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/

Patient preferences and treatment safety for uncomplicated vulvovaginal candidiasis in primary health care

<p>Abstract</p> <p>Background</p> <p>Vaginitis is a common complaint in primary care. In uncomplicated candidal vaginitis, there are no differences in effectiveness between oral or vaginal treatment. Some studies describe that the preferred treatment is the oral one, but a Cochrane's review points out inconsistencies associated with the report of the preferred way that limit the use of such data. Risk factors associated with recurrent vulvovaginal candidiasis still remain controversial.</p> <p>Methods/Design</p> <p>This work describes a protocol of a multicentric prospective observational study with one year follow up, to describe the women's reasons and preferences to choose the way of administration (oral vs topical) in the treatment of not complicated candidal vaginitis. The number of women required is 765, they are chosen by consecutive sampling. All of whom are aged 16 and over with vaginal discharge and/or vaginal pruritus, diagnosed with not complicated vulvovaginitis in Primary Care in Madrid.</p> <p>The main outcome variable is the preferences of the patients in treatment choice; secondary outcome variables are time to symptoms relief and adverse reactions and the frequency of recurrent vulvovaginitis and the risk factors. In the statistical analysis, for the main objective will be descriptive for each of the variables, bivariant analysis and multivariate analysis (logistic regression).. The dependent variable being the type of treatment chosen (oral or topical) and the independent, the variables that after bivariant analysis, have been associated to the treatment preference.</p> <p>Discussion</p> <p>Clinical decisions, recommendations, and practice guidelines must not only attend to the best available evidence, but also to the values and preferences of the informed patient.</p

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Meeldauwprogrammeringstudie : studie mangaan en elicitor/groene gewasbeschermingsmiddelen over verschillende plant-meeldauwsystemen heen

Powdery mildew is still a major problem in greenhouse cultivation. This research was focused on preventive treatments of powdery mildew using nutrition en elicitors/green plant protection products to increase plant resilience. Investigations were conducted using different plant-mildew systems: 1. Pot gerbera - Podosphaera xanthii, 2. Sweet pepper – Leveillula taurica, 3. Kalanchoe - Oidium neolycopersicon en 4. Rose – Podosphaera pannosa. Plants were treated with manganese (Mn) concentrations of 5, 10, 20 en 40 μmol/l in the nutrient solution. Next to these preventive treatments were conducted with the elicitors/green plant protection products Hicure, SilicaPower and Inssimo. A concentration of 10 μmol/l Mn had a preventive effect against powdery mildew in sweet pepper and rose in contrast to gerbera and kalanchoe. Treatment with Inssimo had a preventive effect on powdery mildew in all plant-mildew systems. Combinations of Mn treatments with Inssimo did not lead to synergistic effects. To maximise the use of Inssimo for plant resilience treatment should be coupled to an early warning system which indicates on time whether preventive treatment is necessary

Involvement of the ABC transporter BcAtrB and the laccase BcLCC2 in defence of Botrytis cinerea against the broad-spectrum antibiotic 2,4-diacetylphloroglucinol

The genetic and biochemical basis of defence mechanisms in plant pathogenic fungi against antifungal compounds produced by antagonistic microorganisms is largely unknown. The results of this study show that both degradative and non-degradative defence mechanisms enable the plant pathogenic fungus Botrytis cinerea to resist the broad-spectrum, phenolic antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). The efflux pump BcAtrB provides the first line of defence for B. cinerea, preventing accumulation of 2,4-DAPG in the cell to toxic concentrations, whereas the extracellular laccase BcLCC2 mediates, via conversion of tannic acid, subsequent degradation of 2,4-DAPG. Expression of BcatrB is induced by 2,4-DAPG and efflux gives B. cinerea sufficient time to more effectively initiate the process of BcLCC2-mediated antibiotic degradation. This is supported by the observations that the BcatrB mutant is significantly more sensitive to 2,4-DAPG than its parental strain, and is substantially less effective in 2,4-DAPG degradation. The results of this study further showed that BcLCC2 itself is not able to degrade 2,4-DAPG, but requires tannic acid as a mediator for 2,4-DAPG degradation. To our knowledge, this is the first time that the laccase-mediator system is shown to play a role in the detoxification of a broad-spectrum antibiotic compound from bacterial origin. We postulate that yet unknown constituents present in tannic acid act as substrate(s) of BcLCC2, thereby generating radicals that mediate 2,4-DAPG degradation