Induction of camalexin biosynthesis in Arabidopsis thaliana in response to elicitation by lipopolysaccharides
- Authors: Beets, Caryn Ann
- Date: 2011-06-29T06:37:33Z
- Subjects: Phytoalexins , Endotoxins , Arabidopsis thaliana , Biosynthesis
- Type: Thesis
- Identifier: uj:7137 , http://hdl.handle.net/10210/3729
- Description: M.Sc. , On exposure to abiotic or biotic stresses, plants initiate a cascade of metabolic reactions, some of which lead to the biosynthesis of secondary metabolites with roles in self defense. Phytoalexins are a class of secondary metabolites synthesized de novo in response to microbial attack by activation of certain biosynthetic pathways. Cruciferae phytoalexins are all indole based with a carbon, nitrogen and sulfur containing constituent on the 3’ position of the indole ring. This common similarity of all Cruciferae phytoalexins suggests that the plants all share a common indole precursor. Camalexin is the primary phytoalexin of Arabidopsis thaliana. De novo synthesis of camalexin upon infection, as well as its antimicrobial nature supports its role in disease resistance. Evidence exists that suggests the inducible biosynthesis of camalexin involves steps of the tryptophan pathway, along with an increase in transcript and protein levels of the tryptophan pathway enzymes after microbial infection. Bacterial LPS (lipopolysaccharide) has been described as one of the microbe/pathogenassociated molecular patterns (M/PAMPs) capable of eliciting the activation of the plant innate immune system. LPS is an integral component of the cell surface of Gram-negative bacteria. It is a complex which is exposed to the external environment, and is thus involved with external interactions of the bacteria. The hypothesis investigated in this dissertation is that LPS, as a lipoglycan PAMP, results in activation of signal transduction pathways involved in defense that lead to the production of the defense metabolite, camalexin. Furthermore, that the genes CYP71B15, CYP79B2 and TSB are up-regulated in response to LPS during camalexin biosynthesis via the tryptophan pathway. To test this hypothesis, camalexin production was investigated through a combination of analytical techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), ultra pressure liquid chromatography-mass spectrometry (UPLC-MS) and fluorescence spectroscopy. Genes in the camalexin biosynthetic pathway were investigated by two-step reverse transcription polymerase chain reaction (PCR), GUS reporter gene assays and quantitative real time PCR (RT-qPCR).
- Full Text:
- Authors: Beets, Caryn Ann
- Date: 2011-06-29T06:37:33Z
- Subjects: Phytoalexins , Endotoxins , Arabidopsis thaliana , Biosynthesis
- Type: Thesis
- Identifier: uj:7137 , http://hdl.handle.net/10210/3729
- Description: M.Sc. , On exposure to abiotic or biotic stresses, plants initiate a cascade of metabolic reactions, some of which lead to the biosynthesis of secondary metabolites with roles in self defense. Phytoalexins are a class of secondary metabolites synthesized de novo in response to microbial attack by activation of certain biosynthetic pathways. Cruciferae phytoalexins are all indole based with a carbon, nitrogen and sulfur containing constituent on the 3’ position of the indole ring. This common similarity of all Cruciferae phytoalexins suggests that the plants all share a common indole precursor. Camalexin is the primary phytoalexin of Arabidopsis thaliana. De novo synthesis of camalexin upon infection, as well as its antimicrobial nature supports its role in disease resistance. Evidence exists that suggests the inducible biosynthesis of camalexin involves steps of the tryptophan pathway, along with an increase in transcript and protein levels of the tryptophan pathway enzymes after microbial infection. Bacterial LPS (lipopolysaccharide) has been described as one of the microbe/pathogenassociated molecular patterns (M/PAMPs) capable of eliciting the activation of the plant innate immune system. LPS is an integral component of the cell surface of Gram-negative bacteria. It is a complex which is exposed to the external environment, and is thus involved with external interactions of the bacteria. The hypothesis investigated in this dissertation is that LPS, as a lipoglycan PAMP, results in activation of signal transduction pathways involved in defense that lead to the production of the defense metabolite, camalexin. Furthermore, that the genes CYP71B15, CYP79B2 and TSB are up-regulated in response to LPS during camalexin biosynthesis via the tryptophan pathway. To test this hypothesis, camalexin production was investigated through a combination of analytical techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), ultra pressure liquid chromatography-mass spectrometry (UPLC-MS) and fluorescence spectroscopy. Genes in the camalexin biosynthetic pathway were investigated by two-step reverse transcription polymerase chain reaction (PCR), GUS reporter gene assays and quantitative real time PCR (RT-qPCR).
- Full Text:
Induksie van B-1,3 glukanase en chitinase iso-ensieme in katoenplante deur elisitormolekule van die patogeen, Verticillium dahliae
- Authors: Slater, Vernon
- Date: 2014-04-02
- Subjects: Verticillium dahliae , Cotton verticillium wilt , Phytoalexins , Lignin
- Type: Thesis
- Identifier: uj:4589 , http://hdl.handle.net/10210/9935
- Description: M.Sc. , Inducible defence responses in both a susceptible cotton cultivar ( Acala ) and a resistant cotton cultivar ( OR-19 ) in response to elicitors fromVerticillium dahliae were investigated. These oligosaccharin elicitors represent the heat solubilized, non-dialyzable fraction of the pathogen cell wall. This elicitorfraction consist of 6.57 % protein and 68 % carbohydrate and represent merely a discrete portion of the cell wall. Moreover, symptoms such as chlorosis and necrosis were induced by the elicitor in both cultivars, but the timing and magnitute of symptom development differed in that the symptoms occurred much faster and were more intense ( hypersensitive response) in the resistant cultivar. An effective elicitor concentration of 30 ug/ ml ( = 21 ug glucose equivalents) was determined and used throughout this study. Inducible defence responses i.e, the accumulation of PR-proteins and specifically activities of B-1,3-glucanase and chitinase were investigated in the intercellular environment as well as cellular extractions of both cultivars. An attempt was also made to analyse the expression of B-1 ,3-glucanase and chitinase genes at them RNA level ( level of transcription) and to correlate it to the determined levels of enzyme activities. Lignification as well as smaller plant metabolites i.e, sesquiterpenoid-phytoalexins relating to pathogenesis that are induced by the elicitor, were also investigated in both cultivars. In this study it is shown that differences are found between Acala and OR-19 and that some of these differences can be correlated to plant resistance. These differences found in the time studies ( intensity, time of response, qualitative and quantitative differences ) of the defence responses induced in both cultivars is discussed against the background of disease resistance.
- Full Text:
- Authors: Slater, Vernon
- Date: 2014-04-02
- Subjects: Verticillium dahliae , Cotton verticillium wilt , Phytoalexins , Lignin
- Type: Thesis
- Identifier: uj:4589 , http://hdl.handle.net/10210/9935
- Description: M.Sc. , Inducible defence responses in both a susceptible cotton cultivar ( Acala ) and a resistant cotton cultivar ( OR-19 ) in response to elicitors fromVerticillium dahliae were investigated. These oligosaccharin elicitors represent the heat solubilized, non-dialyzable fraction of the pathogen cell wall. This elicitorfraction consist of 6.57 % protein and 68 % carbohydrate and represent merely a discrete portion of the cell wall. Moreover, symptoms such as chlorosis and necrosis were induced by the elicitor in both cultivars, but the timing and magnitute of symptom development differed in that the symptoms occurred much faster and were more intense ( hypersensitive response) in the resistant cultivar. An effective elicitor concentration of 30 ug/ ml ( = 21 ug glucose equivalents) was determined and used throughout this study. Inducible defence responses i.e, the accumulation of PR-proteins and specifically activities of B-1,3-glucanase and chitinase were investigated in the intercellular environment as well as cellular extractions of both cultivars. An attempt was also made to analyse the expression of B-1 ,3-glucanase and chitinase genes at them RNA level ( level of transcription) and to correlate it to the determined levels of enzyme activities. Lignification as well as smaller plant metabolites i.e, sesquiterpenoid-phytoalexins relating to pathogenesis that are induced by the elicitor, were also investigated in both cultivars. In this study it is shown that differences are found between Acala and OR-19 and that some of these differences can be correlated to plant resistance. These differences found in the time studies ( intensity, time of response, qualitative and quantitative differences ) of the defence responses induced in both cultivars is discussed against the background of disease resistance.
- Full Text:
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