1,240 research outputs found
Functional analysis of the tomato Ve resistance locus against Verticillium wilt
No full text Verticillium dahliae, V. albo-atrum and V. longisporum are soil-borne plant pathogens that are responsible for Verticillium wilt diseases in temperate and subtropical regions. Collectively they can infect over 200 hosts, including many economically important crops. Chapter 1 is a “pathogen profile” which describes the most important aspects of the biology of the Verticillium wilt pathogens. They colonize the xylem vessels of their host plants and cause symptoms such as wilting, chlorosis, stunting, necrosis and vein clearing. Verticillium species are notoriously difficult to control as there are no fungicides available to cure plants once they are infected. Therefore, genetic resistance is the preferred method for disease control. Chapter 2 describes the functional characterization of the tomato (Solanum lycopersicum) Ve locus. This locus is responsible for resistance against race 1 strains of V. dahliae and V. albo-atrum and comprises two closely linked inversely oriented genes, Ve1 and Ve2. Both genes encode cell surface receptor proteins of the extracellular leucine-rich repeat (eLRR) receptor-like protein (RLP) class of disease resistance proteins. In chapter 2, it is demonstrated that Ve1, but not Ve2, provides resistance in tomato against race 1 but not against race 2 strains of V. dahliae and V. albo-atrum. Using virus-induced gene silencing in tomato, the signaling cascade downstream of Ve1 was shown to require both EDS1 (enhanced disease susceptibility1) and NDR1 (non-race-specific disease resistance1). In addition, also NRC1 (NB-LRR protein required for hypersensitive response-associated cell death1), ACIF (Avr9/Cf-9–induced F-box1), MEK2 (MAP/ERK kinase2), and SERK3/BAK1 (somatic embryogenesis receptor kinase 3/brassinosteroid-associated kinase 1) act as positive regulators of Ve1 in tomato. In conclusion, Ve1-mediated resistance signaling only partially overlaps with signaling mediated by Cf proteins, type members of the eLRR-RLP-class of resistance proteins. In chapter 3 an attempt to introduce Nicotiana benthamiana as a model to facilitate the study of Ve1-mediated resistance is described. Challenge of wild type plants with several race 1 and race 2 strains of V. dahliae and V. albo-atrum demonstrated that N. benthamiana is susceptible to both Verticillium species. To obtain Verticillium wilt resistant plants, N. benthamiana was engineered to express the tomato Ve1 coding sequence. However, out of thirteen transgenic lines, six showed clear phenotypic aberrancies that included severe stunting and malformed leaves when compared to wild type plants. The seven Ve1-transgenic lines that did not show any phenotypic alterations were challenged with race 1 and race 2 strains. Although the pathogenicity assays indicated that in few lines Ve1 expression temporarily reduced disease development, most lines were as susceptible as wild type parental line. In conclusion, in chapter 3 it is demonstrated that the Ve1-transgenic N. benthamiana lines could not be used to study Ve1-mediated resistance signaling. In chapter 4, the use of Arabidopsis (Arabidopsis thaliana) as model to facilitate the study of Ve1-mediated resistance is presented. To this end, tomato Ve1 was expressed in susceptible Arabidopsis plants. Upon challenge with race 1 strains of V. dahliae or V. albo-atrum, Ve1-expressing plants were found to be resistant. In contrast, Ve1-expressing plants were susceptible to race 2 strains of both V. dahliae and V. albo-atrum. Furthermore, expression of Ve1 in Arabidopsis plants did not prevent colonization by V. longisporum strains. Through Ve1-expression in Arabidopsis defense signaling mutants, it was demonstrated that signaling downstream of Ve1 is highly conserved between tomato and Arabidopsis. In previous chapters it was shown that the receptor kinase SERK3/BAK1 is required for Ve1-mediated resistance in tomato as well as in Arabidopsis. In Arabidopsis, SERK3/BAK1 belongs to a gene family consisting of five members. In chapter 5, the requirement of the different SERK family members in Ve1-mediated resistance in Arabidopsis is investigated, revealing the requirement of SERK1 and, although to a lesser extent, SERK4 for resistance. Using virus-induced gene silencing, it was subsequently shown that SERK1 is also required for Ve1-mediated resistance in tomato. In conclusion, the results of chapter 5 demonstrate that Arabidopsis can be used as model to unravel the molecular mechanisms of Ve1-mediated resistance. In chapter 6, the recognition specificity of Ve1 was further investigated by performing domain-swaps with Ve2 and expressing the chimeric Ve proteins in Arabidopsis. Various domain swaps in which eLRRs from Ve1 were replaced by those of Ve2 suggest that the region between eLRR22 and eLRR35 is required for full Ve1-mediated resistance. However, plants expressing a Ve chimera in which eLRR1 to eLRR30 of Ve1 was replaced with those of Ve2 were resistant against Verticillium. Overall, these results suggest that Ve2 may still bind the elicitor in the eLRR domain, but its C-terminal domain is not able to activate a successful defense response. Finally in Chapter 7, highlights of this thesis are discussed and placed in a broader perspective. </p
The Cladosporium fulvum Avr2 protein behaves both as a virulence and an avirulence factor
No full textPlants are not able to move or escape and have to confront environmental challenges like nutrient and water deprivation, low and high temperatures, and biotic stress imposed by pathogens like viruses, bacteria, fungi, nematodes and insects that all compete for plant nutrient sources. The outcome of a plant-pathogen interaction can vary from mild symptoms that are hardly harmful to the host to complete destruction of the host plant. Plants have evolved various mechanisms to counter-attack infections by pathogens. Mechanisms of evasion or suppression of basal host defense by pathogens on the one hand, and specific recognition of a pathogen by its host and activation of downstream defense signaling on the other hand, are complex and both organisms have to come up with sophisticated strategies to survive their encounters. In principle these encounters have two possible outcomes: (i) a pathogen successfully infects the host plant, which is also referred to as a compatible interaction (the pathogen is virulent and the host plant is susceptible), or (ii) the pathogen cannot successfully infect the host plant which stays healthy, also referred to as an incompatible interaction (the pathogen is avirulent and the host plant is resistant). Nearly 70 years ago, Harold Flor (1942) studied the genetics of the interaction between the flax rust fungus Melampsora lini and flax, Linum usitatissimum. Based on these studies he postulated the so-called gene-for-gene hypothesis (Flor 1942) which states that for each dominant resistance (R) gene in the host there is a matching dominant avirulence (Avr) gene in the pathogen. Co-occurrence and expression of both genes leads to an incompatible interaction that is often associated with a hypersensitive response (HR). The interaction between the fungus Cladosporium fulvum (syn. Passalora fulva) and the host tomato (Solanum lycopersicum) is an excellent model to study plant-pathogen interactions and obeys to the gene-for-gene hypothesis. C. fulvum is a biotrophic pathogen that causes leaf mold of tomato, avoids breaching the cell wall and exclusively colonizes the tomato leaf apoplast while establishing a long-term feeding relationship with the living cells of the host. During the infection process, the fungus secretes several effector molecules, relatively small, cysteine–rich proteins. They are likely to contribute to pathogen fitness and play a role in pathogen virulence. According to the 'Zig-Zag' model that explains the evolutionary development of plant-pathogen interactions, effectors are required for ETS (effector -triggered susceptibility). Tomato plants that carry cognate Cf resistance genes recognize the effector and elicit a defense response known as the hypersensitive response (HR), nowadays known as effector-triggered immunity (ETI). In this thesis I have focused on several molecular and biochemical aspects of the Avr2 and Cf-2 gene pair and on an additional gene, Rcr3 (required for Cladosporium resistance), that is required for Cf-2-mediated resistance with an emphasis on the role of Avr2 in ETS and ETI in the C. fulvum-tomato interaction. The gene-for-gene hypothesis postulated by Harrold Flor has inspired many plant pathologists and initiated numerous plant-pathogen studies as discussed in chapter 1. This hypothesis has lead to the characterization of various host plant R genes and cognate pathogen Avr genes from fungi, bacteria and oomycetes. Plant resistance proteins are the basic molecules that mediate a defense reaction, triggered by cognate effectors directed against the pathogens, are found extracellularly as well as intracellularly and are divided in classes based on the composition of different subdomains that may have various functions. Particularly the LRR domain(s) are involved in recognition, regulating protein activation and signal transduction and are highly adjustable in diverse binding specificities to self and non-self molecules. The nucleotide binding (NB) domain acts a switch for activation of downstream host defenses, often resulting in HR. Inappropriate R protein folding and activation is controlled by intramolecular interactions between the various domains and by hetero-multimeric protein complexes. Studies on interactions of plants, especially Arabidopsis thaliana, with prokaryotic pathogens have resulted in major scientific breakthroughs with respect to the gene-for-gene hypothesis. Research on the bacterial Type Three Secretion System and the delivery of the effectors has indentified sophisticated mechanisms for perception and recognition of pathogens and regulation of host resistance. The functions of effectors of eukaryotic plant pathogens remain largely unknown so far. Oomycete pathogens such as Phytophthora infestans produce various types of effectors during infection of their hosts. One class of oomycete effectors localizes to, and operates in, the extracellular matrix while the other class acts inside the host plant cell. Recent studies on the interaction of the flax rust fungus Melampsora lini with flax (Linum usitatissimum) has revealed a number of direct Avr-R protein interactions in vitro. These interactions are expected to occur in the haustorial matrix which is produced by the fungus during host infection. Secreted Avr proteins of C. fulvum interact exclusively with the corresponding extracellular Cf proteins of tomato. The C. fulvum-tomato pathosystem is one of the most well-studied plant pathogen interactions and revealed important insights in perception and recognition of Avr proteins. For many years it was assumed that the interactions beween C. fulvum Avrs and tomato R proteins occurred in a direct manner, but proof for such interactions has never been obtained. Indirect interactions were more likely and obeyed to the guard hypothesis wherein the Avr protein interacts with a host target and this interaction is monitored, or guarded, by the Cf- protein. Chapter 2 reports on the avirulence function of Avr2 in the Cf-2-mediated resistance that also requires the extracellular tomato cysteine protease Rcr3. The interaction between Avr2, Cf-2 and Rcr3 obeys to the guard hypothesis. Purified heterologously expressed and affinity-tagged Rcr3 and Avr2 were applied in co-immunoprecipitation assays and revealed a physical interaction between Avr2 and Rcr3 independent of additional plant and or fungal factors. It is shown that Avr2 binds and inhibits Rcr3, and blocking of the active site of Rcr3 by the irreversible cysteine protease inhibitor E-64 eliminates this interaction. The interaction with and the inhibition of Rcr3 by Avr2 occurs in a pH-dependent fashion and the pH optimum for Rcr3 activity and its inhibition by Avr2 coincides with the pH of the tomato apoplast. Cysteine protease activity profiling showed that, in addition to Rcr3, Avr2 inhibits several other apoplastic cysteine proteases in tomato, but this inhibition did not lead to Cf-2-mediated HR. Infiltration of purified active Rcr3, or E-64-inactivated Rcr3, in combination with Avr2 in Cf-2/rcr3 tomato leaves revealed that only the Avr2-Rcr3 inhibition complex triggers Cf-2-dependent HR. It is proposed that Avr2 modifies Rcr3 which is recognized by Cf-2 and initiates the HR. This study represents the first indirect fungus-plant gene-for-gene interaction that obeys to the guard hypothesis. In chapter 3 the focus is on the virulence function of Avr2, and it is demonstrated that Avr2 has an indisputable intrinsic biological virulence function for C. fulvum during infection of tomato. Silencing of the Avr2 gene in C. fulvum significantly compromised fungal virulence on tomato. Heterologous expression of Avr2 in tomato resulted in enhanced susceptibility towards natural Avr2-defective C.fulvum strains, but also towards Botrytis cinerea and Verticillium dahliae. In A. thaliana, Avr2 expression resulted in enhanced susceptibility to various extracellular fungal pathogens including Botrytis cinerea and Verticillium dahliae. Microarray analysis of unchallenged A. thaliana plants showed that Avr2 expression induced a global transcription profile that is comparable to the profile upon pathogen challenge. Cysteine protease activity profiling and LC-MS/MS analyses showed that Avr2 inhibits multiple extracellular A. thaliana cysteine proteases. Similar results were obtained for tomato, showing that Avr2 inhibits multiple cysteine proteases including Rcr3 and its close relative Pip1. This all shows that Avr2 is a genuine virulence factor of C. fulvum that inhibits several cysteine proteases that are required for basal host defense. In chapter 4 the emphasis is on Avr2 protein features and the mode of inhibition of Rcr3. Like many other Avr genes, Avr2 lacks homology with sequences deposited in public databases. The mature Avr2 protein contains 8 cysteine residues and biochemical analyses revealed that all of these are involved in disulphide bridging, showing a unique disulphide bridge pattern. Based on a bioinformatics analysis, site-specific mutations were made in the Avr2 protein and affinity-tagged wild-type and mutant proteins were produced by heterologous expression in the yeast Pichia pastoris. After affinity purification, all proteins were infiltrated in tomato Cf-2 plants, and proteins with altered HR inducing activity were tested for their ability to inhibit Rcr3. From these assays it became evident that especially the C-terminal six amino acids that also include one disulphide bridge are essential for the interaction with and inhibition of Rcr3. All these data show that Avr2 is a novel type of cysteine protease inhibitor. Chapter 5 is a general discussion about the role of plant cysteine proteases and cysteine protease inhibitors in plant-pathogen interactions. Microbial pathogens and host plants both employ cysteine proteases and cysteine protease inhibitors as weapons for attack and defence. This so-called arms race has led to multiple attacks and counter-attacks that have shaped co-evolution between pathogens and their host plants. Examples of some prokaryotic plant pathogens that employ cysteine proteases as effector proteins to suppress plant defense will be discussed, in addition to some eukaryotic pathogens that use cysteine protease inhibitors for the same purpose. Examples of plant cysteine proteases will be discussed that are involved in multiple processes including plant development, plant defense and processes in programmed cell death. <br/
Phosphorylation and proteome dynamics in pathogen-resistant tomato plants
No full textMicrobial plant pathogens impose a continuous threat on global food production. Similar to disease resistance in mammals, an innate immune system allows plants to recognise pathogens and swiftly activate defence. For the work described in this thesis, the interaction between tomato and the extracellular fungal pathogen Cladosporium fulvum serves as a model system to study host resistance and susceptibility in plant-pathogen interactions. Resistance to C. fulvum in tomato plants follows the gene-for-gene hypothesis, which requires the presence of a Cf resistance gene in tomato and presence of the cognate avirulence gene (Avr) in C. fulvum. Upon perception of the Avr by a tomato plant, a typical hypersensitive response (HR) is induced that renders the plant resistant to C. fulvum. In the years preceding this thesis work, most research was focussed on understanding which Avrs are produced by C. fulvum and how these Avrs are actually perceived by resistant plants (Chapter 1). The goal of the work described in this thesis is to reveal downstream signalling cascades triggered upon Avr perception. Therefore, the HR was studied by using a model system in which the Cf-4 protein of tomato and the Avr4 protein from C. fulvum were simultaneously expressed in tomato seedlings. Since the Cf-4/Avr-induced responses are inhibited at 33°C and high humidity, these Cf-4/Avr4 seedlings initiate a synchronized and reproducible HR after incubation at 33°C and a subsequent shift to 20°C, which allows studying downstream responses. To prevent pathogen proliferation in the resistant plant, defence signalling cascades need to be activated extremely fast upon pathogen recognition. Therefore, many downstream signalling cascades depend on post-translational modifications (PTMs) that allow a rapid, reversible, controlled and highly specific transduction of perceived signals. An overview of the various types of PTMs and their role in the resistance response of plants to pathogens is provided in Chapter 2. In addition, examples are provided of successful pathogens that manipulate PTMs. Protein phosphorylation seems to play an important role in the Cf-4/Avr4-triggered HR, since Avr4 perception leads to the specific activation of at least three mitogen-activated protein kinases, LeMPK1, -2 and -3, which requires phosphorylation by an upstream kinase (Chapter 3). Each of these three kinases seems to have a different role in downstream defence signalling, since the kinases were shown to have different phosphorylation specificities and therefore most likely have different downstream target substrates. Furthermore, these kinases appear to play a different role with regard to HR and full resistance to C. fulvum in tomato (Chapter 3). Since protein phosphorylation was shown to play an important role in Cf-4/Avr4-induced defence signalling, the phosphoproteome of Cf-4/Avr4 and control seedlings after HR initiation was studied using a new approach (Chapter 4). This approach led to the identification of 50 phosphoproteins, most of which have not been described in tomato before. Quantification revealed 13 phosphoproteins with an altered abundance in the Cf-4/Avr4 seedlings as compared to the control, which implies HR-induced differential phosphorylation of these proteins. Phosphorylation-mediated regulation of the activity of these proteins pointed to a swift decrease in photosynthetic activity upon HR-initiation, which was confirmed by experiments in which the actual efficiency of the photosynthesis in the Cf-4/Avr4 seedlings was determined upon induction of the HR. Furthermore, a shift from aerobic to anaerobic respiration, which possibly results from oxygen depletion caused by a massive oxidative burst consuming large amounts of oxygen, seems to take place upon initiation of the HR. Finally, differential phosphorylation of the four cytoplasmic isoforms of the Hsp90 chaperone protein was observed, suggesting that they play distinct roles during defence signalling (Chapter 4). In addition to the HR, other associated defence responses are initiated upon recognition of C. fulvum. One of these responses is the secretion of defence-related proteins into the apoplast, which is the environment where C. fulvum operates. Therefore, the dynamics of the apoplastic proteome of resistant, Cf-4-expressing plants and susceptible tomato plants lacking Cf-4, were studied after inoculation with a strain of C. fulvum that secretes Avr4 (Chapter 5). Analysis of the apoplastic proteome revealed a slow accumulation of defence proteins in the apoplast of susceptible plants, which is most likely the result of perception of general elicitors of C. fulvum by tomato. In resistant plants, the same set of proteins accumulates in the apoplast, but this occurs much faster and to higher levels. The accelerated response is caused by the Cf-4/Avr4-initiated HR that also leads to cell death. The HR, in combination with the accelerated protein secretion, renders the plants resistant to C. fulvum. In addition, in susceptible plants C. fulvum seems to specifically downregulate genes encoding cell wall proteins of which the accumulation possibly hampers nutrient and water uptake and thereby proliferation of the pathogen in the tomato apoplast. Possibly, an effector of C. fulvum targets a receptor for general elicitors, thereby suppressing transcription of these genes (Chapter 5). Most data described in this thesis have been obtained from Cf-4/Avr4 seedlings in which the HR can be inhibited by incubating the plants at 33°C. The present data suggest that this temperature-sensitivity occurs at the site of signal perception. Possibly, cytoplasmic Hsp90 stabilizes R protein complexes localized at the plasma membrane. Upon high temperature stress, an increased demand for Hsp90 occurs in the cells to stabilize unfolding proteins that play a role in basal cellular processes, which could lead to the release and subsequent degradation of R protein complexes, rendering defence signalling temperature-sensitive (Chapter 6). The temperature-sensitivity of the Cf-4/Avr4-initiated HR provides a very clean and reproducible tool to study the HR, in the absence of the fungus that produces the Avr. Furthermore, the data described in this thesis provide evidence that the Cf-4/Avr4 seedlings recover from the temperature stress before the specific Cf-4/Avr4-triggered HR is initiated. The possibility to separate the events directly associated with the HR from the full resistance response of the plant to the invading fungus, provides new insight into the complexity of plant defence responses and their specific suppression upon successful colonization by C. fulvum (Chapter 6). Comparison of the defence response to other processes that occur in the cell underlines that resistance and HR execution cannot be seen as an independent and separate process in resistant plants that have recognized a pathogen. On the contrary, signalling cascades seem to depend on similar components and on cascades that possibly converge, eventually leading to a similar response (Chapter 6). Finally, an up to date model for the Cf-4/Avr4-triggered HR and resistance is proposed, based on data that have been published before and the results obtained with the research described in this thesis (Chapter 6). <br/
Measuring industry-science links through inventor-author relations: A profiling method
No full textIn this pilot study we examine the performance of text-based profiling in recovering a set of validated inventor-author links. In a first step we match patents and publications solely based on their similarity in content. Next, we compare inventor and author names on the highest ranked matches for the occurrence of name matches. Finally, we compare these candidate matches with the names listed in a validated set of inventor-author names. Our text-based profile methodology performs significantly better than a random matching of patents and publications, suggesting that text-based profiling is a valuable complementary tool to the name searches used in previous studies.innovation; industry-science links; text-based profiling;
Samenwerken in productontwikkeling : een onderzoek naar de optimalisatie van productontwikkelingsprocessen bij DAF Trucks Eindhoven
No full textMin-max control of fuel-cell-car-based smart energy systems
No full textRecently, the idea of using fuel cell vehicles as the future way of producing electricity has emerged. A fuel cell car has all the necessary devices on board to convert the chemical energy of hydrogen into electricity. This paper considers a scenario where a parking lot for fuel cell cars acts as a virtual power plant. In order to describe the system behavior from the energy point of view, a hybrid (mixed logical dynamical) model is constructed. With this model, a control system is designed to determine the production profile for both the fuel cell and battery of each car in the parking lot subject to minimizing the operational cost. In order to deal with both the uncertainty in the demand profile and the power balance constraint, a robust min-max model predictive control algorithm is developed. The effectiveness of the proposed approach is illustrated in a numerical example.Accepted Author ManuscriptTeam Bart De Schutte
BArt: Trading digital contents through digital assets
No full textSince digital artworks are indeed digital content, they face the inherent problem digital content has: the link between content and its original author is very difficult
to keep. Additionally, retaining control over digital copies of the content is also a challenging task. Digital coins have solved this very same problem through cryptocurrencies (for instance, Bitcoin) and have opened the door to apply the same techniques to other similar scenarios where ownership of digital assets needs to be preserved. In this paper, we propose BArt, a transparent and distributed mechanism for artists to commercialize their digital artwork, keeping control of the copies, monetizing its usage, and maintaining ownership. BArt allows artists to publicly register
their work in the Bitcoin blockchain and sell usage rights in exchange for bitcoins. Buyers are allowed to exert the acquired rights. Proper behaviour from all parties is
enforced by the system with cryptography and economic incentives.Government of Catalonia, Grant/Award Number: 2017SGR00705Martí i Franquès research grants programme, Grant/Award Number:2017PMF-POST2-06Spanish Government, Grant/Award Number: RTI2018-095094-B-C22 CONSENT and TIN2014-57364-C2-2-RSMARTGLACI
Prescribed-performance tracking for high-power nonlinear dynamics with time-varying unknown control coefficients
No full textPrescribed-performance control (PPC) for high-power dynamics with time-varying unknown control coefficients requires to address two open problems: (a) given a Nussbaum function, which properties hold for the power of the Nussbaum function? (b) to avoid high gains, how to design a switching gain that increases only when the tracking error is close to violate the performance bounds? To address the first problem, we show with a counterexample and a positive example that only some Nussbaum functions are suited to handle time-varying unknown control coefficients for high-power dynamics. To address the second problem, we propose a new switching conditional inequality.Green Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Team Bart De Schutte
Adaptive synchronization in networks with heterogeneous uncertain Kuramoto-like units
No full textWe analyze adaptive synchronization capabilities in networks with Kuramoto-like units whose dynamical features are unknown and thus synchronization protocols must exhibit co-evolution capabilities. In the presence of heterogeneous and uncertain units, synchronization should be enabled by appropriate adaptive protocols that counteract the effect of heterogeneity. An interaction protocol is presented that is used by the units to communicate with each other: the protocol is based on a distributed disagreement measure. The aim of the protocol is to adapt feedback and coupling gains, so as to guarantee the emergence of a synchronous solution. The adaptive strategy is distributed, i.e. each unit self-determines the strength of its gains by using only neighboring measurements. Convergence of the synchronization error to zero is shown via Lyapunov analysis, and numerical examples demonstrate the effectiveness of the proposed protocol.Accepted Author ManuscriptTeam Bart De Schutte
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