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.