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Identification of Core Genes Regulating Plant Programmed Cell Death (PCD)

ABSTRACT: PCD is a highly organised process that is involved in development and in an organisms response to biotic stresses (toxins and avirulent pathogens) and abiotic stresses (such as temperature, water availability, etc.). It is a genetically regulated form of cellular suicide, however in plants the underlying process is poorly understood. Although PCD may occur in response to different stimuli; we believe once it is triggered, one core mechanism is responsible for the cellular demise. It is our aim to identify the elements of this mechanism. We will do this by expanding on the work of a previous user of GARNet's GeneChip microarray facility, Dr. Jodi Swidzinski. She utilised an Arabidopsis cell suspension system; performing microarray analysis on both senescing, and heat shock induced PCD samples. This resulted in data showing that a large number of genes were upregulated (or downregulated) in response to both treatments. We are working under the premise that some of the genes that were similarly regulated under both treatments must be core PCD genes. However because of the large amount of data generated it is difficult to choose appropriate candidate genes (with any degree of confidence that they are core genes) for further study. We propose to use a third PCD-inducing treatment, involving a mycotoxin, to generate another population of microarray results. The mycotoxin we intend to use is Fumonisin B1 (FB1). This is an extremely potent compound that induces PCD by disrupting ceramide synthesis. We have found Arabidopsis protoplasts to be much more sensitive than cells to the toxin at low concentrations. Protoplasts are treated with 20mM FB1 and RNA is extracted at time points when 0%, 20% and 40% of protoplasts have died. This RNA is then pooled. RNA from methanol treated protoplasts is used as a control. We intend for these RNA samples to be subjected to GeneChip microarray analysis. This would identify genes differentially regulated due to FB1 treatment, which would be interesting in itself. However, by combining this data with that from previous work by Swidzinski (2002) we will be able to decrease the pool of possible core genes further, and increase the chances of selecting an appropriate candidate gene. This will both improve upon existing work and add value to an existing GARNet data set. Experimenter name = Mark Diamond Experimenter phone = 017162251 Experimenter fax = 017161153 Experimenter address = Botany Department Experimenter address = University College Dublin Experimenter address = Belfield Experimenter zip/postal_code = Dublin 4 Experimenter country = Ireland Keywords: compound_treatment_design

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE5735 | GEO | 2007/01/08

SECONDARY ACCESSION(S): PRJNA97043

REPOSITORIES: GEO

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Project description:PCD is a highly organised process that is involved in development and in an organisms response to biotic stresses (toxins and avirulent pathogens) and abiotic stresses (such as temperature, water availability, etc.). It is a genetically regulated form of cellular suicide, however in plants the underlying process is poorly understood. Although PCD may occur in response to different stimuli; we believe once it is triggered, one core mechanism is responsible for the cellular demise. It is our aim to identify the elements of this mechanism. We will do this by expanding on the work of a previous user of GARNet's GeneChip microarray facility, Dr. Jodi Swidzinski. She utilised an Arabidopsis cell suspension system; performing microarray analysis on both senescing, and heat shock induced PCD samples. This resulted in data showing that a large number of genes were upregulated (or downregulated) in response to both treatments. We are working under the premise that some of the genes that were similarly regulated under both treatments must be core PCD genes. However because of the large amount of data generated it is difficult to choose appropriate candidate genes (with any degree of confidence that they are core genes) for further study. We propose to use a third PCD-inducing treatment, involving a mycotoxin, to generate another population of microarray results. The mycotoxin we intend to use is Fumonisin B1 (FB1). This is an extremely potent compound that induces PCD by disrupting ceramide synthesis. We have found Arabidopsis protoplasts to be much more sensitive than cells to the toxin at low concentrations. Protoplasts are treated with 20mM FB1 and RNA is extracted at time points when 0%, 20% and 40% of protoplasts have died. This RNA is then pooled. RNA from methanol treated protoplasts is used as a control. We intend for these RNA samples to be subjected to GeneChip microarray analysis. This would identify genes differentially regulated due to FB1 treatment, which would be interesting in itself. However, by combining this data with that from previous work by Swidzinski (2002) we will be able to decrease the pool of possible core genes further, and increase the chances of selecting an appropriate candidate gene. This will both improve upon existing work and add value to an existing GARNet data set. Experimenter name = Mark Diamond; Experimenter phone = 017162251; Experimenter fax = 017161153; Experimenter address = Botany Department; Experimenter address = University College Dublin; Experimenter address = Belfield; Experimenter zip/postal_code = Dublin 4; Experimenter country = Ireland Experiment Overall Design: 8 samples were used in this experiment

Project description:Fungal secondary metabolites can not only cause toxic effects in animals and humans, but also serve as virulence factors of the producing fungi for causing plant diseases.Thus, the severity of plant diseases associated with mycotoxins depend on the sensitivity towards the toxin. In previous experiments, we have evaluated the phytotoxic effect ofa mycotoxin on root growth of Arabidopsis wild-type and mutant seedlings. Mycotoxin treatment of a new conditional root expansion mutant partially restores the expansion phenotype (JE100; Werner et al., unpublished). AIM: This experiment aims to identify genes, in early and later phases after mycotoxin treatment in wild-type and mutant seedlings. EXPERIMENTAL PLAN: Eight Affymetrix chips are needed for this experiment. RNA preparation will be provided from wild-type, accession Columbia, and mutant seedlings after different time points of mycotoxin treatment. As control, separate seedlings will be treated with the same concentration of solvent (DMSO). Briefly, seeds will be sterilized, stratified for 48 hours and germinated on MS agar plates containing 4.5% sucrose at 22Â°C and 16h/8h light/dark cycles. 10 days after germination, seedlings will be transferred to liquid MS medium and shaken for another 3 days for acclimatization. Seedlings will be harvested after 2 and 24 hours of treatment with a single concentration (50 ÂµM) of mycotoxin. To account for experimental variations (i.e. time needed for freezing the tissues, circadian clock,...), the experiment will be repeated three times and RNA samples will be pooled. EXPECTED RESULTS: The experiment should identify genes differentially expressed:; 1) between wild-type and mutant seedlings,; 2) upon mycotoxin treatment in wild-type,; 3) upon mycotoxin treatment of mutant seedlings and; 4) upon solvent treatment. The results will allow us to pinpoint the mode of action of this mycotoxin. They will also allow us to better understand the function of the mutated gene which affects the sensitivity towards the mycotoxin. Furthermore, we expect to identify the signaling pathway by which the plant responses towards the mycotoxinis triggered. Experimenter name = Ulrike Werner; Experimenter phone = +43-1-36006-6371; Experimenter fax = +43-1-36006-6392; Experimenter department = Institute of Applied Genetics and Cell Biology; Experimenter institute = BOKU; Experimenter address = Center of Applied Genetics; Experimenter address = University of Agricultural Sciences Vienna; Experimenter address = Muthgasse 18; Experimenter address = Vienna; Experimenter zip/postal_code = 1190; Experimenter country = Austria Experiment Overall Design: 8 samples were used in this experiment

Project description:Fungal secondary metabolites can not only cause toxic effects in animals and humans, but also serve as virulence factors of the producing fungi for causing plant diseases.Thus, the severity of plant diseases associated with mycotoxins depend on the sensitivity towards the toxin. In previous experiments, we have evaluated the phytotoxic effect ofa mycotoxin on root growth of Arabidopsis wild-type and mutant seedlings. Mycotoxin treatment of a new conditional root expansion mutant partially restores the expansion phenotype (JE100; Werner et al., unpublished). AIM: This experiment aims to identify genes, in early and later phases after mycotoxin treatment in wild-type and mutant seedlings. EXPERIMENTAL PLAN: Eight Affymetrix chips are needed for this experiment. RNA preparation will be provided from wild-type, accession Columbia, and mutant seedlings after different time points of mycotoxin treatment. As control, separate seedlings will be treated with the same concentration of solvent (DMSO). Briefly, seeds will be sterilized, stratified for 48 hours and germinated on MS agar plates containing 4.5% sucrose at 22°C and 16h/8h light/dark cycles. 10 days after germination, seedlings will be transferred to liquid MS medium and shaken for another 3 days for acclimatization. Seedlings will be harvested after 2 and 24 hours of treatment with a single concentration (50 µM) of mycotoxin. To account for experimental variations (i.e. time needed for freezing the tissues, circadian clock,...), the experiment will be repeated three times and RNA samples will be pooled. EXPECTED RESULTS: The experiment should identify genes differentially expressed: 1) between wild-type and mutant seedlings, 2) upon mycotoxin treatment in wild-type, 3) upon mycotoxin treatment of mutant seedlings and 4) upon solvent treatment. The results will allow us to pinpoint the mode of action of this mycotoxin. They will also allow us to better understand the function of the mutated gene which affects the sensitivity towards the mycotoxin. Furthermore, we expect to identify the signaling pathway by which the plant responses towards the mycotoxinis triggered. Experimenter name = Ulrike Werner Experimenter phone = +43-1-36006-6371 Experimenter fax = +43-1-36006-6392 Experimenter department = Institute of Applied Genetics and Cell Biology Experimenter institute = BOKU Experimenter address = Center of Applied Genetics Experimenter address = University of Agricultural Sciences Vienna Experimenter address = Muthgasse 18 Experimenter address = Vienna Experimenter zip/postal_code = 1190 Experimenter country = Austria Keywords: genetic_modification_design; compound_treatment_design; time_series_design

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Identification of Core Genes Regulating Plant Programmed Cell Death (PCD)

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