro - oxidative burst - storage proteins - oxalate oxidase - stress - culture - arabidopsis - infection - tobacco
Microspore embryogenesis (ME) is a process in which the gametophytic pollen programme of the microspore is reorientated towards a new embryo sporophytic programme. This process requires a stress treatment, usually performed in the anther or isolated microspores for several days. Despite the universal use of stress to induce ME, very few studies have addressed the physiological processes that occur in the anther during this step. To further understand the processes triggered by stress treatment, we followed the response of anthers by measuring the expression of stress-related genes in two barley (Hordeum vulgare L.) cultivars differing in their ME response. Genes encoding enzymes involved in oxidative stress (glutathione-S-transferase, GST; oxalate oxidase, OxO), in the synthesis of jasmonic acid (13-lipoxygenase, Lox; allene oxide cyclase, AOC; allene oxide synthase, AOS) and in the phenylpropanoid pathway (phenylalanine ammonia lyase, PAL), as well as those encoding PR proteins (Barwin, chitinase 2b, Chit 2b; glucanase, Gluc; basic pathogenesis-related protein 1, PR1; pathogenesis-related protein 10, PR10) were up-regulated in whole anthers upon stress treatment, indicating that anther perceives stress and reacts by triggering general plant defence mechanisms. In particular, both OxO and Chit 2b genes are good markers of anther reactivity owing to their high level of induction during the stress treatment. The effect of copper sulphate appeared to limit the expression of defence-related genes, which may be correlated with its positive effect on the yield of microspore
Tomato spotted wilt virus nucleocapsid protein interacts with both viral glycoproteins Gn and Gc in planta
Ribeiro, D.M.O.G. ; Borst, J.W. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2009
Virology 383 (2009)1. - ISSN 0042-6822 - p. 121 - 130.
endoplasmic-reticulum - homotypic interaction - cells - golgi - localization - microscopy - infection - tobacco
Recently, the Tomato Spotted Wilt Virus (TSWV) Gn and Gc glycoproteins were shown to induce the formation of (pseudo-) circular and pleomorphic membrane structures upon transient expression in plant cells. Furthermore, when singly expressed, Gc retains in the ER, while Gn is able to further migrate to the Golgi. Upon co-expression, Gn rescues Gc and co-migrates to the Golgi complex. Here, we have studied the behavior of the glycoproteins in the presence of the viral nucleocapsid (N) protein and in vivo analyzed the occurrence of protein¿protein interactions by fluorescence life time imaging microscopy (FLIM). The analysis demonstrated that N co-localizes and interacts with both glycoproteins, with a preference for Gn. Additionally, it is shown that N causes a dramatic change in the distribution of Gc within the ER, from reticular to punctate spots. The observations are discussed in the context of the virus particle formation during the infection process.
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