Plant growth promotion by Pseudomonas fluorescens : mechanisms, genes and regulation
Cheng, X. - \ 2016
Wageningen University. Promotor(en): Francine Govers; J.M. Raaijmakers, co-promotor(en): M. van der Voort. - Wageningen : Wageningen University - ISBN 9789462578753 - 192
soil bacteria - pseudomonas fluorescens - plants - growth stimulators - soil suppressiveness - plant diseases - induced resistance - biochemistry - biosynthesis - plant-microbe interactions - transcriptomics - bodembacteriën - pseudomonas fluorescens - planten - groeistimulatoren - bodemweerbaarheid - plantenziekten - geïnduceerde resistentie - biochemie - biosynthese - plant-microbe interacties - transcriptomica
Pseudomonas fluorescens is a Gram-negative rod shaped bacterium that has a versatile metabolism and is widely spread in soil and water. P. fluorescens strain SBW25 (Pf.SBW25) is a well-known model strain to study bacterial evolution, plant colonization and biocontrol of plant diseases. It produces the biosurfactant viscosin, a lipopeptide that plays a key role in motility, biofilm formation and activity against zoospores of Phytophthora infestans and other oomycete pathogens. In addition to viscosin, Pf.SBW25 produces other metabolites with activity against plant pathogens. The production of these yet unknown metabolites appeared to be regulated by the GacS/GacA two-component regulatory system (the Gac-system). The second P. fluorescens strain SS101 (Pf.SS101) studied in this thesis is known for its plant growth-promoting activities but the underlying mechanisms and genes are largely unknown. Therefore, in this study, we aimed to identify novel metabolites and biosynthetic genes in Pf.SBW25 and Pf.SS101, and to investigate their role in plant growth promotion and biocontrol. To this end, a multidisciplinary approach involving bioinformatic analysis of the genome sequences of strains Pf.SBW25 and Pf.SS101, microarray-based expression profiling, screening of genomic libraries, bioactivity assays, mass spectrometric image analysis (MALDI-IMS) and GC/MSMS analysis was adopted. In conclusion, we showed that the GacS/GacA two-component system as a global regulator of the expression of genes play important roles in antagonism of Pseudomonas fluorescens toward plant pathogenic microbes as well as in plant growth promotion and ISR. Growth promotion by P. fluorescens is associated with alterations in auxin biosynthesis and transport, steroid biosynthesis, carbohydrate metabolism and sulfur assimilation. Moreover, advanced chemical profiling allowed us to compare the metabolite profiles of free-living P. fluorescens and P. fluorescens living in association with plant roots. A better understanding of yet unknown mechanisms exploited by the various Pseudomonas fluorescens strains will lead to new opportunities for the discovery and application of natural bioactive compounds for both industrial and agricultural purposes.
Regulation of cucumber (Cucumis sativus) induced defence against the two-spotted spider mite (Tetranychus urticae
He, J. - \ 2016
Wageningen University. Promotor(en): Harro Bouwmeester; Marcel Dicke, co-promotor(en): Iris Kappers. - Wageningen : Wageningen University - ISBN 9789462576810 - 211
cucumis sativus - cucumbers - induced resistance - plant pests - tetranychus urticae - mites - defence mechanisms - herbivore induced plant volatiles - herbivory - metabolomics - terpenoids - genomics - cucumis sativus - komkommers - geïnduceerde resistentie - plantenplagen - tetranychus urticae - mijten - verdedigingsmechanismen - herbivoor-geinduceerde plantengeuren - herbivorie - metabolomica - terpenen - genomica
Plants have evolved mechanisms to combat herbivory. These mechanisms can be classified as direct defences which have a negative influence on the herbivores and indirect defence that attracts natural enemies of the attacking herbivores. Both direct and indirect defences can be constantly present or induced upon attack. This study, using cucumber (Cucumis sativus) and the two-spotted spider mite (Tetranychus urticae) as model, aimed to reveal the molecular mechanisms underlying the induced defence during herbivory, with emphasis on transcriptional changes and the involved TFs, the enzymatic function of the genes associated with volatile biosynthesis, and their promoters which regulate their expression.
Getting prepared for future attack : induction of plant defences by herbivore egg deposition and consequences for the insect community
Pashalidou, F.G. - \ 2015
Wageningen University. Promotor(en): Marcel Dicke; Joop van Loon, co-promotor(en): Nina Fatouros. - Wageningen : Wageningen University - ISBN 9789462574120 - 168
insect-plant relaties - planten - insectenplagen - herbivorie - verdedigingsmechanismen - geïnduceerde resistentie - herbivoor-geinduceerde plantengeuren - ovipositie - natuurlijke vijanden - brassica - pieris brassicae - trofische graden - sluipwespen - hyperparasitoïden - insectengemeenschappen - insect plant relations - plants - insect pests - herbivory - defence mechanisms - induced resistance - herbivore induced plant volatiles - oviposition - natural enemies - brassica - pieris brassicae - trophic levels - parasitoid wasps - hyperparasitoids - insect communities
Plants have evolved intriguing defences against insect herbivores. Compared to constitutive Plants have evolved intriguing defences against insect herbivores. Compared to constitutive defences that are always present, plants can respond with inducible defences when they are attacked. Insect herbivores can induce phenotypic changes in plants and consequently these changes may differentially affect subsequent attackers and their associated insect communities. Many studies consider herbivore-feeding damage as the first interaction between plants and insects. The originality of this study was to start with the first phase of herbivore attack, egg deposition, to understand the consequences of plant responses to eggs on subsequently feeding caterpillars and their natural enemies. The main plant species used for most of the experiments was Brassica nigra (black mustard), which occurs naturally in The Netherlands. The main herbivore used was the lepidopteran Pieris brassicae, which lays eggs in clusters and feeds on plants belonging to the Brassicaceae family. This study investigated plant-mediated responses to oviposition and their effects on different developmental stages of the herbivore, such as larvae and pupae. Furthermore, the effects of oviposition were extended to four more plant species of the same family, and to higher trophic levels including parasitoids and hyperparasitoids. The experiments were conducted under laboratory, semi-field and field conditions. This study shows that B. nigra plants recognize the eggs of P. brassicae and initiate resistance against subsequent developmental stages of the herbivore. Interestingly, plant responses to oviposition were found to be species specific. Plants did not respond to egg deposition by another herbivore species, the generalist moth Mamestra brassicae. Moreover, most of the Brassicaceae species tested were found to respond to P. brassicae eggs, which indicates that plant responses against oviposition are more common among the family of Brassicaceae. To assess effects on other members of the food chain, the effects of oviposition on plant volatile emission and the attraction of parasitic wasps, such as the larval parasitoid Cotesia glomerata, were tested. It was shown that the wasps were able to use the blend of plant volatiles, altered by their hosts’ oviposition, to locate young caterpillars just after hatching from eggs. The observed behaviour of the wasps was associated with higher parasitism success and higher fitness in young hosts. Similar results were obtained in a field experiment, where plants infested with eggs and caterpillars attracted more larval parasitoids and hyperparasitoids and eventually produced more seeds compared to plants infested with caterpillars only. This study shows that an annual weed like B. nigra uses egg deposition as reliable information for upcoming herbivory and responds accordingly with induced defences. Egg deposition could influence plant-associated community members at different levels in the food chain and benefit seed production. As the importance of oviposition on plant-herbivore interactions is only recently discovered, more research is needed to elucidate the mechanisms that underlie such plant responses and how these interactions affect the structure of insect communities in nature.
Induction of indirect plant defense in the context of multiple herbivory : gene transcription, volatile emission, and predator behavior
Menzel, T.R. - \ 2014
Wageningen University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462571297 - 146
planten - plaagresistentie - geïnduceerde resistentie - verdedigingsmechanismen - multitrofe interacties - phaseolus lunatus - mijten - tetranychus urticae - roofmijten - phytoseiulus persimilis - voedingsgedrag - genen - transcriptie - genexpressie - herbivoor-geinduceerde plantengeuren - plants - pest resistance - induced resistance - defence mechanisms - multitrophic interactions - phaseolus lunatus - mites - tetranychus urticae - predatory mites - phytoseiulus persimilis - feeding behaviour - genes - transcription - gene expression - herbivore induced plant volatiles
Plants live in complex environments and are under constant threat of being attacked by herbivorous arthropods. Consequently plants possess an arsenal of sophisticated mechanisms in order to defend themselves against their ubiquitous attackers. Induced indirect defenses involve the attraction of natural enemies of herbivores, such as predators and parasitoids. Predators and parasitoids use odors emitted by damaged plants that serve as a “cry for help” to find their respective prey or host herbivore. The aim of this thesis was to use a multidisciplinary approach, with focus on molecular and chemical methods, combined with behavioral investigations, to elucidate the mechanisms of plant responses to multiple herbivory that affect a tritrophic system consisting of a plant, an herbivore and a natural enemy.
Induced plant defenses are regulated by a network of defense signaling pathways in which phytohormones act as signaling molecules. Accordingly, simulation of herbivory by exogenous application of phytohormones and actual herbivory by the two-spotted spider mite Tetranychus urticae affected transcript levels of a defense gene involved in indirect defense in Lima bean. However, two other genes involved in defense were not affected at the time point investigated. Moreover, application of a low dose of JA followed by minor herbivory by T. urticae spider mites affected gene transcript levels and emissions of plant volatiles commonly associated with herbivory. Only endogenous phytohormone levels of jasmonic acid (JA), but not salicylic acid (SA), were affected by treatments. Nevertheless, the low-dose JA application resulted in a synergistic effect on gene transcription and an increased emission of a volatile compound involved in indirect defense after herbivore infestation.
Caterpillar feeding as well as application of caterpillar oral secretion on mechanically inflicted wounds are frequently used to induce plant defense against biting-chewing insects, which is JA-related. Feeding damage by two caterpillar species caused mostly identical induction of gene transcription, but combination of mechanical damage and oral secretions of caterpillars caused differential induction of the transcription of defense genes. Nevertheless, gene transcript levels for plants that subsequently experienced an infestation by T. urticae were only different for a gene potentially involved in direct defense of plants that experienced a single event of herbivory by T. urticae. Indirect defense was not affected. Also sequential induction of plant defense by caterpillar oral secretion and an infestation by T. urticae spider mites did not interfere with attraction of the specialist predatory mite P. persimilis in olfactometer assays. The predator did distinguish between plants induced by spider mites and plants induced by the combination of mechanical damage and caterpillar oral secretion but not between plants with single spider mite infestation and plants induced by caterpillar oral secretion prior to spider mite infestation. The composition of the volatile blends emitted by plants induced by spider mites only or by the sequential induction treatment of caterpillar oral secretion followed by spider mite infestation were similar. Consequently, the induction of plant indirect defense as applied in these experiments was not affected by previous treatment with oral secretion of caterpillars. Moreover, herbivory by conspecific T. urticae mites did not affect gene transcript levels or emission of volatiles of plants that experienced two bouts of herbivore attack by conspecific spider mites compared to plants that experienced only one bout of spider mite attack. This suggests that Lima bean plants do no increase defense in response to sequential herbivory by T. urticae.
In conclusion, using a multidisciplinary approach new insights were obtained in the mechanisms of induction of indirect plant defense and tritrophic interactions in a multiple herbivore context, providing helpful leads for future research on plant responses to multiple stresses.
Endophytic control of Cosmopolites sordidus and Radopholus similis using Fusarium oxysporum V5w2 in tissue culture banana
Ochieno, D.M.W. - \ 2010
Wageningen University. Promotor(en): Marcel Dicke; Arnold van Huis, co-promotor(en): T. Dubois. - [S.l. : S.n. - ISBN 9789085856375 - 209
musa paradisiaca - bananen - weefselkweek - endofyten - fusarium oxysporum - nuttige organismen - plantenparasitaire nematoden - radopholus similis - insectenplagen - cosmopolites sordidus - biologische bestrijding - geïnduceerde resistentie - musa paradisiaca - bananas - tissue culture - endophytes - fusarium oxysporum - beneficial organisms - plant parasitic nematodes - radopholus similis - insect pests - cosmopolites sordidus - biological control - induced resistance
Banana plants are being inoculated with Fusarium oxysporum V5w2 and Beauveria bassiana G41 for endophytic control of pests. The effects of F. oxysporum V5w2 and B. bassiana G41, soil sterility, fertilizer, and mulching, on Cosmopolites sordidus and Radopholus similis in banana plants, are investigated. Cosmopolites sordidus has low preference for plants inoculated with the two endophytes; corm damage is low on F. oxysporum V5w2-treated plants. High root damage and growth suppression are evident in R. similis-treated plants. Under N-deficiency, R. similis-treated plants are larger than those without the nematode. Compared to plants treated with complete nutrient solution (CNS), those under P-deficiency have higher root damage, but lower under K-deficiency, and not different under N-deficiency and when only water was applied. Plants under CNS have lower R. similis density than those under N-deficiency and those treated with water only. Also, C. sordidus larvae from plants under CNS are smaller than those given only water. Under N-deficiency and supply of only water, potted plants in non-sterile soil are smaller than those from sterile soil, but are larger under CNS, P or K-deficiencies when N is present. Radopholus similis densities are lower in roots from non-sterile soil, compared to those from sterile soil. Mulched plants are larger with bigger bunches than those without mulch, but are more prone to toppling when R. similis is present. Plants treated with F. oxysporum V5w2 have lower R. similis density under N-deficiency but higher under P-deficiency, and are smaller in size under K-deficiency, than endophyte-free ones. Fusarium oxysporum V5w2-treated plants are small and take short time to harvest. With mulch, R. similis-induced toppling is less in F. oxysporum V5w2-treated plants, possibly due to their smaller size during growth. In conclusion, data on the effect of nutrients, soil microorganisms and mulching do not support the transfer of F. oxysporum V5w2-treated banana plants to farmers, because the plants suffer from reduced performance. Understanding endophytic mechanisms of action and establishing successful inoculation is necessary for drawing a final valid conclusion.
Molecular contest between potato and the potato cyst nematode Globodera pallida: modulation of Gpa2-mediated resistance
Koropacka, K.B. - \ 2010
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Aska Goverse; Geert Smant. - [S.l. : S.n. - ISBN 9789085856061 - 131
solanum tuberosum - globodera pallida - ziekteresistentie - geïnduceerde resistentie - genen - genetische analyse - loci voor kwantitatief kenmerk - genetische kartering - solanum tuberosum - globodera pallida - disease resistance - induced resistance - genes - genetic analysis - quantitative trait loci - genetic mapping
Gpa2 recognition specificity
Among all the multicellular animals, nematodes are the most numerous. In soil, a high variety
of free living nematodes feeding on bacteria can be found as well as species that parasitize
insects, animals or plants. The potato cyst nematode (PCN) Globodera pallida is an important
pest of cultivated potato. Upon infection of the roots, the nematode induces a feeding cell
complex or so-called syncytium, on which the immobilized nematode fully depends for its
development and reproduction. Due to the sophisticated feeding manner and ability to survive
for a long time in the absence of a host plant, the best way to control these soil-born
pathogens is the exploitation host resistance. Natural resistance to nematodes is based on
single dominant resistance genes (R) or quantitative trait loci (QTL). Several nematode
resistance genes have been identified and mapped. This includes the potato gene Gpa2 (Van
der Vossen et al., 2000) that confers resistance against the population D383 of G. pallida. The
Gpa2 gene is highly homologous to Rx1, which confers resistance against potato virus X
(Bendahmane et al., 1999). Both genes encode a protein with a nucleotide-binding leucinerich
repeat (NB-LRR) domains and a short coiled-coil domain at the N-terminus, which are in
88% identical at the amino acid level. The vast majority of the differences between Gpa2 and
Rx1 is found in the predicted solvent exposed regions of the LRR domain. In chapter 2, we
have shown that the LRR domain is essential for the recognition specificities of Gpa2 and
Rx1, whereas the CC-NBS domains can be exchanged without affecting the specificity. In
chapter 5, we have used a series of chimeric constructs in which segments of the Gpa2 LRR
were replaced by the corresponding segments from Rx1. These constructs allowed us to
narrow down the region required for nematode recognition to a stretch of residues between
808 and 912 amino acid residues in Gpa2, including 10 amino acids that differ between Gpa2
and Rx1. Furthermore, a computer-aided 3D model of the LRR domain is presented in which
7 of the Gpa2 specific amino acid residues map in a cluster onto the concave surface of the
horseshoe-like structure of the LRR domain.
Gpa2-mediated nematode resistance
The research described in chapter 3 aimed to understand the mechanisms underlying Gpa2-
mediated resistance to the potato cyst nematode G. pallida. The extreme resistance response
conferred by the close homologue Rx1 results in the blocking of the potato virus X (PVX) at
the infection sites and hence, the prevention of systemic spreading throughout the plant.
Surprisingly, an entirely different defense mechanism was observed for resistant potato plants
infected with juveniles of the avirulent Globodera pallida population D383. In susceptible
plants, both the virulent population Rookmaker and the avirulent population D383 formed
normal developing syncytia and nematodes were able to complete their life cycle as described
in previous studies. Infection of resistant plants with the avirulent population showed no
differences between susceptible and resistant potato plants in the early stages of G.pallida
parasitism (root entering, migration, syncytium initiation). Syncytium induction took place in
parenchyma cells, but rarely in other tissues. In samples collected 7 days later, however, the
first necrotic cells in the surrounding of the syncytium were noticed including symptoms of
degradation in the ultra structure of the syncytium itself in case of resistant plants infected
with avirulent nematodes. Samples collected 10 days post infection had already a layer of
necrotic cells, which separates the syncytium from the vascular bundle. At 14 days post
infection, it was observed that the parenchyma cells not incorporated directly in the syncytia
started to divide fast. Groups of hyperplastic cells surrounding the degrading syncytium
resulted in pushing it away to the outer part of the root. This unique phenomenon, which was
not observed before, can be part of the Gpa2-mediated defense response or a secondary
reaction to the presence of necrotic, dead cells and a way to exclude them from the healthy
conductive tissue of the root.
Transcriptional regulation of the Gpa2 promoter
To look in more details into the transcriptional regulation and expression of Gpa2, the native
promoter was fused to the reporter gene GUS and this construct was introduced into
susceptible potato. In chapter 3, the activity of the Gpa2 promoter was observed and shown to
be restricted to the vascular system and the root tips in uninfected plants. Roots were
challenged with G.pallida and the localization of the GUS expression was observed at the
infection sites at different parasitic stages. During infection with virulent nematodes - but not
the avirulent ones - this activity seems to be down regulated in vicinity of the syncytium.
Such a local inhibition of Gpa2 promoter activity is in line with observations made on
resistant roots when necrotic cells were only present around the feeding cell complex,
distantly from the feeding nematode.
The effector protein RBP-1 elicits a Gpa2 dependent HR
Recently, a RBP-1protein with strong similarity to the SPRY domain of the Ran-binding
protein RanBPM in juveniles of G. pallida was identified as a putative Gpa2 elicitor.
Transient expression of RBP-1 in N. benthamiana leaves elicits a Gpa2-dependent cell death
typical for the R-gene associated hypersensitive response (HR). Total RNA isolated from two
populations of G.pallida, D383 (avr to Gpa2) and Rookmaker (vir to Gpa2) was converted
into cDNA and screened for the presence of RBP-1s. This screening allowed the identification
of in total 10 classes of closely related homologs of RBP-1. All identified classes were tested
for their ability to elicit the Gpa2-dependent HR in an agroinfiltration assay. The capacity to
induce an Gpa2-dependent HR was shown to correlate with a single amino acid substitution in
RBP-1. No response was observed for two classes, which were obtained from the virulent
population (RBP-1ROOK2, RBP-1ROOK4). For the other homologous RBP-1 classes – both
deriving from the virulent and avirulent population - the response was ranging from a mild to
a strong and fast HR. Both in-active RBP-1 variants have a serine substitution at position 166
(S166P) within the SPRY domain. When this residue was projected on a computer aided 3D
model of RBP, we noticed that this amino acid is in a loop extending from the protein core.
Replacing the proline into a serine is predicted to change the shape of the loop and hence, to
affect the potential surface for protein-protein interactions.
Non-eliciting RBP-1 variants suppress RBP-induced Gpa2 activation
It was shown that the non-eliciting variants (RBP-1ROOK2 and RBP-1ROOK4) can suppress the
activation of a Gpa2-mediated HR by the eliciting RBP-1 variants. This effect was specific
for the Gpa2-mediated HR, and not observed with a Rx1-induced HR. As autoactive mutants
of Gpa2 and Rx1-mediated cell death are not blocked by the inactive variants of RBP-1, the
mechanism of suppression or inhibition likely operates on a functional Gpa2 protein, instead
of downstream Gpa2-activated signaling pathways. Further research is required to resolve the
mechanism underlying the possible competitive interactions of the active and the inactive
RBP-1 variants on the Gpa2-mediated HR. Essentially, two possible models that could
explain this phenomenon. First, the inactive variants could physically out compete the active
RBP-1s. The binding target of active and inactive variants of RBP-1 variants could be directly
in the Gpa2 protein or in the virulence target monitored by Gpa2. Alternatively, the inactive
variants of RBP-1 may intercept active RBP-1 variants by forming an inactive heterodimer
complex rendering it essentially undetectable for the Gpa2 protein.
Engineering disease resistance in plants
Custers, J. - \ 2007
Gewasbescherming 38 (2007)2. - ISSN 0166-6495 - p. 57 - 60.
gewasbescherming - genetisch bepaalde resistentie - geïnduceerde resistentie - ziekteresistentie - immuunsysteem - genetische modificatie - karakteristieken - aardappelen - gastheer parasiet relaties - onderzoek - plant protection - genetic resistance - induced resistance - disease resistance - immune system - genetic engineering - characteristics - potatoes - host parasite relationships - research
Op 8 januari 2007 promoveerde Jerome Custers aan Wageningen Universiteit op het proefschrift getiteld “Engineering disease resistance in plants”. Promotor was Prof. Dr.Ir.P.J.G.M. de Wit, verbonden aan de leerstoelgroep Fytopathologie, Wageningen Universiteit. Co-promotor was Dr. M.H. Stuiver, Head of Genomics, BASF Plant Science GmbH, Limburgerhof, Duitsland. Het werk beschreven in dit proefschrift werd uitgevoerd bij Syngenta Mogen B.V. (voorheen MOGEN International NV) in Leiden. Een reportage over gen-om-gen resistentie genereren, plantenpathogenen, afweerreacties tegen infecties en interacties tussen de gastheer en het plantenpathogeen
Induced pathogen and insect resistance in Arabidopsis: transcriptomics and specificity of defense
Oosten, V.R. van - \ 2007
Wageningen University. Promotor(en): Marcel Dicke; C.M.J. Pieterse; L.C. van Loon. - [S.l.] : S.n. - ISBN 9789085046271 - 160
arabidopsis - geïnduceerde resistentie - plaagresistentie - pathogenen - verdediging - insecten - genexpressieanalyse - arabidopsis - induced resistance - pest resistance - pathogens - defence - insects - genomics
An important question in plant defense signaling research is: how are plants capable of integrating signals induced by pathogenic micro-organisms and herbivorous insects into defenses that are specifically active against the attacker encountered? Plant defenses against pathogens and insects are differentially regulated by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling and genome-wide transcriptome changes in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium ( Pseudomonas syringae pv. tomato ), a pathogenic leaf fungus ( Alternaria brassicicola ), tissue-chewing caterpillars ( Pieris rapae ), cell-content-feeding thrips ( Frankliniella occidentalis ), or phloem-feeding aphids ( Myzus persicae ). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. The transcriptional alterations were predominantly attacker-specific, but the processes affected surprisingly similar. This indicates that the different attackers induce changes in similar plant processes through largely non-overlapping transcriptional alterations. Yet, infestation by M. persicae induced a transcriptional response that was opposite to those induced by the other attackers or exogenous application of MeJA. We concluded that SA, JA, and ET play a primary role in the orchestration of the plant's defense response, but other regulatory mechanisms, such as pathway cross-talk and additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.Little is known about the spectrum of effectiveness of the different types of induced resistance that are expressed upon attack by pathogens or insects. Because the signaling pathways that control induced resistance against pathogens and insects partly overlap, we decided to investigate the effectiveness of microbially induced resistance against the tissue-chewing herbivorous insects Pieris rapae and Spodoptera exigua in Arabidopsis. Two types of microbially induced resistance were studied: systemic acquired resistance (SAR), which is induced upon predisposal infection by necrotizing pathogens, and rhizobacteria-mediated induced systemic resistance (ISR), which is triggered by selected strains of non-pathogenic, root-colonizingrhizobacteria. No effect of SAR or ISR was evident on herbivore-induced attractiveness of the parasitic wasp Cotesia rubecula , indicating that neither type of induced resistance influenced indirect defense against these insects. In feeding experiments on whole plants, induction of SAR and ISR significantly reduced growth and development of the generalist herbivore S. exigua , whereas the performance of the specialist P. rapae was unaffected. The JA- and ET-responsive genes PDF1.2 and HEL , which were activated upon feeding by either of the two herbivores, showed a strongly potentiated expression pattern in SAR- and ISR-expressing plants upon feeding by S. exigua , but not upon feeding by P. rapae . This differential priming for enhanced herbivore-induced gene expression was confirmed in microarray experiments using a dedicated cDNA microarray containing 111 insect-responsive Arabidopsis genes. These results suggest that the effectiveness of microbially induced SAR and ISR against S. exigua feeding is associated with priming for enhanced defense-related gene expression.In conclusion, this thesis highlights the complexity of the defense signaling interactions between plants, pathogens and insect herbivores.
Micro-organismen beschermen planten tegen rupsenvraat
Oosten, V.R. van; Pelt, J.A. van; Loon, L.C. van; Pieterse, C.M.J. ; Dicke, M. - \ 2005
Gewasbescherming 36 (2005)6. - ISSN 0166-6495 - p. 267 - 267.
gewasbescherming - biologische bestrijding - geïnduceerde resistentie - insectenbestrijding - arabidopsis - experimenten - onderzoek - genen - rupsen - plant protection - biological control - induced resistance - insect control - arabidopsis - experiments - research - genes - caterpillars
Samenvatting van de voordracht te houden op 30 november 2005 tijdens de Najaarsvergadering van de KNPV (Koninklijke Nederlandse Plantenziektekundige Vereniging). Onderzoek naar inductie van resistentie in Arabidopsis tegen vraat van rupsen
Occurence of Cucumber Mosaic Virus in Ornamental Plants and Perspectives of Transgenic Control
Chen, Y.K. - \ 2003
Wageningen University. Promotor(en): R.W. Goldbach, co-promotor(en): M.W. Prins. - Wageningen : S.n. - ISBN 9789058087997 - 152
sierplanten - plantenvirussen - komkommermozaïekvirus - geïnduceerde resistentie - ziekteresistentie - genetische modificatie - transgene planten - ornamental plants - plant viruses - cucumber mosaic virus - induced resistance - disease resistance - genetic engineering - transgenic plants
This thesis described the characterization of a range of ornamental-infecting Cucumber mosaic virus strains and the development of novel transgene constructs to improve the efficiency of obtaining resistant transformants which is essential for most ornamental plants that are difficult to transform and regenerate. Nucleotide sequence-based grouping of ornamental-infecting CMV was carried out and found that within the ornamental-infecting CMV viruses both subgroups were represented. Moreover, the analyzed nucleotide sequences of CMV isolates obtained from lily showed remarkably high homology despite different origins. A recombination phenomenon was found in alstroemeria-infecting CMV isolates. This newly discovered event and its beneficial effects on biological fitness have been described. A survey of CMV genome for RNA or protein based transgenic resistance demonstrated that resistance against CMV was feasible. Inverted repeat sequences based on CMV RNA 2 and CP gene have been constructed and transformed into plants. These plants were subsequently tested for high efficiency to obtain resistance. The resistance levels conferred by RNA 2 and CP sequences were greatly improved by using inverted repeat constructs. This efficient approach to obtain CMV resistant transgenic plants opened the way for the use of these transformation constructs for the production of virus resistant ornamental plants.
Cf-afhankelijke afweerreacties, geinduceerd door avirulentie-eiwitten van het tomatenpathogeen Cladosporium fulvum ofwel: Koude fataal voor zelfmoordtomaat
Jong, C.F. de - \ 2003
Gewasbescherming 34 (2003)2. - ISSN 0166-6495 - p. 50 - 53.
tomaten - solanum lycopersicum - tabak - nicotiana tabacum - passalora fulva - plantenziekteverwekkende schimmels - pathogenesis-gerelateerde eiwitten - geïnduceerde resistentie - ziekteresistentie - tomatoes - solanum lycopersicum - tobacco - nicotiana tabacum - passalora fulva - plant pathogenic fungi - pathogenesis-related proteins - induced resistance - disease resistance
Uitgebreide samenvatting met afbeeldingen van een promotieonderzoek over het resistentie-gen in tomaat tegen Cladosporium fulvum en hoe de tomaat zich dan beschermt tegen indringers
Cf-dependent early defence responses induced by avirulence proteins of the tomato pathogen : Cladosporium fulvum
Jong, C.F. de - \ 2002
Wageningen University. Promotor(en): P.J.G.M. de Wit; M.H.A.J. Joosten. - S.l. : S.n. - ISBN 9789058086778 - 109
tomaten - solanum lycopersicum - tabak - nicotiana tabacum - passalora fulva - plantenziekteverwekkende schimmels - pathogenesis-gerelateerde eiwitten - geïnduceerde resistentie - verdedigingsmechanismen - ziekteresistentie - signaaltransductie - tomatoes - solanum lycopersicum - tobacco - nicotiana tabacum - passalora fulva - plant pathogenic fungi - pathogenesis-related proteins - induced resistance - defence mechanisms - disease resistance - signal transduction
The outcome of a plant-pathogen interaction is determined by both the presence of resistance ( R ) genes in the plant and matching avirulence ( Avr ) genes in the pathogen. According to the gene-for-gene concept, for a dominant R gene in the host plant resistant to a specific strain of a pathogen, a corresponding dominant Avr gene exists in that strain of the pathogen. R gene-mediated recognition of an Avr gene product triggers a signal transduction cascade, eventually resulting in a hypersensitive response (HR). This HR consists of a collapse of plant cells at the primary site of infection, resulting in an arrest of growth of the pathogen. As it is impractical to study defense signaling responses in whole plants, often cell suspensions are used for this purpose. Directly after treatment of cell suspensions with elicitors, activation of signal transduction processes, such as ion fluxes over the plasma membrane (detectable as alkalization of the extra cellular medium), phospholipid signaling and protein phosphorylation occur. Also reactive oxygen species, which are thought to play a role both in defense signaling and in direct defense against the pathogen, are produced after AVR perception.
The interaction between Cladosporium fulvum and tomato is a well-studied plant-pathogen interaction that obeys the gene-for-gene concept. From this pathosystem several resistance ( Cf ) and Avr genes have been cloned, from which the matching gene pairs Cf-4/Avr4 and Cf-9/Avr9 are the best studied. Although many efforts were undertaken to study defense signaling in cell suspensions derived from Cf- carrying tomato plants, they were not responsive to the matching AVRs. Therefore, Cf4 +-and Cf-9 +- tobacco cell suspensions, which are responsive to the matching AVR protein, were used to study Cf/Avr- mediated defense signaling.
In Chapter 2, defense responses in Cf-9 +- tobacco leaves and Cf-9 +- tobacco cell suspensions induced by both wild-type (WT-AVR9) and mutant AVR9 analogues were studied. Upon injection into leaves of both tomato MM-Cf9 and Cf-9 +- tobacco leaves, the mutant AVR9 peptides R08K, F10A and F21A showed higher, lower and no necrosis-inducing activity, respectively, as compared to WT-AVR9. Similar relative activities were found for these 4 peptides when assayed in Cf-9 +- tobacco cell suspensions. R08K showed a stronger, whereas F10A and F21A showed a lower oxidative burst-inducing activity as compared to WT-AVR9. In a medium alkalization assay equal activities were observed for R08K and WT-AVR9, whereas F10A showed a lower activity and no medium alkalization activity at all was observed for F21A. Surprisingly, the oxidative burst was induced at peptide concentrations that were 100 times lower as compared to those inducing medium alkalization. Concentrations inducing a full medium alkalization response are similar to peptide concentrations that induce necrosis in leaves of Cf-9- carrying tomato or tobacco plants. Treatment of Cf-9 +- tobacco cell suspensions with WT-AVR9 resulted in the activation of a MAP kinase, whereas F21A activated the MAP kinase only to a small extent. WT-AVR9 also induced massive cell death at 18 hr after addition to Cf-9 +- tobacco cell suspensions, whereas tobacco cells not expressing Cf-9 remained viable, illustrating the specificity of this response.
In Chapter 3 we have shown that, upon AVR4 treatment of Cf-4 +-tobacco cells, levels of the second messenger phosphatidic acid (PA) increased dramatically. This response occurred within 2 min after addition of AVR4 and was highly specific. The PA conversion product diacylglycerol pyrophosphate (DGPP) accumulated between 4-8 minutes after addition of AVR4. Whether DGPP is a second messenger in its own right or serves as a negative regulator for PA signaling is still unclear. A differential labeling strategy showed that AVR4-induced PA accumulation resulted predominantly from the conversion of diacylglycerol (DAG) into PA by diacylglycerol kinase (DGK). DAG can be generated during signaling events by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by phospholipase C (PLC). Pretreatment of Cf-4 +- tobacco cells with the PLC inhibitors neomycin and U73122 blocked AVR4-induced PA accumulation, indicating that PA is indeed generated via PLC activity. The AVR4-induced oxidative burst was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), whereas it did not block AVR4-induced PA accumulation. Conversely, the PLC inhibitor U73122 blocked both AVR4-induced PA accumulation and oxidative burst in a dose-dependent way. Treatment with a synthetic, water-soluble PA derivative induced a small, and transient oxidative burst in Cf-4 +- tobacco cells. These data demonstrate the importance of phospholipid signaling in the AVR4-induced oxidative burst. Additional studies showed that AVR4-induced medium alkalization and MAP kinase activation were also blocked by PLC inhibitors, suggesting that these responses are also PLC-dependent.
During the experiments described in Chapter 2 it became clear that AVR9-induced defense responses are temperature sensitive. The temperature-sensitivity of Cf/Avr- mediated defense responses is studied in Chapter 4. Injection of AVR4 or AVR9 in leaves of tobacco and tomato plants carrying Cf - 4 or Cf-9, respectively, resulted in necrosis in the injected area at 20°C, whereas at 33°C this response was suppressed. At 20°C, tomato seedlings expressing both a Cf gene and matching Avr gene germinate but develop systemic HR after unfolding of the cotyledons and subsequently die. These seedlings could be rescued at 33°C but rapidly died after transfer to 20°C. Gel blot analysis of RNA isolated at different time points after transfer of the rescued Cf/Avr seedlings to 20°C, revealed a controlled induction of expression of various typical defense-related genes. This synchronized onset of HR provides an excellent basis for the identification of novel, HR-related genes by cDNA-AFLP analysis.
In cell suspensions we found that both the AVR4- and AVR9-induced medium alkalization response is slowly suppressed at 33°C, but quickly recovers upon transfer to 15°C. For AVR4- and AVR9-induced medium alkalization, differences in the kinetics of the suppression of this response at an elevated temperature were demonstrated. The high affinity binding site for AVR9 is thought to be the AVR9 receptor, which is involved in the initiation of Cf-9/Avr9- mediated defense responses. It was shown that binding of AVR9 to microsomal fractions isolated from cell suspensions incubated at 33°C was decreased by 80%, as compared to microsomal fractions isolated from cell suspensions incubated at 20°C. The decrease of AVR9 binding was caused by a decrease in amount of binding sites rather than by a decrease in the affinity of the binding site for AVR9, providing a molecular basis for the temperature sensitivity of Cf-9/Avr9- mediated defense.
In Chapter 5 the results described in this thesis are discussed and some additional unpublished data are included. A model for AVR-induced signaling in the C. fulvum/ tomato interaction is presented. In addition, new techniques, future experiments and perspectives to further unravel Cf/Avr- mediated signaling are discussed.
|Environmental risks of transgenic multiple herbicide resistance
Kempenaar, C. ; Lotz, L.A.P. - \ 1999
Wageningen : AB - 30
genetische modificatie - recombinant dna - milieueffect - milieubescherming - agronomie - gewassen - plantenveredeling - transgene planten - geïnduceerde resistentie - resistentie tegen herbiciden - risicoschatting - gewasbescherming - genetic engineering - environmental impact - environmental protection - agronomy - crops - plant breeding - transgenic plants - induced resistance - herbicide resistance - risk assessment - plant protection
|Introductie van virusresistentie in lelie door middel van genetische modificatie : eindverslag van het project 'Inbouw van virusresistentie in lelies via genetische manipulatie'
Langeveld, S.A. ; Bol, J.F. ; Boonekamp, P.M. - \ 1996
Lisse : Laboratorium voor Bloembollenonderzoek (Urgentieprogramma Bollenziekte- en veredelingsonderzoek ) - 19
plantenziekten - plantenvirussen - bloembollen - plantenveredeling - ziekteresistentie - plaagresistentie - genetische modificatie - recombinant dna - planten - immunisatie - geïnduceerde resistentie - onderzoek - lilium - plant diseases - plant viruses - ornamental bulbs - plant breeding - disease resistance - pest resistance - genetic engineering - plants - immunization - induced resistance - research
|Het inbrengen van genen coderend voor antibacteriele eiwitten bij wilg ter bescherming tegen de watermerkziekte
Roest, S. ; Dam, B.C. van; Evers, P.W. - \ 1994
Wageningen : IBN (IBN - rapport 082)
bosbouw - genetische modificatie - immunisatie - immunogenetica - geïnduceerde resistentie - plantenziekten - plantenziekteverwekkende bacteriën - planten - recombinant dna - bomen - salix alba - brenneria salicis - forestry - genetic engineering - immunization - immunogenetics - induced resistance - plant diseases - plant pathogenic bacteria - plants - trees
Engineering resistance against potato virus Y
Vlugt, R.A.A. van der - \ 1993
Agricultural University. Promotor(en): R.W. Goldbach; H. Huttinga. - S.l. : Van der Vlugt - ISBN 9789054850847 - 111
plantenziekten - plantenvirussen - solanum tuberosum - aardappelen - potyvirus - planten - immunisatie - geïnduceerde resistentie - genetica - genetische variatie - evolutie - soortvorming - immunogenetica - genetische modificatie - recombinant dna - plant diseases - plant viruses - solanum tuberosum - potatoes - potyvirus - plants - immunization - induced resistance - genetics - genetic variation - evolution - speciation - immunogenetics - genetic engineering - recombinant dna
Potato virus Y is the type species of the potyvirus genus, the largest genus of the plant virus family Potyviridae. The virus causes serious problems in the cultivation of several Solanaceous crops and although certain poly- and monogenic resistances are available, these can not always be employed, e.g. R y genes in potato cv. 'Bintje'. The aim of the research described in this thesis was to establish new forms of resistance against PVY by genetic modification of host plants. One such form of genetic engineered resistance is 'coat protein-mediated resistance', whereby expression of a viral coat protein (CP) in a transgenic plant may confer resistance against infection with the homologous virus, and some closely related viruses.
At the start of this investigation no sequence data on the RNA genome of PVY were available, therefore cDNA synthesis and subsequent sequence determination was performed to obtain the necessary PVY CP gene sequence as well as additional sequences from the 3'-terminal region of the viral genome (Chapter 2 and Van der VIugt et al., 1989). This enabled the determination of the exact taxonomic position of the PVY N('tobacco veinal necrosis strain') isolate used in these experiments, among other PVY isolates from at least two different strains. Detailed comparisons of the PVY NCP and 3'-non translated (3'-NTR) sequences with those from a large number of geographically distinct PVY isolates that became available during the course of this investigation, showed that these sequences, in addition to distinguish between different potyvirus species (Ward and Shukla, 1991; Frenkel et al., 1989), can also be used for the distinction between strains of one potyvirus (Chapter 3, Van der VIugt et al., 1992a). Several strain specific amino acid sequences in the CPs and nucleotide sequences in the 3'-NTRs could be discerned, that are possibly involved in virulence and/or symptom expression. Further experiments are required to elucidate the precise biological significance of these sequence motifs. Interestingly the sequence comparisons as complied in Chapter 3 also confirmed the high levels of CP and 3'-NTR sequence identity between the PVY isolates at one hand and one putative isolate of pepper mottle virus (PepMoV, Dougherty et al., 1985) at the other, as described previously (Van der VIugt et al., 1989; Van der Vlugt, 1992). Initially described as an atypical strain of PVY (PVY-S, Zitter, 1972) PepMoV was later found to be serologically and biologically distinct from PVY (Purcifull et al., 1973, 1975; Zitter and Cook, 1973). Recent determination of the complete genomic RNA sequence of a Californian isolate of pepper mottle virus (PepMoV-C; Bowman-Vance et al, 1992a,b) and comparisons between a Florida isolate of PepMoV and PVY (Hiebert and Purcifull, 1992) however, suggest that PepMoV represents a distinct potyvirus though more closely related to PVY than to any other potyvirus. Additional sequence information of other, biologically well characterized, isolates of PepMoV, like a virus isolate apparently intermediate between PepMoV and PVY (Nelson and Wheeler, 1978), will hopefully aid in establishing the exact taxonomic position of this pepper infecting virus in the genus Potyvirus. Generally it is to be recommended that of all virus isolates whose (partial) sequences are under investigation, precise origin and other relevant biological characteristics are also accurately documented.
Analysis of the transgenic potato lines (Chapter 4) showed that most lines, as the transgenic tobacco lines, expressed CP specific RNA transcripts. Under the given greenhouse conditions, however, in none of the transgenic plants protection to PVY could be determined. In view of the results obtained with the transgenic tobacco lines, it may be anticipated that virus challenging of additional transgenic potato lines, under more optimal greenhouse conditions, will reveal similar levels of RNA-mediated virus resistance as observed in tobacco. For all practical purposes genetically engineered resistance based on the presence of RNA molecules is to be preferred over forms of resistance that are based on the expression of a (foreign) protein. Apart from being energetically more favourable for the plant, it is likely to aid in the acceptance of genetically modified crop plants by both politicians and the public, something which might, in the next few years, turn out to be the major obstacle in the successful application of plant transformation techniques.
At this stage one can only speculate on the mechanism(s) on which this RNAmediated resistance is based. Transformation of plants with partial CP or other PVY Ngenomic sequences will help in identifying the protection mechanism(s) involved and show whether regions other than the CP-encoding domain can be equally effective in conferring virus resistance. If the resistance is based on a 'sense-RNA' effect, i.e. hybridization of the positive sense transgenic RNA to negative-sense viral RNA replication intermediates, thereby blocking further virus replication, the ribozyme technology might prove an efficient expansion of this genetically engineered type of resistance. Ribozymes, RNA sequences capable of specific and catalytic cleavage of other RNA-sequences, are able to cleave target RNAs efficiently and catalytically in vitro . The antiviral application of ribozymes in transgenic plants however has sofar demonstrated not to be very successful and reported protection levels are not yet exceeding those obtained with antisense RNAs (Edington and Nelson, 1992). Chapter 7 describes the design and synthesis of hammerhead ribozymes capable to cleave a highly conserved region from the PVY RNA dependent RNA-polymerase cistron. It was shown that the correct formation of the hammerhead cleavage complex, determined at least in part by the lengths of the antisense arms of the ribozyme, forms an important factor in the efficiency of cleavage. Cellular and full-length viral RNA molecules generally posses extended, unknown secondary structures which are likely to hamper precise formation of hammerhead structures, which requires bimolecular basepairing. Correct hammerhead formation and efficient cleavage of these RNAs will therefore require ribozymes with rather long basepairing arms. These long antisense arms however will make catalytic cleavage rather unlikely since complex dissociation will probably become the rate limiting factor. For this reason one can assume that ribozymes will only be successful when introduced into specific antisense RNA molecules, directed against the less abundant viral complementary strands, rather than as highly efficient RNA cleaving "enzymes".
Duurzame resistentie tegen pathogenen via genetische modificatie; utopie of werkelijkheid voor de gewasbescherming in het jaar 2000?
Wit, P.J.G.M. de - \ 1991
Wageningen : Landbouwuniversiteit Wageningen - 31
plantenziektekunde - plantenziekteverwekkende schimmels - plantenziekten - plantenvirussen - plantenziekteverwekkende bacteriën - planten - immunisatie - geïnduceerde resistentie - plantenveredeling - ziekteresistentie - plaagresistentie - colleges (hoorcolleges) - genetische modificatie - recombinant dna - plant pathology - plant pathogenic fungi - plant diseases - plant viruses - plant pathogenic bacteria - plants - immunization - induced resistance - plant breeding - disease resistance - pest resistance - lectures - genetic engineering - recombinant dna
Heat treatment and meristem culture for the production of virus free plant material
Houten, J.G. ten; Quak, F. ; Meer, F.A. van der - \ 1968
Wageningen : [s.n.] (Mededeling / Instituut voor plantenziektenkundig onderzoek no. 473) - 7
plantkunde - teelt - cultuurmethoden zonder grondbewerking - groei - gewassen - planten - immunisatie - geïnduceerde resistentie - plantenziekten - plantenplagen - gewasbescherming - plantenziektekunde - afwijkingen, planten - warmtebehandeling - bestrijdingsmethoden - temperatuur - experimenten - weefselkweek - celkweek - meristemen - plantenontwikkeling - plantenvirussen - handel - plagenbestrijding - ziektebestrijding - botany - cultivation - no-tillage - growth - crops - plants - immunization - induced resistance - plant diseases - plant pests - plant protection - plant pathology - plant disorders - heat treatment - control methods - temperature - experiments - tissue culture - cell culture - meristems - plant development - plant viruses - trade - pest control - disease control