Ethylene perception and NEP-like protein production by Botrytis cinerea

Botrytis cinerea can infect more than 200 plant species, including a wide range of economically important crops. During pathogen infection, plants release ethylene and it has been hypothesized that ethylene may predispose host tissue for infection by inducing senescence and ripening. This thesis focused on the roles that ethylene production and perception, both by the pathogen and the plant, play in the interaction between B. cinerea and crops, using tomato as a model. Furthermore, functional analysis was performed of B. cinerea Nep1-Like Proteins (NLPs), called BcNEP1 and BcNEP2, with emphasis on their role in virulence and mode of action. Ethylene regulates several developmental processes in plants and plays an important role in plant-pathogen interactions. We investigated possible effects of ethylene on B. cinerea during infection of tomato Solanum lycopersicum (Chapter 2). There were previous reports that ethylene released by the plant could stimulate germination of B. cinerea conidia and affect germ tube growth and infection structure differentiation. Based on growth experiments in vitro in the presence of ethylene, we conclude that ethylene does not affect hyphal development of the fungus. Also the virulence of B. cinerea on tomato genotypes with a reduced or an enhanced ethylene production level was unaltered. Neither did ethylene induce fungal gene expression as was previously reported. We studied a B. cinerea gene encoding a histidine kinase (BcHHK5) with strong structural similarity to plant ethylene receptors. Mutants in which the Bchhk5 was deleted were neither affected in growth in vitro nor in virulence. We propose that the effects of ethylene on B. cinerea disease development are not a direct consequence of an ethylene response in the pathogen, but rather a consequence of induced senescence and ripening processes in the host. These processes provoke softening and disintegration of tissues that facilitate the entry and proliferation of the pathogen. Functional analysis was performed of two B. cinerea NLPs, named BcNEP1 and BcNEP2, produced in Pichia pastoris (Chapter 3). Infiltration of purified proteins into N. benthamiana leads to induction of ethylene in a dose-dependent manner. BcNEP1 was able to induce ethylene and necrosis at lower concentrations as compared to BcNEP2. Transcriptional studies (Chapter 3) showed that Bcnep1 is transiently expressed during early stages of infection when primary lesions develop, while Bcnep2 is expressed when the infection is established and lesions are expanding. Altogether these results suggested that BcNEP1 and BcNEP2 may have different functions or they have a similar function at different stages of the infection process. Single knock-out mutants of either Bcnep1 or Bcnep2 gene showed no reduction of virulence on tomato or N. benthamiana. Ethylene emitted by leaves inoculated with Bcnep mutants was not significantly different from leaves inoculated with the parental wild type strain B05.10. These results demonstrate that BcNEP proteins are not essential in the infection process of B. cinerea and that ethylene produced in B. cinerea-infected tissue does not result from a response to BcNEP proteins (Chapter 3). By transiently expressing site-directed mutant BcNEP proteins in N. benthamiana and N. tabacum through Agrobacterium tumefaciens, we could study structure-function relationships (Chapter 4). The conserved hepta-peptide GHRHWDE, in the central part of the protein sequence, was shown to be essential for the necrosis-inducing activity. Also the first two cysteine residues, C68 and C94, which are predicted to form a disulfide bridge, are important for necrosis-inducing activity. The two proteins contain different post-transcriptional modification motifs, however, none of these motifs is essential for necrosis-inducing activity. Necrosis-inducing activity of BcNEP1 was independent of light, whereas the activity of BcNEP2 was compromised when the protein was infiltrated in leaves of dark-adapted plants and the infiltrated plants were kept in darkness (Chapter 5). We studied the role of the plant in the mode of action of BcNEP proteins using genetic and pharmacological approaches (Chapter 5). In spite of several efforts, we were not able to identify any cellular process or signaling pathway in plants that is required for the necrosis-inducing activity of BcNEP proteins. The target(s) and mode(s) of action of BcNEP proteins remain unresolved. Botrytis cinerea can infect more than 200 plant species, including a wide range of economically important crops. During pathogen infection, plants release ethylene and it has been hypothesized that ethylene may predispose host tissue for infection by inducing senescence and ripening. This thesis focused on the roles that ethylene production and perception, both by the pathogen and the plant, play in the interaction between B. cinerea and crops, using tomato as a model. Furthermore, functional analysis was performed of B. cinerea Nep1-Like Proteins (NLPs), called BcNEP1 and BcNEP2, with emphasis on their role in virulence and mode of action. Ethylene regulates several developmental processes in plants and plays an important role in plant-pathogen interactions. We investigated possible effects of ethylene on B. cinerea during infection of tomato Solanum lycopersicum (Chapter 2). There were previous reports that ethylene released by the plant could stimulate germination of B. cinerea conidia and affect germ tube growth and infection structure differentiation. Based on growth experiments in vitro in the presence of ethylene, we conclude that ethylene does not affect hyphal development of the fungus. Also the virulence of B. cinerea on tomato genotypes with a reduced or an enhanced ethylene production level was unaltered. Neither did ethylene induce fungal gene expression as was previously reported. We studied a B. cinerea gene encoding a histidine kinase (BcHHK5) with strong structural similarity to plant ethylene receptors. Mutants in which the Bchhk5 was deleted were neither affected in growth in vitro nor in virulence. We propose that the effects of ethylene on B. cinerea disease development are not a direct consequence of an ethylene response in the pathogen, but rather a consequence of induced senescence and ripening processes in the host. These processes provoke softening and disintegration of tissues that facilitate the entry and proliferation of the pathogen. Functional analysis was performed of two B. cinerea NLPs, named BcNEP1 and BcNEP2, produced in Pichia pastoris (Chapter 3). Infiltration of purified proteins into N. benthamiana leads to induction of ethylene in a dose-dependent manner. BcNEP1 was able to induce ethylene and necrosis at lower concentrations as compared to BcNEP2. Transcriptional studies (Chapter 3) showed that Bcnep1 is transiently expressed during early stages of infection when primary lesions develop, while Bcnep2 is expressed when the infection is established and lesions are expanding. Altogether these results suggested that BcNEP1 and BcNEP2 may have different functions or they have a similar function at different stages of the infection process. Single knock-out mutants of either Bcnep1 or Bcnep2 gene showed no reduction of virulence on tomato or N. benthamiana. Ethylene emitted by leaves inoculated with Bcnep mutants was not significantly different from leaves inoculated with the parental wild type strain B05.10. These results demonstrate that BcNEP proteins are not essential in the infection process of B. cinerea and that ethylene produced in B. cinerea-infected tissue does not result from a response to BcNEP proteins (Chapter 3). By transiently expressing site-directed mutant BcNEP proteins in N. benthamiana and N. tabacum through Agrobacterium tumefaciens, we could study structure-function relationships (Chapter 4). The conserved hepta-peptide GHRHWDE, in the central part of the protein sequence, was shown to be essential for the necrosis-inducing activity. Also the first two cysteine residues, C68 and C94, which are predicted to form a disulfide bridge, are important for necrosis-inducing activity. The two proteins contain different post-transcriptional modification motifs, however, none of these motifs is essential for necrosis-inducing activity. Necrosis-inducing activity of BcNEP1 was independent of light, whereas the activity of BcNEP2 was compromised when the protein was infiltrated in leaves of dark-adapted plants and the infiltrated plants were kept in darkness (Chapter 5). We studied the role of the plant in the mode of action of BcNEP proteins using genetic and pharmacological approaches (Chapter 5). In spite of several efforts, we were not able to identify any cellular process or signaling pathway in plants that is required for the necrosis-inducing activity of BcNEP proteins. The target(s) and mode(s) of action of BcNEP proteins remain unresolved. <br/

Discrete-time Markov chain approach to contact-based disease spreading in complex networks

Many epidemic processes in networks spread by stochastic contacts among their connected vertices. There are two limiting cases widely analyzed in the physics literature, the so-called contact process (CP) where the contagion is expanded at a certain rate from an infected vertex to one neighbor at a time, and the reactive process (RP) in which an infected individual effectively contacts all its neighbors to expand the epidemics. However, a more realistic scenario is obtained from the interpolation between these two cases, considering a certain number of stochastic contacts per unit time. Here we propose a discrete-time formulation of the problem of contact-based epidemic spreading. We resolve a family of models, parameterized by the number of stochastic contact trials per unit time, that range from the CP to the RP. In contrast to the common heterogeneous mean-field approach, we focus on the probability of infection of individual nodes. Using this formulation, we can construct the whole phase diagram of the different infection models and determine their critical properties.Comment: 6 pages, 4 figures. Europhys Lett (in press 2010

Synchronizability determined by coupling strengths and topology on Complex Networks

We investigate in depth the synchronization of coupled oscillators on top of complex networks with different degrees of heterogeneity within the context of the Kuramoto model. In a previous paper [Phys. Rev. Lett. 98, 034101 (2007)], we unveiled how for fixed coupling strengths local patterns of synchronization emerge differently in homogeneous and heterogeneous complex networks. Here, we provide more evidence on this phenomenon extending the previous work to networks that interpolate between homogeneous and heterogeneous topologies. We also present new details on the path towards synchronization for the evolution of clustering in the synchronized patterns. Finally, we investigate the synchronization of networks with modular structure and conclude that, in these cases, local synchronization is first attained at the most internal level of organization of modules, progressively evolving to the outer levels as the coupling constant is increased. The present work introduces new parameters that are proved to be useful for the characterization of synchronization phenomena in complex networks.Comment: 11 pages, 10 figures and 1 table. APS forma

Paths to Synchronization on Complex Networks

The understanding of emergent collective phenomena in natural and social systems has driven the interest of scientists from different disciplines during decades. Among these phenomena, the synchronization of a set of interacting individuals or units has been intensively studied because of its ubiquity in the natural world. In this paper, we show how for fixed coupling strengths local patterns of synchronization emerge differently in homogeneous and heterogeneous complex networks, driving the process towards a certain global synchronization degree following different paths. The dependence of the dynamics on the coupling strength and on the topology is unveiled. This study provides a new perspective and tools to understand this emerging phenomena.Comment: Final version published in Physical Review Letter

Surgical Blog: An Important Supplement to Traditional Scientific Literature

Introduction: Blogging is a form of social media that has emerged as an important means of information exchange and opinion-building in the academic surgical community. We examined the Association of Women Surgeons (AWS) blog to understand its audience and most read content. Methods: Google Analytics was used to assess the AWS blog site data. A search was performed from the earliest searchable date (February 16, 2018) to February 21, 2019. Demographic data, blog posts, and tags sorted by unique pageviews were recorded. Results: There were 31,221 unique pageviews during the search period. The AWS Blog readership was mostly women (75%), ages 25-44 years (70.3%). The three tags that elicited the most pageviews were “residency” (16.95%), “medical students” (12.12%), and “family life” (10.38%). The most read blog post was titled “Ban the Bouffant,” which was responsible for 9.7% of total pageviews. Conclusion: Most of the AWS Blog readership are young, women, and interested in content related to graduate and postgraduate medical education or family life. Blogging may be a good vehicle for topics not covered in traditional scientific literature

Communication in networks with hierarchical branching

We present a simple model of communication in networks with hierarchical branching. We analyze the behavior of the model from the viewpoint of critical systems under different situations. For certain values of the parameters, a continuous phase transition between a sparse and a congested regime is observed and accurately described by an order parameter and the power spectra. At the critical point the behavior of the model is totally independent of the number of hierarchical levels. Also scaling properties are observed when the size of the system varies. The presence of noise in the communication is shown to break the transition. Despite the simplicity of the model, the analytical results are a useful guide to forecast the main features of real networks.Comment: 4 pages, 3 figures. Final version accepted in PR

Synchronization in a ring of pulsating oscillators with bidirectional couplings

We study the dynamical behavior of an ensemble of oscillators interacting through short range bidirectional pulses. The geometry is 1D with periodic boundary conditions. Our interest is twofold. To explore the conditions required to reach fully synchronization and to invewstigate the time needed to get such state. We present both theoretical and numerical results.Comment: Revtex, 4 pages, 2 figures. To appear in Int. J. Bifurc. and Chao

Explosive Synchronization Transitions in Scale-free Networks

The emergence of explosive collective phenomena has recently attracted much attention due to the discovery of an explosive percolation transition in complex networks. In this Letter, we demonstrate how an explosive transition shows up in the synchronization of complex heterogeneous networks by incorporating a microscopic correlation between the structural and the dynamical properties of the system. The characteristics of this explosive transition are analytically studied in a star graph reproducing the results obtained in synthetic scale-free networks. Our findings represent the first abrupt synchronization transition in complex networks thus providing a deeper understanding of the microscopic roots of explosive critical phenomena.Comment: 6 pages and 5 figures. To appear in Physical Review Letter

How to suppress undesired synchronization

It is delightful to observe the emergence of synchronization in the blinking of fireflies to attract partners and preys. Other charming examples of synchronization can also be found in a wide range of phenomena such as, e.g., neurons firing, lasers cascades, chemical reactions, and opinion formation. However, in many situations the formation of a coherent state is not pleasant and should be mitigated. For example, the onset of synchronization can be the root of epileptic seizures, traffic congestion in communication networks, and the collapse of constructions. Here we propose the use of contrarians to suppress undesired synchronization. We perform a comparative study of different strategies, either requiring local or total knowledge of the system, and show that the most efficient one solely requires local information. Our results also reveal that, even when the distribution of neighboring interactions is narrow, significant improvement in mitigation is observed when contrarians sit at the highly connected elements. The same qualitative results are obtained for artificially generated networks as well as two real ones, namely, the Routers of the Internet and a neuronal network