In silico analysis of cis elements and expression analysis of selected LPS-responsive RLK genes from Arabidopsis thaliana
- Authors: New, Sherrie-Ann
- Date: 2013-07-29
- Subjects: Plant defenses , Plant-pathogen relationships , Arabidopsis thaliana - Disease and pest resistance
- Type: Thesis
- Identifier: uj:7708 , http://hdl.handle.net/10210/8573
- Description: M.Sc. (Biochemistry) , Our comprehension of pathogen perception and defense response mechanisms that play key roles in the resistance of plants against pathogen attack have progressed substantially within the recent years. Recognizing the molecular mechanisms involved in pathogen perception is the basis of understanding the signalling networks that are involved, including the transcriptional regulation of plant defense genes. This has proven to be a great challenge in plant pathology and, as such, has attracted much attention. The receptor-like kinases (RLKs) constitute one of the largest classes of plant defense genes in Arabidopsis thaliana, and contains, inter alia, the well-known leucine-rich repeats-RLKs (LRR-RLK), as well as the S-domain receptor-like kinases (SD-RLKs) that have been shown to be involved in pathogen perception and not only self-incompatibility (SI) as originally discovered. Some members of these RLKs are able to detect pattern-associated molecular patterns (PAMPs), which are conserved pathogen-derived molecules, and trigger a battery of basal defense responses. The transcriptional activation and expression levels of RLKs are dependent on the variation in promoter architecture as a result of the number, location, order and class of cis-elements found in a promoter sequence. It is hypothesized that candidate RLK genes involved in PAMP surveillance are triggered and transcriptionally regulated in response to perception of PAMPs, and that the intensity of response is relative to the promoter architecture. The primary objective was to identify SD-RLKs and LRR-RLKs which demonstrated up-regulation in response to PAMPs. The SD-RLKs (At1g11330, At1g61430 and At1g61610) and LRR-RLKs (At1g51850, At2g19190 and At5g45840) were selected on the basis of microarray data (Nürnberger - TAIR accession set 100808727) and the Genevestigator database, and characterized utilizing bioinformatics tools. Here, molecular techniques were used to show that the selected RLK genes were responsive to PAMP inductions. Furthermore, this study explored which cis-elements and their corresponding transcription factors (TFs) are found in the promoter of plant defense genes and that may be involved in transcriptional regulation thereof...
- Full Text:
- Authors: New, Sherrie-Ann
- Date: 2013-07-29
- Subjects: Plant defenses , Plant-pathogen relationships , Arabidopsis thaliana - Disease and pest resistance
- Type: Thesis
- Identifier: uj:7708 , http://hdl.handle.net/10210/8573
- Description: M.Sc. (Biochemistry) , Our comprehension of pathogen perception and defense response mechanisms that play key roles in the resistance of plants against pathogen attack have progressed substantially within the recent years. Recognizing the molecular mechanisms involved in pathogen perception is the basis of understanding the signalling networks that are involved, including the transcriptional regulation of plant defense genes. This has proven to be a great challenge in plant pathology and, as such, has attracted much attention. The receptor-like kinases (RLKs) constitute one of the largest classes of plant defense genes in Arabidopsis thaliana, and contains, inter alia, the well-known leucine-rich repeats-RLKs (LRR-RLK), as well as the S-domain receptor-like kinases (SD-RLKs) that have been shown to be involved in pathogen perception and not only self-incompatibility (SI) as originally discovered. Some members of these RLKs are able to detect pattern-associated molecular patterns (PAMPs), which are conserved pathogen-derived molecules, and trigger a battery of basal defense responses. The transcriptional activation and expression levels of RLKs are dependent on the variation in promoter architecture as a result of the number, location, order and class of cis-elements found in a promoter sequence. It is hypothesized that candidate RLK genes involved in PAMP surveillance are triggered and transcriptionally regulated in response to perception of PAMPs, and that the intensity of response is relative to the promoter architecture. The primary objective was to identify SD-RLKs and LRR-RLKs which demonstrated up-regulation in response to PAMPs. The SD-RLKs (At1g11330, At1g61430 and At1g61610) and LRR-RLKs (At1g51850, At2g19190 and At5g45840) were selected on the basis of microarray data (Nürnberger - TAIR accession set 100808727) and the Genevestigator database, and characterized utilizing bioinformatics tools. Here, molecular techniques were used to show that the selected RLK genes were responsive to PAMP inductions. Furthermore, this study explored which cis-elements and their corresponding transcription factors (TFs) are found in the promoter of plant defense genes and that may be involved in transcriptional regulation thereof...
- Full Text:
Plasma membrane proteins differentially expressed in response to lps perception in arabidopsis thaliana
- Authors: Baloyi, Nwaxigombe Maureen
- Date: 2015-04-22
- Subjects: Arabidopsis thaliana , Plant gene expression , Plant-pathogen relationships , Cell membranes
- Type: Thesis
- Identifier: uj:13553 , http://hdl.handle.net/10210/13694
- Description: M.Sc. (Biochemistry) , Plant innate immunity occurs in two interconnected branches, the first being the recognition of pathogen conserved surface structures known as pathogen- or microbe-associated molecular patterns (P/MAMPs) by the plant plasma membrane pathogen recognition receptors (PRRs), leading to activation of P/MAMP-triggered immunity (P/MTI). The second branch involves the recognition of pathogen avirulence (Avr) genes by the corresponding plant disease resistance (R) genes, known as the ‘gene-for-gene‘ interaction, and results in a more efficient or stronger defence response, namely effector-triggered immunity (ETI). Lipopolysaccharide (LPS) acts as a P/MAMP that induces an innate immune response in both plants and animals. LPS, especially the lipid A component, has been shown to play a vital role in activating immune responses in animals. Other LPS components such as lipooligosaccharide (LOS) and the core-oligosaccharide have also been shown to trigger an immune response in plants such as Arabidopsis thaliana. In mammalian cells, LPS binds to the LPS-binding protein (LBP) forming a LPS-LBP complex, which binds to a Toll-like receptor 4/myeloid differentiation-2 (TLR4/MD-2) complex together with the co-receptor CD14, a glycosylphosphatidylinositol (GPI)-linked protein, and activates an immune response. To date, there is still no knowledge about the LPS receptor(s) in plants.....
- Full Text:
- Authors: Baloyi, Nwaxigombe Maureen
- Date: 2015-04-22
- Subjects: Arabidopsis thaliana , Plant gene expression , Plant-pathogen relationships , Cell membranes
- Type: Thesis
- Identifier: uj:13553 , http://hdl.handle.net/10210/13694
- Description: M.Sc. (Biochemistry) , Plant innate immunity occurs in two interconnected branches, the first being the recognition of pathogen conserved surface structures known as pathogen- or microbe-associated molecular patterns (P/MAMPs) by the plant plasma membrane pathogen recognition receptors (PRRs), leading to activation of P/MAMP-triggered immunity (P/MTI). The second branch involves the recognition of pathogen avirulence (Avr) genes by the corresponding plant disease resistance (R) genes, known as the ‘gene-for-gene‘ interaction, and results in a more efficient or stronger defence response, namely effector-triggered immunity (ETI). Lipopolysaccharide (LPS) acts as a P/MAMP that induces an innate immune response in both plants and animals. LPS, especially the lipid A component, has been shown to play a vital role in activating immune responses in animals. Other LPS components such as lipooligosaccharide (LOS) and the core-oligosaccharide have also been shown to trigger an immune response in plants such as Arabidopsis thaliana. In mammalian cells, LPS binds to the LPS-binding protein (LBP) forming a LPS-LBP complex, which binds to a Toll-like receptor 4/myeloid differentiation-2 (TLR4/MD-2) complex together with the co-receptor CD14, a glycosylphosphatidylinositol (GPI)-linked protein, and activates an immune response. To date, there is still no knowledge about the LPS receptor(s) in plants.....
- Full Text:
Biochemical characterization of the polygalacturonase inhibiting protein from cotton
- Authors: James, Jacinda Terry
- Date: 2012-08-13
- Subjects: Plant-pathogen relationships , Plants -- Disease and pest resistance , Pectinase
- Type: Thesis
- Identifier: uj:9067 , http://hdl.handle.net/10210/5529
- Description: M.Sc. , Plants have evolved a complex array of biochemical pathways that enable them to recognise and respond to signals from the environment. At present, little is known about the signal transduction pathways that are activated during a plant's response to attack by a pathogen, although this knowledge is central to our understanding of disease susceptibily and resistance. A common form of plant resistance is the restriction of pathogen proliferation to a small zone surrounding the site of infection. In many cases, this restriction is accompanied by localized death of host tissues, known as the hypersensitive response. In addition to local defense responses, many plants respond to infection by activating defenses in uninfected parts of the plant. As a result, the entire plant is more resistant to a secondary infection. This systemic acquired resistance can persist for several weeks or more and often confers crossresistance to unrelated pathogens. Fungal polygalacturonases (PGs) catalyze the fragmentation and the solubilisation of the homogalacturonan in the plant cell wall. These enzymes might have important functions during plant colonization by a fungus. PGs have also been shown to activate plant defense responses, likely because they generate oligogalacturonides with elicitor activity from the plant cell wall. A polygalacturonase inhibiting protein (PGIP), found in the plant cell wall of many plants, forms a specific complex with fungal PGs and favours the accumulation of elicitor-active oligogalacturonides in vitro. An agarose diffusion assay was used to screen the extracts from Verticillium dahliae for PG activity and ensuing inhibition by purified cotton PGIP. Quantitative determination of differences in polygalacturonase activity in the extracts were performed using a reducing sugar assay. There may be more than one isoform of PG present since the polygalacturonases produced by fungi are likely to be to a mixture of exo- and endo-PGs. Polygalacturonase was therefore isolated from 18-day-old culture filtrates of V. dahliae. The enzyme was partially purified by means of ammonium sulphate precipitation and gel chromatography. The band responsible for PG activity was identified and characterized, having a molecular weight of approximately 28-31 kDa, and a pl of 5.1 - 5.9. Kinetic studies indicate a Km of 0.33% and V,„,,of 0.85 pmoles reducing units / min. A commercial preparation of endo-PG from Aspergillus niger was used as a control. This endo-PG had a molecular weight of 68 kDa and a pl point of 3.6 and 5.1, suggesting there were at least two isoforms of endo-PG present. Kinetic studies indicate a K m of 0.33% and V,,„ of 1.07 gmoles reducing units / min.
- Full Text:
- Authors: James, Jacinda Terry
- Date: 2012-08-13
- Subjects: Plant-pathogen relationships , Plants -- Disease and pest resistance , Pectinase
- Type: Thesis
- Identifier: uj:9067 , http://hdl.handle.net/10210/5529
- Description: M.Sc. , Plants have evolved a complex array of biochemical pathways that enable them to recognise and respond to signals from the environment. At present, little is known about the signal transduction pathways that are activated during a plant's response to attack by a pathogen, although this knowledge is central to our understanding of disease susceptibily and resistance. A common form of plant resistance is the restriction of pathogen proliferation to a small zone surrounding the site of infection. In many cases, this restriction is accompanied by localized death of host tissues, known as the hypersensitive response. In addition to local defense responses, many plants respond to infection by activating defenses in uninfected parts of the plant. As a result, the entire plant is more resistant to a secondary infection. This systemic acquired resistance can persist for several weeks or more and often confers crossresistance to unrelated pathogens. Fungal polygalacturonases (PGs) catalyze the fragmentation and the solubilisation of the homogalacturonan in the plant cell wall. These enzymes might have important functions during plant colonization by a fungus. PGs have also been shown to activate plant defense responses, likely because they generate oligogalacturonides with elicitor activity from the plant cell wall. A polygalacturonase inhibiting protein (PGIP), found in the plant cell wall of many plants, forms a specific complex with fungal PGs and favours the accumulation of elicitor-active oligogalacturonides in vitro. An agarose diffusion assay was used to screen the extracts from Verticillium dahliae for PG activity and ensuing inhibition by purified cotton PGIP. Quantitative determination of differences in polygalacturonase activity in the extracts were performed using a reducing sugar assay. There may be more than one isoform of PG present since the polygalacturonases produced by fungi are likely to be to a mixture of exo- and endo-PGs. Polygalacturonase was therefore isolated from 18-day-old culture filtrates of V. dahliae. The enzyme was partially purified by means of ammonium sulphate precipitation and gel chromatography. The band responsible for PG activity was identified and characterized, having a molecular weight of approximately 28-31 kDa, and a pl of 5.1 - 5.9. Kinetic studies indicate a Km of 0.33% and V,„,,of 0.85 pmoles reducing units / min. A commercial preparation of endo-PG from Aspergillus niger was used as a control. This endo-PG had a molecular weight of 68 kDa and a pl point of 3.6 and 5.1, suggesting there were at least two isoforms of endo-PG present. Kinetic studies indicate a K m of 0.33% and V,,„ of 1.07 gmoles reducing units / min.
- Full Text:
Differential gene expression in Nicotiana tabacum cells in response to isonitrosoacetophenone
- Authors: Maake, Mmapula Peggy
- Date: 2013-12-09
- Subjects: Gene expression , Nicotiana tabacum , Tobacco - Diseases and pest resistance , Plant immunology , Plant-pathogen relationships , Plant defenses
- Type: Thesis
- Identifier: uj:7850 , http://hdl.handle.net/10210/8744
- Description: M.Sc. (Biochemistry) , Plants respond to various stress stimuli by activating a broad-spectrum of defence responses that can be expressed locally at the site of pathogen infection (hypersensitive response-HR) as well as systemically in uninfected tissue (systemic acquired resistance-SAR). The ability to continuously respond to both abiotic and biotic stimuli leads to changes in the plants’ physiology, morphology and development. Therefore, there is a need to define and understand the mechanism of the plant defence system, including the mode of recognition, activation of signalling pathways and subsequent defence. In so doing, a long lasting and effective protection against various pathogens may be established. In the current study, the transcriptome status of cultured cells of Nicotiana tabacum was investigated using annealing control primer (ACP)-based differential display (DD) since it is an improved technology to compare patterns of gene expression in RNA samples, isolated from tissue / cells under different biological conditions, using a novel priming system. Here, ACP-DDRT-PCR was used in combination with a next-generation sequencing technology, namely 454 pyro-sequencing, which is the only technique that generates longer reads which are suitable for de novo assembly and annotation of non-model plants like tobacco of which the genome is not yet published in Genbank. SAR occurs following induction by biotrophic or necrotising pathogens. However, it can also be manifested artificially after chemical treatment. In this study, isonitrosoacetophenone (INAP), a novel compound that was originally isolated from extracts of citrus peel undergoing oxidative stress, was used as a chemical inducer and it was hypothesised that this compound induces defence-related responses in plants. In order to investigate this, tobacco cell suspensions were elicited with 1 mM INAP, followed by ACP-DDRT-PCR and subsequent identification of differentially expressed genes using pyro-sequencing.
- Full Text:
- Authors: Maake, Mmapula Peggy
- Date: 2013-12-09
- Subjects: Gene expression , Nicotiana tabacum , Tobacco - Diseases and pest resistance , Plant immunology , Plant-pathogen relationships , Plant defenses
- Type: Thesis
- Identifier: uj:7850 , http://hdl.handle.net/10210/8744
- Description: M.Sc. (Biochemistry) , Plants respond to various stress stimuli by activating a broad-spectrum of defence responses that can be expressed locally at the site of pathogen infection (hypersensitive response-HR) as well as systemically in uninfected tissue (systemic acquired resistance-SAR). The ability to continuously respond to both abiotic and biotic stimuli leads to changes in the plants’ physiology, morphology and development. Therefore, there is a need to define and understand the mechanism of the plant defence system, including the mode of recognition, activation of signalling pathways and subsequent defence. In so doing, a long lasting and effective protection against various pathogens may be established. In the current study, the transcriptome status of cultured cells of Nicotiana tabacum was investigated using annealing control primer (ACP)-based differential display (DD) since it is an improved technology to compare patterns of gene expression in RNA samples, isolated from tissue / cells under different biological conditions, using a novel priming system. Here, ACP-DDRT-PCR was used in combination with a next-generation sequencing technology, namely 454 pyro-sequencing, which is the only technique that generates longer reads which are suitable for de novo assembly and annotation of non-model plants like tobacco of which the genome is not yet published in Genbank. SAR occurs following induction by biotrophic or necrotising pathogens. However, it can also be manifested artificially after chemical treatment. In this study, isonitrosoacetophenone (INAP), a novel compound that was originally isolated from extracts of citrus peel undergoing oxidative stress, was used as a chemical inducer and it was hypothesised that this compound induces defence-related responses in plants. In order to investigate this, tobacco cell suspensions were elicited with 1 mM INAP, followed by ACP-DDRT-PCR and subsequent identification of differentially expressed genes using pyro-sequencing.
- Full Text:
Differential proteomic analysis of Lipopolysaccharide-responsive proteins in Nicotiana tabacum
- Authors: Gerber, Isak B.
- Date: 2008-05-22T07:35:16Z
- Subjects: Tobacco disease and pest resistance , Plant-pathogen relationships , Plant defenses , Plant proteins , Plant proteomics
- Type: Thesis
- Identifier: uj:2083 , http://hdl.handle.net/10210/442
- Description: Prof. I.A. Dubery
- Full Text:
- Authors: Gerber, Isak B.
- Date: 2008-05-22T07:35:16Z
- Subjects: Tobacco disease and pest resistance , Plant-pathogen relationships , Plant defenses , Plant proteins , Plant proteomics
- Type: Thesis
- Identifier: uj:2083 , http://hdl.handle.net/10210/442
- Description: Prof. I.A. Dubery
- Full Text:
Molecular characterization of elicitor-responsive genes in cotton
- Authors: Phillips, Sonia Melanie
- Date: 2012-05-02
- Subjects: Cotton verticillium wilt , Gossypium hirsutum , Cotton diseases and pests , Verticillium dahliae , Cotton disease and pest resistance , Gene expression , Plant-pathogen relationships
- Type: Thesis
- Identifier: uj:2238 , http://hdl.handle.net/10210/4678
- Description: D.Phil. , The fungus, Verticillium dahliae, is the causative agent of Verticillium wilt, which results in significant cotton (Gossypium hirsutum) crop losses worldwide. This study contributes to the elucidation of cotton defence responses against V. dahliae. The identification, cloning and characterization of three genes that were differentially expressed in response to elicitation with a cell wall-derived (CWD) V. dahliae elicitor are described. It was hypothesized that the molecular architectures of the three characterized genes are supportive of a role in cotton defence against V. dahliae. As one of these genes was present as two homoeologous copies, this study also reports on the molecular characterization of both homoeologs, thus providing further insight into the processes of genomic evolution between homoeologous loci in allotetraploid cotton. The three genes were initially represented as expressed sequence tags (ESTs), obtained from a previous differential display reverse transcription polymerase chain reaction (DDRT-PCR) study by Zwiegelaar (2003), as part of an MSc project. These ESTs, designated C1B10, C4B5 and C4B4, were differentially induced upon elicitation with a CWD V. dahliae elicitor (Zwiegelaar, 2003). In the present study, the genes represented by the three ESTs were identified and characterized by genome walking and 5‘/3‘ rapid amplification of cDNA ends (RACE). Additionally, PCR and reverse-transcription PCR (RT-PCR) were utilized, where necessary, to obtain internal sequences, not covered by the genome walking and RACE reactions. Through the use of these molecular techniques, the full transcript and genomic sequences of each of the three genes was obtained, including their promoters. The promoter of each gene was analyzed for cis-elements driving gene transcription, through bioinformatic analysis. Furthermore, the copy number of each gene was determined through Southern blot analysis. The genes were translated to reveal their encoded protein sequences. The amino acid sequences were submitted to a basic local alignment (BLAST) search of the NCBI database to identify, and align them with, homologous proteins from other plant species (and those from G. hirsutum, if any). An in silico analysis of the encoded protein of each gene was also performed. This examination included domain architecture, post-translational modification, subcellular location and tertiary structure predictions. This study also involved the isolation of the elicitor from the cell walls of V. dahliae fungal cultures. The potency of the freshly-isolated elicitor was investigated with a triphenyltetrazolium chloride (TTC) viability assay on cotton cell suspensions. Its potential to induce PR-proteins was also explored but these results were inconclusive. In addition, expression studies were performed with real-time PCR (q-PCR), to confirm the up- or down-regulation of each gene upon elicitation of cotton cell suspensions with the CWD V. dahliae elicitor, and to investigate the time frame/kinetics of induction. The gene corresponding to the C1B10 EST was designated GhLIPN as this study revealed that it encodes a lipin protein. Lipins are novel proteins with phosphatidate phosphatase 1 (PAP1) activity, exclusive to eukaryotes. They play a fundamental role in the lipid metabolism of organisms ranging in complexity from yeast to animals and plants. In plants, this role includes lipid membrane remodelling during phosphate (Pi) deficiency. During the study of the GhLIPN gene, it was discovered that it occurred as two distinct homoeologous copies from the A- and D-co-resident genomes of allopolyploid G. hirsutum. The GhLIPN homoeologs were named GhLIPN I and N for Insert present and No insert, respectively, based on the presence or absence of a 13 base pair (bp) insertion/deletion (indel) site in intron 6.
- Full Text:
- Authors: Phillips, Sonia Melanie
- Date: 2012-05-02
- Subjects: Cotton verticillium wilt , Gossypium hirsutum , Cotton diseases and pests , Verticillium dahliae , Cotton disease and pest resistance , Gene expression , Plant-pathogen relationships
- Type: Thesis
- Identifier: uj:2238 , http://hdl.handle.net/10210/4678
- Description: D.Phil. , The fungus, Verticillium dahliae, is the causative agent of Verticillium wilt, which results in significant cotton (Gossypium hirsutum) crop losses worldwide. This study contributes to the elucidation of cotton defence responses against V. dahliae. The identification, cloning and characterization of three genes that were differentially expressed in response to elicitation with a cell wall-derived (CWD) V. dahliae elicitor are described. It was hypothesized that the molecular architectures of the three characterized genes are supportive of a role in cotton defence against V. dahliae. As one of these genes was present as two homoeologous copies, this study also reports on the molecular characterization of both homoeologs, thus providing further insight into the processes of genomic evolution between homoeologous loci in allotetraploid cotton. The three genes were initially represented as expressed sequence tags (ESTs), obtained from a previous differential display reverse transcription polymerase chain reaction (DDRT-PCR) study by Zwiegelaar (2003), as part of an MSc project. These ESTs, designated C1B10, C4B5 and C4B4, were differentially induced upon elicitation with a CWD V. dahliae elicitor (Zwiegelaar, 2003). In the present study, the genes represented by the three ESTs were identified and characterized by genome walking and 5‘/3‘ rapid amplification of cDNA ends (RACE). Additionally, PCR and reverse-transcription PCR (RT-PCR) were utilized, where necessary, to obtain internal sequences, not covered by the genome walking and RACE reactions. Through the use of these molecular techniques, the full transcript and genomic sequences of each of the three genes was obtained, including their promoters. The promoter of each gene was analyzed for cis-elements driving gene transcription, through bioinformatic analysis. Furthermore, the copy number of each gene was determined through Southern blot analysis. The genes were translated to reveal their encoded protein sequences. The amino acid sequences were submitted to a basic local alignment (BLAST) search of the NCBI database to identify, and align them with, homologous proteins from other plant species (and those from G. hirsutum, if any). An in silico analysis of the encoded protein of each gene was also performed. This examination included domain architecture, post-translational modification, subcellular location and tertiary structure predictions. This study also involved the isolation of the elicitor from the cell walls of V. dahliae fungal cultures. The potency of the freshly-isolated elicitor was investigated with a triphenyltetrazolium chloride (TTC) viability assay on cotton cell suspensions. Its potential to induce PR-proteins was also explored but these results were inconclusive. In addition, expression studies were performed with real-time PCR (q-PCR), to confirm the up- or down-regulation of each gene upon elicitation of cotton cell suspensions with the CWD V. dahliae elicitor, and to investigate the time frame/kinetics of induction. The gene corresponding to the C1B10 EST was designated GhLIPN as this study revealed that it encodes a lipin protein. Lipins are novel proteins with phosphatidate phosphatase 1 (PAP1) activity, exclusive to eukaryotes. They play a fundamental role in the lipid metabolism of organisms ranging in complexity from yeast to animals and plants. In plants, this role includes lipid membrane remodelling during phosphate (Pi) deficiency. During the study of the GhLIPN gene, it was discovered that it occurred as two distinct homoeologous copies from the A- and D-co-resident genomes of allopolyploid G. hirsutum. The GhLIPN homoeologs were named GhLIPN I and N for Insert present and No insert, respectively, based on the presence or absence of a 13 base pair (bp) insertion/deletion (indel) site in intron 6.
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Development of analytical procedures for the quantification and evaluation of the roles of plant hormones involved in defense and induced resistance in plants
- Authors: Riet, Katlego Botlhaleng
- Date: 2015
- Subjects: Plant hormones , Plant defenses , Plant-pathogen relationships
- Language: English
- Type: Masters (Thesis)
- Identifier: http://hdl.handle.net/10210/58825 , uj:16496
- Description: Abstract: Please refer to full text to view abstract , M.Sc. (Biochemistry)
- Full Text:
- Authors: Riet, Katlego Botlhaleng
- Date: 2015
- Subjects: Plant hormones , Plant defenses , Plant-pathogen relationships
- Language: English
- Type: Masters (Thesis)
- Identifier: http://hdl.handle.net/10210/58825 , uj:16496
- Description: Abstract: Please refer to full text to view abstract , M.Sc. (Biochemistry)
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Determination of biological activity of Celtis africana extracts and its endophytic microflora and mycoflora
- Authors: Nchabeleng, Evonia Kanyane
- Date: 2017
- Subjects: Plant metabolites , Plant biotechnology , Medicinal plants , Plant-pathogen relationships , Endophytes , Celtis africana
- Language: English
- Type: Masters (Thesis)
- Identifier: http://hdl.handle.net/10210/235616 , uj:24098
- Description: M.Tech. (Biotechnology) , Abstract: With the rapid rise in untreatable diseases, researchers are compelled to search for new drugs that can combat these diseases. Plants are recognized as unlimited sources of bioactive compounds that can be used to treat different ailments and also be applied in various sectors including agriculture, pharmaceutical and food. In addition, plants are also known to be associated with endophytes such as bacteria and fungi, which are also regarded as fertile sources of bioactive constituents. Celtis africana is an ornamental and medicinal plant that is used to treat different ailments. In the study reported herein, the aims were to identify and characterize secondary metabolites produced by both the plant and its endophytes and to further investigate the antibacterial activity of C. africana extracts against fourteen pathogenic bacteria. To achieve these aims, endophytes were isolated from fresh and apparently healthy aerial parts (leaves, stems and fruit) of C. africana. The isolated endophytes were then cultivated and secondary metabolites extracted sequentially with hexane, dichloromethane and ethyl acetate. Dried plant aerial parts were screened for the presence of phytochemicals, extracted successively with hexane, ethyl acetate and dichloromethane: methanol (1:1 v/v) and the crude extracts tested for antibacterial activity. The crude extracts of both the plant parts and endophytes were analyzed using two dimensional gas chromatography coupled with time of flight mass spectrometry(GC×GC-TOF/MS ) to determine their volatile secondary metabolites constituents. From the results obtained, it is evident that C. africana has endophytic bacteria and fungi in the stem and fruit, but not in the leaves. Seven bacteria (Kocuria sp., Micrococcus luteus, Staphylococcus hominis, Bacillus sp., Staphylococcus saprophyticus, Brachybacterium conglomeratum and Arthrobacter sp) were isolated and identified. Four fungal endophytes, all belonging to the same genus (Aspergillus) were also isolated and identified, of which two were identified to genus level and two were found to be A. niger and A. flavus. The plant crude extracts showed antibacterial activity against seven of the test microorganisms, viz., Escherichia coli, Proteus mirabilis, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter aerogenes. These microorganisms are causal agents of various ailments ranging from food-borne illnesses to skin infections, urinary tract infections and pneumonia....
- Full Text:
- Authors: Nchabeleng, Evonia Kanyane
- Date: 2017
- Subjects: Plant metabolites , Plant biotechnology , Medicinal plants , Plant-pathogen relationships , Endophytes , Celtis africana
- Language: English
- Type: Masters (Thesis)
- Identifier: http://hdl.handle.net/10210/235616 , uj:24098
- Description: M.Tech. (Biotechnology) , Abstract: With the rapid rise in untreatable diseases, researchers are compelled to search for new drugs that can combat these diseases. Plants are recognized as unlimited sources of bioactive compounds that can be used to treat different ailments and also be applied in various sectors including agriculture, pharmaceutical and food. In addition, plants are also known to be associated with endophytes such as bacteria and fungi, which are also regarded as fertile sources of bioactive constituents. Celtis africana is an ornamental and medicinal plant that is used to treat different ailments. In the study reported herein, the aims were to identify and characterize secondary metabolites produced by both the plant and its endophytes and to further investigate the antibacterial activity of C. africana extracts against fourteen pathogenic bacteria. To achieve these aims, endophytes were isolated from fresh and apparently healthy aerial parts (leaves, stems and fruit) of C. africana. The isolated endophytes were then cultivated and secondary metabolites extracted sequentially with hexane, dichloromethane and ethyl acetate. Dried plant aerial parts were screened for the presence of phytochemicals, extracted successively with hexane, ethyl acetate and dichloromethane: methanol (1:1 v/v) and the crude extracts tested for antibacterial activity. The crude extracts of both the plant parts and endophytes were analyzed using two dimensional gas chromatography coupled with time of flight mass spectrometry(GC×GC-TOF/MS ) to determine their volatile secondary metabolites constituents. From the results obtained, it is evident that C. africana has endophytic bacteria and fungi in the stem and fruit, but not in the leaves. Seven bacteria (Kocuria sp., Micrococcus luteus, Staphylococcus hominis, Bacillus sp., Staphylococcus saprophyticus, Brachybacterium conglomeratum and Arthrobacter sp) were isolated and identified. Four fungal endophytes, all belonging to the same genus (Aspergillus) were also isolated and identified, of which two were identified to genus level and two were found to be A. niger and A. flavus. The plant crude extracts showed antibacterial activity against seven of the test microorganisms, viz., Escherichia coli, Proteus mirabilis, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter aerogenes. These microorganisms are causal agents of various ailments ranging from food-borne illnesses to skin infections, urinary tract infections and pneumonia....
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Comparison between conventional and quantum dot labeling strategies for LPS binding studies in Arabidopsis thaliana
- Authors: Mgcina, Londiwe Siphephise
- Date: 2013-12-09
- Subjects: Arabidopsis thaliana - Disease and pest resistance , Plant-pathogen relationships , Plant defenses , Plant protoplasts , Binding sites (Biochemistry) , Gram-negative bacteria , Quantum dots
- Type: Thesis
- Identifier: uj:7844 , http://hdl.handle.net/10210/8739
- Description: M.Sc. (Biochemistry) , Lipopolysaccharide (LPS) is a complex lipoglycan that is found in the outer membrane of Gram-negative bacteria and is composed of three regions namely the fatty acid Lipid A, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides. When LPS is recognized as a microbe-/pathogen-associated molecular pattern (M/PAMP), it not only induces an innate immune response in plants but also stimulates the development of defence responses such as the immediate release of reactive oxygen species/intermediates (ROS/I), pathogen-related (PR) gene expression and activation of the hypersensitive response (HR), resulting in stronger subsequent pathogen interactions. The identification and characterisation of the elusive LPS receptor/receptor complex in plants is thus of importance, since understanding the mechanism of perception and specific signal transduction pathways will clarify, and lead to the advancement of, basal resistance in plants in order to decrease crop plant losses due to pathogen attack. In mammals, LPS binds to a LPS binding protein (LBP) to form a LPS-LBP complex which is translocated to myeloid differentiation 2 (MD2) with the presence/absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14. The interaction occurs on the host membrane and triggers an inflammatory defence response through the signalling cascade activated by the interaction with Toll-like receptor 4 (TLR4). A similar LPS-receptor interaction is, however, unknown in plants. To address the LPS perception mechanism in plants, biological binding studies with regard to concentration, incubation time and temperature, affinity, specificity and saturation were conducted in Arabidopsis thaliana protoplasts using LPS labeled with Alexa 488 hydrazide. Quantum dots (Qdots), which allow non-covalent hydrophobic labeling of LPS, were further also employed in similar Arabidopsis protoplast binding studies. These studies were conducted by fluorescence determination through the use of a BD FACS Aria flow cytometer. Although Alexa-labeling does not affect the biological activity in mammalian studies, the same cannot necessarily be said for plant systems, and hence Qdots were included to address this question. The conjugation of Qdots to LPS was confirmed by transmission electron microscopy (TEM) and results illustrated higher fluorescence values as compared to Alexa-LPS fluorescence analysis. Furthermore, inhibition of the perception process is also reported using Wortmannin and Brefeldin A as suitable endo- and exocytosis inhibitors. Affinity, specificity and saturability as well as the role of endo- and exocytosis inhibition in LPS binding to protoplasts was ultimately demonstrated by both fluorophores, with the use of Qdots as a label proving to be a more sensitive strategy in comparison to the conventional Alexa 488 hydrazide label.
- Full Text:
- Authors: Mgcina, Londiwe Siphephise
- Date: 2013-12-09
- Subjects: Arabidopsis thaliana - Disease and pest resistance , Plant-pathogen relationships , Plant defenses , Plant protoplasts , Binding sites (Biochemistry) , Gram-negative bacteria , Quantum dots
- Type: Thesis
- Identifier: uj:7844 , http://hdl.handle.net/10210/8739
- Description: M.Sc. (Biochemistry) , Lipopolysaccharide (LPS) is a complex lipoglycan that is found in the outer membrane of Gram-negative bacteria and is composed of three regions namely the fatty acid Lipid A, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides. When LPS is recognized as a microbe-/pathogen-associated molecular pattern (M/PAMP), it not only induces an innate immune response in plants but also stimulates the development of defence responses such as the immediate release of reactive oxygen species/intermediates (ROS/I), pathogen-related (PR) gene expression and activation of the hypersensitive response (HR), resulting in stronger subsequent pathogen interactions. The identification and characterisation of the elusive LPS receptor/receptor complex in plants is thus of importance, since understanding the mechanism of perception and specific signal transduction pathways will clarify, and lead to the advancement of, basal resistance in plants in order to decrease crop plant losses due to pathogen attack. In mammals, LPS binds to a LPS binding protein (LBP) to form a LPS-LBP complex which is translocated to myeloid differentiation 2 (MD2) with the presence/absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14. The interaction occurs on the host membrane and triggers an inflammatory defence response through the signalling cascade activated by the interaction with Toll-like receptor 4 (TLR4). A similar LPS-receptor interaction is, however, unknown in plants. To address the LPS perception mechanism in plants, biological binding studies with regard to concentration, incubation time and temperature, affinity, specificity and saturation were conducted in Arabidopsis thaliana protoplasts using LPS labeled with Alexa 488 hydrazide. Quantum dots (Qdots), which allow non-covalent hydrophobic labeling of LPS, were further also employed in similar Arabidopsis protoplast binding studies. These studies were conducted by fluorescence determination through the use of a BD FACS Aria flow cytometer. Although Alexa-labeling does not affect the biological activity in mammalian studies, the same cannot necessarily be said for plant systems, and hence Qdots were included to address this question. The conjugation of Qdots to LPS was confirmed by transmission electron microscopy (TEM) and results illustrated higher fluorescence values as compared to Alexa-LPS fluorescence analysis. Furthermore, inhibition of the perception process is also reported using Wortmannin and Brefeldin A as suitable endo- and exocytosis inhibitors. Affinity, specificity and saturability as well as the role of endo- and exocytosis inhibition in LPS binding to protoplasts was ultimately demonstrated by both fluorophores, with the use of Qdots as a label proving to be a more sensitive strategy in comparison to the conventional Alexa 488 hydrazide label.
- Full Text:
Stress protein expression and cell survival in tomato in response to Ralstonia solanacearum exposure
- Authors: Byth, Heather-Anne
- Date: 2012-08-20
- Subjects: Ralstonia solanacearum , Plant-pathogen relationships , Plant defenses , Heat shock proteins , Tomato wilts
- Type: Thesis
- Identifier: uj:2813 , http://hdl.handle.net/10210/6250
- Description: M.Sc. , Plants are in constant conflict with pathogens and have evolved intricate mechanisms to protect themselves against pathogens. The gene-for-gene response is regarded as the first line of defence when plant and pathogen meet. This interaction leads to the induction of defence proteins such as PR proteins that protect the plant from invading pathogens. A seemingly unrelated topic to plants and pathogens is heat shock proteins (HSP). HSP are a highly conserved group of defence proteins induced in all organisms in response to a variety of environmental stresses to provide protection from, and adaptation to cellular stress. HSP are in general not considered to be part of the defence response classically induced by avirulent pathogens and whether they are induced and play a role in plant-pathogen interactions is controversial. The protective chaperoning capacity of HSP makes them ideal proteins to exploit to target as endogenous defence proteins in the search for new strategies in the management of infectious diseases. In humans, HSP induction during infection is a complex phenomenon depending on the pathogen, whether the infection is acute or chronic, the host cell type and its differentiative state as well as environmental factors. In this investigation the expression of the inducible and constitutive isoforms of the 70kDa HSP (Hsp70/Hsc70) was investigated in tomato, Lycopersicon esculentum in response to virulent and avirulent strains of Ralstonia solanacearum, the causative agent of bacterial wilt. Expression of Hsp70 was studied in conjunction with the accumulation of PR-la and host cell viability. A quick, non-toxic, tetrazolium-based assay was developed from the Alamar Blue assay, commonly used in mammalian cells, and applied for the evaluation of host cell viability. The results shown suggest Hsp70/Hsc70 is significantly induced in tomato cell suspensions during an incompatible interaction 24h to 48 h following co-cultivation with the avirulent R. solanacearum strain compared to normal levels at this interval in cells exposed to the virulent strain. In both compatible and incompatible interactions Hsp70/Hsc70 levels eventually (72 h) accumulated correlating significantly with decreased viability. PR-la accumulation was significantly induced from 6 h to 18 h by the virulent as well as the avirulent R. solanacearum strains. In general, comparable results were obtained using leaf discs as an in vivo model. Based upon the differential induction of Hsp70/Hsc70 by virulent and avirulent pathogens it is proposed that HSP may play an important role in determining the outcome of the interaction between tomato and R. solanacearum. Successful defence may not only involve a limited number of defence genes but may result from a concerted action of a large number of defence genes.
- Full Text:
Stress protein expression and cell survival in tomato in response to Ralstonia solanacearum exposure
- Authors: Byth, Heather-Anne
- Date: 2012-08-20
- Subjects: Ralstonia solanacearum , Plant-pathogen relationships , Plant defenses , Heat shock proteins , Tomato wilts
- Type: Thesis
- Identifier: uj:2813 , http://hdl.handle.net/10210/6250
- Description: M.Sc. , Plants are in constant conflict with pathogens and have evolved intricate mechanisms to protect themselves against pathogens. The gene-for-gene response is regarded as the first line of defence when plant and pathogen meet. This interaction leads to the induction of defence proteins such as PR proteins that protect the plant from invading pathogens. A seemingly unrelated topic to plants and pathogens is heat shock proteins (HSP). HSP are a highly conserved group of defence proteins induced in all organisms in response to a variety of environmental stresses to provide protection from, and adaptation to cellular stress. HSP are in general not considered to be part of the defence response classically induced by avirulent pathogens and whether they are induced and play a role in plant-pathogen interactions is controversial. The protective chaperoning capacity of HSP makes them ideal proteins to exploit to target as endogenous defence proteins in the search for new strategies in the management of infectious diseases. In humans, HSP induction during infection is a complex phenomenon depending on the pathogen, whether the infection is acute or chronic, the host cell type and its differentiative state as well as environmental factors. In this investigation the expression of the inducible and constitutive isoforms of the 70kDa HSP (Hsp70/Hsc70) was investigated in tomato, Lycopersicon esculentum in response to virulent and avirulent strains of Ralstonia solanacearum, the causative agent of bacterial wilt. Expression of Hsp70 was studied in conjunction with the accumulation of PR-la and host cell viability. A quick, non-toxic, tetrazolium-based assay was developed from the Alamar Blue assay, commonly used in mammalian cells, and applied for the evaluation of host cell viability. The results shown suggest Hsp70/Hsc70 is significantly induced in tomato cell suspensions during an incompatible interaction 24h to 48 h following co-cultivation with the avirulent R. solanacearum strain compared to normal levels at this interval in cells exposed to the virulent strain. In both compatible and incompatible interactions Hsp70/Hsc70 levels eventually (72 h) accumulated correlating significantly with decreased viability. PR-la accumulation was significantly induced from 6 h to 18 h by the virulent as well as the avirulent R. solanacearum strains. In general, comparable results were obtained using leaf discs as an in vivo model. Based upon the differential induction of Hsp70/Hsc70 by virulent and avirulent pathogens it is proposed that HSP may play an important role in determining the outcome of the interaction between tomato and R. solanacearum. Successful defence may not only involve a limited number of defence genes but may result from a concerted action of a large number of defence genes.
- Full Text:
Characterisation of gene sequences induced in barley after pathogen infection
- Authors: Janse van Vuuren, Natasha
- Date: 2011-10-11T08:15:05Z
- Subjects: Barley diseases and pests , Plant-pathogen relationships
- Type: Thesis
- Identifier: uj:7247 , http://hdl.handle.net/10210/3900
- Description: M.Sc. , Barley (Hordeum vulgare) production is a vital constituent of the South African economy. Many pathogens reside on barley, which lead to low quality and yield. One of the most prominent barley pathogens, Fusarium graminearum, is the causal agent of small grain scab. F. graminearum resistance to barley is regulated by multiple genes referred to as quantitative trait loci (QTL), which makes it difficult to breed for resistance in new cultivars. Each of these genes contributes to a specific defence area and collectively counteracts Fusarium infection and spread in the barley plant. The aim of this project was to isolate and identify induced genes after infection of three leave stage barley with F. graminearum. These genes were isolated through the use of Suppression subtractive hybridisation (SSH), cloned and then sequenced. From this data set three transcript derived fragments (TDFs) sharing homology to known genes were selected and their expression profiles were studied through Northern blot analysis. Three TDFs shared homology with known genes namely a putative protease inhibitor-related protein, a senescence associated gene, and a manganese superoxide dismutase (MnSOD). These TDFs were previously also recognised for their function in host pathogen interactions. The expression analysis done using Northern blots showed up-regulation of the three fragments after inoculation. These results indicated that all the TDFs studied may play a role in the defence reaction of barley infected with F. graminearum, where both senescence and proteinase inhibitors could limit infection as well as spread and MnSODs might be a protective enzyme against oxidative stress. The results of this study indicated that all of the identified TDFs had database matches to proteins identified during stress responses. Furthermore, the Northern blot results indicated that all the TDFs studied could play a role in the defence reaction of F. graminearum infected barley. These TDFs will form the basis of further studies into the interaction between barley and F. graminearum. The results form this study will add to our knowledge of the interaction between barley and a necrotrophic pathogen. This will aid in understanding how cereal pathogens deal with pathogen attack and will aid in development of new more tolerant barley cultivars.
- Full Text:
- Authors: Janse van Vuuren, Natasha
- Date: 2011-10-11T08:15:05Z
- Subjects: Barley diseases and pests , Plant-pathogen relationships
- Type: Thesis
- Identifier: uj:7247 , http://hdl.handle.net/10210/3900
- Description: M.Sc. , Barley (Hordeum vulgare) production is a vital constituent of the South African economy. Many pathogens reside on barley, which lead to low quality and yield. One of the most prominent barley pathogens, Fusarium graminearum, is the causal agent of small grain scab. F. graminearum resistance to barley is regulated by multiple genes referred to as quantitative trait loci (QTL), which makes it difficult to breed for resistance in new cultivars. Each of these genes contributes to a specific defence area and collectively counteracts Fusarium infection and spread in the barley plant. The aim of this project was to isolate and identify induced genes after infection of three leave stage barley with F. graminearum. These genes were isolated through the use of Suppression subtractive hybridisation (SSH), cloned and then sequenced. From this data set three transcript derived fragments (TDFs) sharing homology to known genes were selected and their expression profiles were studied through Northern blot analysis. Three TDFs shared homology with known genes namely a putative protease inhibitor-related protein, a senescence associated gene, and a manganese superoxide dismutase (MnSOD). These TDFs were previously also recognised for their function in host pathogen interactions. The expression analysis done using Northern blots showed up-regulation of the three fragments after inoculation. These results indicated that all the TDFs studied may play a role in the defence reaction of barley infected with F. graminearum, where both senescence and proteinase inhibitors could limit infection as well as spread and MnSODs might be a protective enzyme against oxidative stress. The results of this study indicated that all of the identified TDFs had database matches to proteins identified during stress responses. Furthermore, the Northern blot results indicated that all the TDFs studied could play a role in the defence reaction of F. graminearum infected barley. These TDFs will form the basis of further studies into the interaction between barley and F. graminearum. The results form this study will add to our knowledge of the interaction between barley and a necrotrophic pathogen. This will aid in understanding how cereal pathogens deal with pathogen attack and will aid in development of new more tolerant barley cultivars.
- Full Text:
The effect of xenobiotics on the expression of a cytochrome p450 gene in Phaseolus vulgaris
- Authors: Basson, Adriaan Erasmus
- Date: 2012-08-08
- Subjects: Plant defenses , Cytochrome P-450 , Common bean , Plant-pathogen relationships , Phaseolus vulgaris
- Type: Thesis
- Identifier: uj:8976 , http://hdl.handle.net/10210/5446
- Description: M.Sc. , Plant cells have evolved the ability to detect pathogen ingress and subsequently activate defense-related functions as part of the plant pathogen response. One or more poorly defined signal transduction pathways, initiated upon recognition of the pathogen by the plant host, regulate expression of plant defense genes. Acquired resistance (AR) is an inducible defense mechanism exhibited by many plants that provides protection against a broad range of pathogens.Many chemical and environmental cues can elicit the same defenses or subsets therein. Cytochrome P450 enzymes are heme-dependent, mixed function oxidase systems that utilize dioxygen to produce a functionalized organic substrate and a molecule of water. They play important biosynthetic and detoxicative roles. They have been identified as being involved in the activation (e.g. allene oxide synthase) and execution of plant defense responses. To investigate the involvement of cytochromes P450 in the plant defense response - mainly through the activation of allene oxide synthases in the jamonate signaling pathway — Phaseolus vulgaris L.cv. Contender leaves were treated with chemical elicitors to mimic the plant-pathogen interaction and thereby activate plant defense responses. Through the use of differential display reverse transcription polymerase chain reaction and denaturing polyacrylamide gel electrophoresis, differentially expressed cDNA bands were isolated, cloned and sequenced. One of the cloned cDNA fragment proved to be a previously unreported cytochrome P450 cDNA, and was named CYP98A5. Dot blot analysis of bean leaves treated with various chemicals showed an enhanced expression of CYP98A5 in leaves treated with 3,5- dichlorosalicylic acid. Northern blot analysis of a time dependent induction study of CYP98A5 in bean leaves treated with this chemical compound indicated that 3,5-dichlorosalicylic acid induces CYP98A5 transcripts earlier than it is induced in control leaves. This might be indicative of a possible conditioning and sensitizing effect of 3,5- dichlorosalicylic acid on bean leaves to a more rapid and effective response with defense reactions once attacked by pathogens. CYP98A5, however, did not appear to be an allene oxide synthase when sequence comparison with other allene oxide synthases was performed; isolation and comparison of the complete CYP98A5 sequence could prove this wrong. It is not possible to assign any function to CYP98A5 at this stage; elucidation of the function of this enzyme in plants would provide more insight into this study.
- Full Text:
- Authors: Basson, Adriaan Erasmus
- Date: 2012-08-08
- Subjects: Plant defenses , Cytochrome P-450 , Common bean , Plant-pathogen relationships , Phaseolus vulgaris
- Type: Thesis
- Identifier: uj:8976 , http://hdl.handle.net/10210/5446
- Description: M.Sc. , Plant cells have evolved the ability to detect pathogen ingress and subsequently activate defense-related functions as part of the plant pathogen response. One or more poorly defined signal transduction pathways, initiated upon recognition of the pathogen by the plant host, regulate expression of plant defense genes. Acquired resistance (AR) is an inducible defense mechanism exhibited by many plants that provides protection against a broad range of pathogens.Many chemical and environmental cues can elicit the same defenses or subsets therein. Cytochrome P450 enzymes are heme-dependent, mixed function oxidase systems that utilize dioxygen to produce a functionalized organic substrate and a molecule of water. They play important biosynthetic and detoxicative roles. They have been identified as being involved in the activation (e.g. allene oxide synthase) and execution of plant defense responses. To investigate the involvement of cytochromes P450 in the plant defense response - mainly through the activation of allene oxide synthases in the jamonate signaling pathway — Phaseolus vulgaris L.cv. Contender leaves were treated with chemical elicitors to mimic the plant-pathogen interaction and thereby activate plant defense responses. Through the use of differential display reverse transcription polymerase chain reaction and denaturing polyacrylamide gel electrophoresis, differentially expressed cDNA bands were isolated, cloned and sequenced. One of the cloned cDNA fragment proved to be a previously unreported cytochrome P450 cDNA, and was named CYP98A5. Dot blot analysis of bean leaves treated with various chemicals showed an enhanced expression of CYP98A5 in leaves treated with 3,5- dichlorosalicylic acid. Northern blot analysis of a time dependent induction study of CYP98A5 in bean leaves treated with this chemical compound indicated that 3,5-dichlorosalicylic acid induces CYP98A5 transcripts earlier than it is induced in control leaves. This might be indicative of a possible conditioning and sensitizing effect of 3,5- dichlorosalicylic acid on bean leaves to a more rapid and effective response with defense reactions once attacked by pathogens. CYP98A5, however, did not appear to be an allene oxide synthase when sequence comparison with other allene oxide synthases was performed; isolation and comparison of the complete CYP98A5 sequence could prove this wrong. It is not possible to assign any function to CYP98A5 at this stage; elucidation of the function of this enzyme in plants would provide more insight into this study.
- Full Text:
Release of volatile compounds by Arabidopsis thaliana cells in response to elicitation by lipopolysaccharides
- Authors: Le Noury, Denise Anne
- Date: 2011-08-31T07:15:12Z
- Subjects: Arabidopsis thaliana , Plant gene expression , Plant defenses , Plant-pathogen relationships , Endotoxins , Lipopolysaccharides
- Type: Thesis
- Identifier: uj:7177 , http://hdl.handle.net/10210/3788
- Description: M.Sc. , Plants produce volatile organic compounds in response to certain elicitors and environments. These compounds have a variety of functions, including the attraction of insects for pollination and seed dispersal, responses to both abiotic and biotic stresses and the priming or sensitizing of neighbouring plants for subsequent attack. The majority of the volatile blend is made up of terpenoid compounds and these compounds are formed through the action of an important class of enzymes termed Terpene Synthases. Lipopolysaccharides form part of the cell surface of Gram-negative bacteria and they are classed as “pathogen-associated molecular pattern molecules” and are thought to induce defence responses in plants by influencing different metabolic pathways that could ultimately result in the production of defence volatiles. LPS from Burkholderia cepacia that has been reported to induce the oxidative burst, the nitric oxide burst and changes in cytosolic calcium concentrations, was used in this study. In order to analyse the volatiles, Single-Drop Microextraction and Solid-Phase Microextraction were used as static headspace sampling techniques that allow the preconcentration of volatile analytes prior to analysis. Both these techniques are fast, simple and equilibrium based and both allow for minimal sample size and preparation. Luminometry was performed in order to test the efficacy of LPS and to determine if LPS is able to induce the oxidative burst in Arabidopsis thaliana. Histochemical staining of transgenic plants containing the PR1:GUS and PDF:GUS reporter gene constructs was performed in order to determine which signalling pathway LPS follows, either the jasmonic acid pathway or the salicylic acid pathway. SPME was then used to extract samples from both time and concentration studies. The time studies involved incubation times of 0 h, 2 h, 4 h and 6 h and 0 d, 1 d, 2 d and 3 d respectively, while the concentration studies involved using LPS concentrations of 0, 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml. SPME was also used for the comparision of two A. thaliana ecotypes (Columbia and C24) as well as two A. thaliana knock-out lines (At5g44630 – multi-product sesquiterpene synthase and At5g23960 – (E)-β-caryophyllene synthase), and finally it was used for the sampling of A. thaliana leaf tissue. SDME was used to compare two solvents, namely octane and toluene and these results were compared to the SPME results. GC-MS was used only for the identification of volatiles with both SPME and SDME. Finally, GC-MS was used with SPME to identify volatiles that are produced by leaf tissue after priming.
- Full Text:
- Authors: Le Noury, Denise Anne
- Date: 2011-08-31T07:15:12Z
- Subjects: Arabidopsis thaliana , Plant gene expression , Plant defenses , Plant-pathogen relationships , Endotoxins , Lipopolysaccharides
- Type: Thesis
- Identifier: uj:7177 , http://hdl.handle.net/10210/3788
- Description: M.Sc. , Plants produce volatile organic compounds in response to certain elicitors and environments. These compounds have a variety of functions, including the attraction of insects for pollination and seed dispersal, responses to both abiotic and biotic stresses and the priming or sensitizing of neighbouring plants for subsequent attack. The majority of the volatile blend is made up of terpenoid compounds and these compounds are formed through the action of an important class of enzymes termed Terpene Synthases. Lipopolysaccharides form part of the cell surface of Gram-negative bacteria and they are classed as “pathogen-associated molecular pattern molecules” and are thought to induce defence responses in plants by influencing different metabolic pathways that could ultimately result in the production of defence volatiles. LPS from Burkholderia cepacia that has been reported to induce the oxidative burst, the nitric oxide burst and changes in cytosolic calcium concentrations, was used in this study. In order to analyse the volatiles, Single-Drop Microextraction and Solid-Phase Microextraction were used as static headspace sampling techniques that allow the preconcentration of volatile analytes prior to analysis. Both these techniques are fast, simple and equilibrium based and both allow for minimal sample size and preparation. Luminometry was performed in order to test the efficacy of LPS and to determine if LPS is able to induce the oxidative burst in Arabidopsis thaliana. Histochemical staining of transgenic plants containing the PR1:GUS and PDF:GUS reporter gene constructs was performed in order to determine which signalling pathway LPS follows, either the jasmonic acid pathway or the salicylic acid pathway. SPME was then used to extract samples from both time and concentration studies. The time studies involved incubation times of 0 h, 2 h, 4 h and 6 h and 0 d, 1 d, 2 d and 3 d respectively, while the concentration studies involved using LPS concentrations of 0, 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml. SPME was also used for the comparision of two A. thaliana ecotypes (Columbia and C24) as well as two A. thaliana knock-out lines (At5g44630 – multi-product sesquiterpene synthase and At5g23960 – (E)-β-caryophyllene synthase), and finally it was used for the sampling of A. thaliana leaf tissue. SDME was used to compare two solvents, namely octane and toluene and these results were compared to the SPME results. GC-MS was used only for the identification of volatiles with both SPME and SDME. Finally, GC-MS was used with SPME to identify volatiles that are produced by leaf tissue after priming.
- Full Text:
Promoter analysis of members of a plant defense-related LRR-RLK gene cluster in Arabidopsis thaliana
- Authors: Mumm, Anina
- Date: 2014-07-15
- Subjects: Arabidopsis thaliana , Plant-pathogen relationships , Plant genome mapping , Plant defenses
- Type: Thesis
- Identifier: uj:11692 , http://hdl.handle.net/10210/11414
- Description: M.Sc. (Biochemistry) , A 14-member, closely-spaced cluster of genes coding for leucine-rich repeat receptor-like kinases (LRR-RLKs) is located on chromosome 1 of Arabidopsis thaliana. Following on from previous microarray studies that found some of the members of this cluster to be upregulated in response to biotic stressors, including the bacterial elicitor flg22, the present study sought to confirm, using a luciferase-based protoplast assay, that flg22 does in fact induce the expression of the genes, and then to investigate the promoters of the genes. The promoters of At1g51790, At1g51850 and At1g51890 responded positively in this particular assay, and bioinformatic analyses determined that W-boxes are over-represented in the cloned regions. Mutational inactivation of individual W-boxes in the promoter of At1g51790 drastically reduced the flg22 response, except for the W-box closest to the start site, which seemed to increase both basal and flg22-inducible expression. In the promoter of At1g51850, mutational inactivation of either or both of its W-box dyads resulted in virtually no flg22 inducibility. The deletion of 6 W-boxes in the promoter of At1g51890, done via truncation, drastically reduced both its basal expression and its inducible response to flg22. These results provide evidence that W-box cis-elements are responsible for the upregulation of these LRR-RLKs in response to flg22. WRKYs -7, -11, -22,and -26 were found bioinformatically to have similar expression patterns to some of the genes in the cluster, and are thus good candidates to investigate as transcriptional regulators of the cluster in future studies.
- Full Text:
Promoter analysis of members of a plant defense-related LRR-RLK gene cluster in Arabidopsis thaliana
- Authors: Mumm, Anina
- Date: 2014-07-15
- Subjects: Arabidopsis thaliana , Plant-pathogen relationships , Plant genome mapping , Plant defenses
- Type: Thesis
- Identifier: uj:11692 , http://hdl.handle.net/10210/11414
- Description: M.Sc. (Biochemistry) , A 14-member, closely-spaced cluster of genes coding for leucine-rich repeat receptor-like kinases (LRR-RLKs) is located on chromosome 1 of Arabidopsis thaliana. Following on from previous microarray studies that found some of the members of this cluster to be upregulated in response to biotic stressors, including the bacterial elicitor flg22, the present study sought to confirm, using a luciferase-based protoplast assay, that flg22 does in fact induce the expression of the genes, and then to investigate the promoters of the genes. The promoters of At1g51790, At1g51850 and At1g51890 responded positively in this particular assay, and bioinformatic analyses determined that W-boxes are over-represented in the cloned regions. Mutational inactivation of individual W-boxes in the promoter of At1g51790 drastically reduced the flg22 response, except for the W-box closest to the start site, which seemed to increase both basal and flg22-inducible expression. In the promoter of At1g51850, mutational inactivation of either or both of its W-box dyads resulted in virtually no flg22 inducibility. The deletion of 6 W-boxes in the promoter of At1g51890, done via truncation, drastically reduced both its basal expression and its inducible response to flg22. These results provide evidence that W-box cis-elements are responsible for the upregulation of these LRR-RLKs in response to flg22. WRKYs -7, -11, -22,and -26 were found bioinformatically to have similar expression patterns to some of the genes in the cluster, and are thus good candidates to investigate as transcriptional regulators of the cluster in future studies.
- Full Text:
Responses in barley gene expression after pathogen infection
- Authors: Bezuidenhout, Nelanie
- Date: 2009-07-29T07:46:40Z
- Subjects: Barley diseases and pests , Fungal diseases of plants , Plant-pathogen relationships
- Type: Mini-Dissertation
- Identifier: uj:8600 , http://hdl.handle.net/10210/2860
- Description: M.Sc. , A variety of micro-organisms are constantly exploiting plants. Barley is no exception and various pathogens attack it. This interaction between the plant and the pathogen can be divided into two groups. A compatible interaction occurs when the plant shows susceptibility to the pathogen. An incompatible interaction occurs when the plant shows resistance to the pathogen. This incompatible interaction is usually associated with necrotic lesions at the infected area. The aim of this project was to study the interaction between Hordeum vulgare (barley) and Fusarium graminearum (causal agent of head blight / scab). Further objectives were to look at the expression of defence genes in the cultivar Puma 15 when infected with F. graminearum. Juvenile plants were used in this study and infected at the third leaf stage. Suppression subtractive hybridisation (SSH) was performed on inoculated and uninoculated plants to enrich for differentially transcribed genes. The resulting transcript derived fragments (TDF) were cloned and sequenced. Several stress related (biotic or abiotic) genes were identified that included putative selenium-binding proteins (SSH10), glutamine-dependent asparagine synthetase 1 (SSH11) and hypothetical proteins. Upon inspection of the homology at nucleotide level of one of the hypothetical proteins (SSH16), it became evident that it was from the 26S ribosomal gene. A selection of the identified genes was further evaluated to the role that they play during this specific interaction by Northern blot analyses. Northern blot analysis of the putative selenium binding homologue (SSH10) showed low levels of induced gene expression in the inoculated plants as compared to the control at 24 hpi. For the asparagine synthetase homologue (SSH11) there was a significant up-regulation in the inoculated sample at 24 hpi. From the results obtained in this study it was found that there are certain defence related, stress related, and unknown genes that are expressed during infection of the barley cultivar Puma15 with F. graminearum. A correlation could be made between the results presented here and literature on F. graminearum infection studies. When F. graminearum infects the host it will respond to that infection between 24 hpi and 72 hpi. This response includes the expression of defence genes and other antifungal mechanisms. The results from this study complements previous studies and will in future aid in the understanding of barley responses after infection by necrotrophic pathogens.
- Full Text:
- Authors: Bezuidenhout, Nelanie
- Date: 2009-07-29T07:46:40Z
- Subjects: Barley diseases and pests , Fungal diseases of plants , Plant-pathogen relationships
- Type: Mini-Dissertation
- Identifier: uj:8600 , http://hdl.handle.net/10210/2860
- Description: M.Sc. , A variety of micro-organisms are constantly exploiting plants. Barley is no exception and various pathogens attack it. This interaction between the plant and the pathogen can be divided into two groups. A compatible interaction occurs when the plant shows susceptibility to the pathogen. An incompatible interaction occurs when the plant shows resistance to the pathogen. This incompatible interaction is usually associated with necrotic lesions at the infected area. The aim of this project was to study the interaction between Hordeum vulgare (barley) and Fusarium graminearum (causal agent of head blight / scab). Further objectives were to look at the expression of defence genes in the cultivar Puma 15 when infected with F. graminearum. Juvenile plants were used in this study and infected at the third leaf stage. Suppression subtractive hybridisation (SSH) was performed on inoculated and uninoculated plants to enrich for differentially transcribed genes. The resulting transcript derived fragments (TDF) were cloned and sequenced. Several stress related (biotic or abiotic) genes were identified that included putative selenium-binding proteins (SSH10), glutamine-dependent asparagine synthetase 1 (SSH11) and hypothetical proteins. Upon inspection of the homology at nucleotide level of one of the hypothetical proteins (SSH16), it became evident that it was from the 26S ribosomal gene. A selection of the identified genes was further evaluated to the role that they play during this specific interaction by Northern blot analyses. Northern blot analysis of the putative selenium binding homologue (SSH10) showed low levels of induced gene expression in the inoculated plants as compared to the control at 24 hpi. For the asparagine synthetase homologue (SSH11) there was a significant up-regulation in the inoculated sample at 24 hpi. From the results obtained in this study it was found that there are certain defence related, stress related, and unknown genes that are expressed during infection of the barley cultivar Puma15 with F. graminearum. A correlation could be made between the results presented here and literature on F. graminearum infection studies. When F. graminearum infects the host it will respond to that infection between 24 hpi and 72 hpi. This response includes the expression of defence genes and other antifungal mechanisms. The results from this study complements previous studies and will in future aid in the understanding of barley responses after infection by necrotrophic pathogens.
- Full Text:
Thermotolerance and Ralstonia solanacearum infection: implications for phenylpropanoid metabolism in Lycopersicon esculentum
- Authors: Kuun, Karolina
- Date: 2012-08-28
- Subjects: Plants, Effect of heat on , Plant defenses , Plant-pathogen relationships , Heat shock proteins , Plants - Metabolism , Ralstonia solanacearum
- Type: Thesis
- Identifier: uj:3372 , http://hdl.handle.net/10210/6771
- Description: M.Sc. , Field grown plants are constantly challenged with a variety of stressful factors, such as high temperatures, drought and pathogen infection that adversely affect crop production and quality. These stresses seldom occur as single entities in plants and in warm climates, heat stress is often a common dominator in combinatorial stress. The heat shock (HS) response in plants has priority over other stress responses, including the pathogen-induced stress response. Activation of the HS response prevents the normal plant defence strategy, leaving the plant vulnerable to pathogen attack. However, prior exposure to elevated temperatures confers protection from subsequent, otherwise lethal, temperatures (thermotolerance) and a variety of other stress conditions including heavy-metals, chilling injury and certain pathogens (cross tolerance). In general, litterature supports a central role for heat shock proteins (HSP), in particular the 70 kDa HSP (Hsp70), in thermotolerance. Incompatible host-pathogen interactions lead to the activation of an array of defence mechanisms, including the promotion of phenylpropanoid metabolism. Phenylalanine ammonia-lyase is a key regulator of this metabolic pathway, influencing the production of salicylic acid, lignin and phytoalexins among other essential defence products. In this study it was hypothesised that prior exposure to non-lethal HS confers protection from subsequent heat-related suppression of the phenylpropanoid pathway, induced as a defence mechanism during an incompatible plant-pathogen interaction. This hypothesis was verified by analysing the effect of thermotolerance on pathogen-related stimulation of PAL promoter activity, enzyme activity and lignin deposition. The tomato, Lycopersicon esculentum cultivar UC82B and Ralstonia solanacearum, the causative agent of bacterial wilt, were used as host-pathogen model. Specific objectives in the study were: (1) Development of PAL promoter-GUS reporter transformed Lycopersicon esculentum. (2) Establishment of a thermotolerance protocol that ensures optimal Hsp70 levels at subsequent HS. (3) Evaluation of the influence of prior heat treatment on phenylpropanoid metabolism after exposure to HS in combination with Ralstonia solanacearum. Results obtained support the hypothesis indicating that thermotolerance protects phenylpropanoid metabolism, in particular PAL promoter and enzyme activity, and to a certain extent lignin production, induced by avirulent Ralstonia solanacearum during a second severe HS. In contrast, HS without a prior heat treatment, suppressed phenylpropanoid metabolism. The protective potential of prior heat treatment during subsequent infection under hyperthermic conditions support the application of HSP in the development of novel plant protection strategies.
- Full Text:
- Authors: Kuun, Karolina
- Date: 2012-08-28
- Subjects: Plants, Effect of heat on , Plant defenses , Plant-pathogen relationships , Heat shock proteins , Plants - Metabolism , Ralstonia solanacearum
- Type: Thesis
- Identifier: uj:3372 , http://hdl.handle.net/10210/6771
- Description: M.Sc. , Field grown plants are constantly challenged with a variety of stressful factors, such as high temperatures, drought and pathogen infection that adversely affect crop production and quality. These stresses seldom occur as single entities in plants and in warm climates, heat stress is often a common dominator in combinatorial stress. The heat shock (HS) response in plants has priority over other stress responses, including the pathogen-induced stress response. Activation of the HS response prevents the normal plant defence strategy, leaving the plant vulnerable to pathogen attack. However, prior exposure to elevated temperatures confers protection from subsequent, otherwise lethal, temperatures (thermotolerance) and a variety of other stress conditions including heavy-metals, chilling injury and certain pathogens (cross tolerance). In general, litterature supports a central role for heat shock proteins (HSP), in particular the 70 kDa HSP (Hsp70), in thermotolerance. Incompatible host-pathogen interactions lead to the activation of an array of defence mechanisms, including the promotion of phenylpropanoid metabolism. Phenylalanine ammonia-lyase is a key regulator of this metabolic pathway, influencing the production of salicylic acid, lignin and phytoalexins among other essential defence products. In this study it was hypothesised that prior exposure to non-lethal HS confers protection from subsequent heat-related suppression of the phenylpropanoid pathway, induced as a defence mechanism during an incompatible plant-pathogen interaction. This hypothesis was verified by analysing the effect of thermotolerance on pathogen-related stimulation of PAL promoter activity, enzyme activity and lignin deposition. The tomato, Lycopersicon esculentum cultivar UC82B and Ralstonia solanacearum, the causative agent of bacterial wilt, were used as host-pathogen model. Specific objectives in the study were: (1) Development of PAL promoter-GUS reporter transformed Lycopersicon esculentum. (2) Establishment of a thermotolerance protocol that ensures optimal Hsp70 levels at subsequent HS. (3) Evaluation of the influence of prior heat treatment on phenylpropanoid metabolism after exposure to HS in combination with Ralstonia solanacearum. Results obtained support the hypothesis indicating that thermotolerance protects phenylpropanoid metabolism, in particular PAL promoter and enzyme activity, and to a certain extent lignin production, induced by avirulent Ralstonia solanacearum during a second severe HS. In contrast, HS without a prior heat treatment, suppressed phenylpropanoid metabolism. The protective potential of prior heat treatment during subsequent infection under hyperthermic conditions support the application of HSP in the development of novel plant protection strategies.
- Full Text:
Identification of MAMP-responsive plasma membrane proteins in Arabidopsis thaliana following challenge by mutant LPS chemotypes
- Authors: Hussan, Hoosen Raeesa
- Date: 2019
- Subjects: Plants - Disease and pest resistance , Arabidopsis thaliana|xDefenses , Plant-pathogen relationships , Cell membranes , Plant proteins
- Language: English
- Type: Masters (Thesis)
- Identifier: http://hdl.handle.net/10210/401039 , uj:33497
- Description: Abstract : Please refer to full text to view abstract. , M.Sc. (Biochemistry)
- Full Text:
- Authors: Hussan, Hoosen Raeesa
- Date: 2019
- Subjects: Plants - Disease and pest resistance , Arabidopsis thaliana|xDefenses , Plant-pathogen relationships , Cell membranes , Plant proteins
- Language: English
- Type: Masters (Thesis)
- Identifier: http://hdl.handle.net/10210/401039 , uj:33497
- Description: Abstract : Please refer to full text to view abstract. , M.Sc. (Biochemistry)
- Full Text:
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