Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): Identify cotton plastid promoters that demonstrate high expression levels in both green and non-green plant tissues for use in development of cotton plastid transformation vectors. Determine levels of expression of reporter genes in both green (leaf and outer boll) and non-green (cottonseed and root) cotton tissues under control of select, engineered plastid promoters. Generate cotton plastid transformation vectors that place antifungal genes and selectable marker genes under control of selected cotton plastid promoters and transform cotton. Test transformed tissues for expression of antifungal genes and selectable marker genes under both light and dark growth conditions. Perform in planta bioassays for antifungal activity in transplastomic cotton plants. Approach (from AD-416): Total RNA isolated from developing cotton plants and cottonseed will be hybridized with PCR-generated probes for selected cotton plastid genes using standard Northern hybridization technology. The promoters from those genes that demonstrate high levels of expression in green and/or non-green cotton tissues based on Northern hybridization results will be cloned and characterized using standard molecular biological methods. Promoter fragments of select plastid genes will be fused to reporter genes (GUS, GFP, etc.) and transformed into tobacco and cotton plastids in order to identify the minimal functional promoter sequences. While tobacco plastid transformation protocols have been developed, the same cannot be said of cotton, and protocols will have to be optimized for this plant. Once an efficient cotton plastid transformation system has been developed, cotton plastid will be transformed with transformation vectors in which reporter, antifungal, and selectable marker genes are placed under control of selected cotton plastid gene promoters. Transplastomic cotton plants will be analyzed for expression and production of reporter, antifungal, and selectable marker genes by standard molecular biological techniques (PCR, Northern and Western blotting). In planta antifungal bioassays will be performed to determine levels of resistance to A. flavus, as well as other cotton fungal pathogens. Important information has been obtained over the five years of this project and a number of the experimental approaches will be continued under the new project agreement. Suitable promoters (regions of deoxyribonucleic acid (DNA) near genes that control their expression) for use in cotton plastid (DNA containing structures in green plant tissues) transformation vectors were identified and the research results have been published in Biologia Plantarum. This work addressed research outlined in Objective 3 of Project Plan 6435-42000-019-00D. Unfortunately, research on production of plastid transformed cotton cells has been terminated due to lack of proper selectable (antibiotic resistance) markers. A selection procedure with the antibiotic spectinomycin, which works well in tobacco, does not work at all in cotton or corn. Currently work in progress includes studies designed to allow a better understanding of where in the seed is the product of the antifungal transgene (gene producing the antifungal compound inserted into the plant�s DNA) localizing. We have transformed nuclei or chloroplasts of tobacco plants with a special tagged version of the antifungal peptide D4E1. This tag, called hemagglutinin (HA), makes it possible to localize the expression of this transgenic peptide in plant cells and to determine how the peptide damages the invading fungi. To monitor this process, we have developed an optimized procedure for localizing this peptide using Transmission Electron Microscopy (a type of microscopy that allows one to see minute details inside of a cell). Studies are in progress to determine where and how much of the lytic (ability to break open cells) peptide is expressed in the plastid-transformed plants compared to expression in the nuclear transformants. Another approach we are pursuing is to identify cotton cultivars with enhanced resistance to Aspergillus (A.) flavus infection. Based on our first bioassay of 26 different cotton cultivars, we have selected a panel of candidate cultivars for further study. Our goal is to perform these assays in triplicate, and most seed of interest have been screened at least twice. These studies are ongoing and will be completed as soon as enough seed become available (plants are currently growing in the greenhouse). The mechanism of action for the lytic peptide D4E1 is unknown. To determine the mechanism of action of this peptide, we are performing a series of assays using a special dye called "SYTOX green." The ability of a fungal cell to take up SYTOX green is a means to determine the integrity of the cell�s outer membrane. SYTOX green cannot penetrate healthy, intact cells but it can penetrate into cells that have damaged membranes. If SYTOX green is able to enter a cell it will bind nucleic acids and the level of fluorescence of the dye will increase by more than 500-fold. We have developed a SYTOX green microtiter plate assay (a plate with many small wells for multiple assays) to determine the effect D4E1 has on membrane integrity. In this assay, Aspergillus flavus spores are incubated with varying concentrations of D4E1 in the presence of SYTOX green and the fluorescence values quantified. If D4E1 is causing damage to cell membranes we will see a significant increase in fluorescence of the sample. These studies are currently in progress and replicates will be completed by the end of August 2013. Microscopic analysis of germinating A. flavus spores in the presence of different concentrations of D4E1 will be done to determine if there are any observable effects on the cells at sub-lethal concentrations of the peptide. The State of Louisiana Board of Regents has recently funded an equipment proposal (BORSF Enhancement, High Throughput Rapid Analysis of Plant Tissues for Agricultural Applications, LEQSF (2013-14)-ENH-TR-20, Principal Investigator $65,000) to purchase a high throughput tissue homogenizer and stereomicroscope with the capability to detect fluorescence that will be used in these studies. These studies address research outlined in Objective 3 of Project Plan 6435-42000-021-00D.
Impacts
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Identify cotton plastid promoters that demonstrate high expression levels in both green and non-green plant tissues for use in development of cotton plastid transformation vectors. Determine levels of expression of reporter genes in both green (leaf and outer boll) and non-green (cottonseed and root) cotton tissues under control of select, engineered plastid promoters. Generate cotton plastid transformation vectors that place antifungal genes and selectable marker genes under control of selected cotton plastid promoters and transform cotton. Test transformed tissues for expression of antifungal genes and selectable marker genes under both light and dark growth conditions. Perform in planta bioassays for antifungal activity in transplastomic cotton plants. Approach (from AD-416): Total RNA isolated from developing cotton plants and cottonseed will be hybridized with PCR-generated probes for selected cotton plastid genes using standard Northern hybridization technology. The promoters from those genes that demonstrate high levels of expression in green and/or non-green cotton tissues based on Northern hybridization results will be cloned and characterized using standard molecular biological methods. Promoter fragments of select plastid genes will be fused to reporter genes (GUS, GFP, etc.) and transformed into tobacco and cotton plastids in order to identify the minimal functional promoter sequences. While tobacco plastid transformation protocols have been developed, the same cannot be said of cotton, and protocols will have to be optimized for this plant. Once an efficient cotton plastid transformation system has been developed, cotton plastid will be transformed with transformation vectors in which reporter, antifungal, and selectable marker genes are placed under control of selected cotton plastid gene promoters. Transplastomic cotton plants will be analyzed for expression and production of reporter, antifungal, and selectable marker genes by standard molecular biological techniques (PCR, Northern and Western blotting). In planta antifungal bioassays will be performed to determine levels of resistance to A. flavus, as well as other cotton fungal pathogens. To understand how and where in the seed the transgene is expressing an antifungal peptide that may inhibit Aspergillus flavus growth in the plant, we have transformed nuclei or chloroplasts of tobacco plants with a special tagged version of the antifungal peptide D4E1. This tag, called hemagglutinin (HA), makes it possible to visualize where this transgenic peptide is expressed in plant cells and how they act on invading fungi. We have also developed and optimized several procedures for localizing this peptide using Transmission Electron Microscopy. We used a bioassay of 26 different cotton cultivars to identify which cultivars or species exhibited enhanced resistance to Aspergillus flavus infection. Based on our first assay, we have chosen candidate cultivars and species for further study. These studies are ongoing. The mechanism of action for the lytic (ability to break open cells) peptide d4E1 is unknown. To determine the mechanism of action of the peptide, we are performing a series of assays using the dye SYTOX green. SYTOX green can not penetrate intact cells, and its uptake is a measure of membrane integrity. When SYTOX green binds nucleic acids, the fluorescence emission increases by more than 500-fold. We are in the process of developing a microtiter plate (a plate with many small wells for multiple assays) assay to determine the effect d4E has on membranes. In this assay, Aspergillus flavus spores are incubated with varying concentrations of d4E in the presence of SYTOX green and the emission values quantified. These studies are currently in progress. An extension of these studies that will be performed this summer is to perform microscopic analysis of germinating Aspergillus flavus spores in the presence of different concentrations of d4E followed by staining the dyes with calcafluor white and SYTOX green. This will enable us to visualize SYTOX green that has permeated the Aspergillus flavus cell membranes which should be elevated relative to the peptide dose, as well as to determine if there are any effects on the morphology of the cells at sub-lethal concentrations of the peptide.
Impacts
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Publications
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Identify cotton plastid promoters that demonstrate high expression levels in both green and non-green plant tissues for use in development of cotton plastid transformation vectors. Determine levels of expression of reporter genes in both green (leaf and outer boll) and non-green (cottonseed and root) cotton tissues under control of select, engineered plastid promoters. Generate cotton plastid transformation vectors that place antifungal genes and selectable marker genes under control of selected cotton plastid promoters and transform cotton. Test transformed tissues for expression of antifungal genes and selectable marker genes under both light and dark growth conditions. Perform in planta bioassays for antifungal activity in transplastomic cotton plants. Approach (from AD-416) Total RNA isolated from developing cotton plants and cottonseed will be hybridized with PCR-generated probes for selected cotton plastid genes using standard Northern hybridization technology. The promoters from those genes that demonstrate high levels of expression in green and/or non-green cotton tissues based on Northern hybridization results will be cloned and characterized using standard molecular biological methods. Promoter fragments of select plastid genes will be fused to reporter genes (GUS, GFP, etc.) and transformed into tobacco and cotton plastids in order to identify the minimal functional promoter sequences. While tobacco plastid transformation protocols have been developed, the same cannot be said of cotton, and protocols will have to be optimized for this plant. Once an efficient cotton plastid transformation system has been developed, cotton plastid will be transformed with transformation vectors in which reporter, antifungal, and selectable marker genes are placed under control of selected cotton plastid gene promoters. Transplastomic cotton plants will be analyzed for expression and production of reporter, antifungal, and selectable marker genes by standard molecular biological techniques (PCR, Northern and Western blotting). In planta antifungal bioassays will be performed to determine levels of resistance to A. flavus, as well as other cotton fungal pathogens. Introduction of the new antifungal genes into chloroplast, the green pigment organelles in plant cells, is being studied as a means to reduce Aspergillus flavus invasion and subsequent aflatoxin contamination of cottonseed. Successful production of the antifungal protein/peptide will depend in large part on the ability of the promoter sequence of the gene, which serves as trigger mechanism, controlling expression of the antifungal gene to generate high levels of gene expression in the chloroplast genomes present in cells of both green and non-green tissues. This work addresses objective 3 of the project plan to identify potential antifungal genes and optimize their expression in transgenic crops such as cotton. We have identified two promoters called psbA and rrn16 that direct high levels of expression (more than 200 fold then that of the reference gene) in at least one sample. Other promoters have been identified as likely candidates, if preferential expression in specific tissues is required. The goal of this study has been met and the research published. We have also developed transgenic tobacco plants that express an antifungal gene D4E1 tagger with another master gene (called HA g hemoglutenin) in either nuclei or chloroplasts. These plants are currently being studied using microscopy and a protein tracking procedure called immunolocalization to determine the sites and levels of expression of the antifungal peptide. We are in the final stages of developing and optimizing protocols that are appropriate for visualization of the introduced proteins in tobacco leaf tissues. In another study, we are growing 34 cotton cultivars in the greenhouse to generate sufficient seeds for a kernel screening assay (KSA). At this point, we have sufficient seeds (>30) for a preliminary assay of 26 of these cotton varieties (namely, 2 Gossypium (G.) barbadense, 5 G. arboreum and 19 G. hirsutum). The KSA will monitor the progression and growth of Aspergillus flavus on these seeds using a green fluorescent protein (GFP)-expressing strain of Aspergillus flavus. Research progress was monitored through teleconferencing, frequent email communications and reports.
Impacts
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Identify cotton plastid promoters that demonstrate high expression levels in both green and non-green plant tissues for use in development of cotton plastid transformation vectors. Determine levels of expression of reporter genes in both green (leaf and outer boll) and non-green (cottonseed and root) cotton tissues under control of select, engineered plastid promoters. Generate cotton plastid transformation vectors that place antifungal genes and selectable marker genes under control of selected cotton plastid promoters and transform cotton. Test transformed tissues for expression of antifungal genes and selectable marker genes under both light and dark growth conditions. Perform in planta bioassays for antifungal activity in transplastomic cotton plants. Approach (from AD-416) Total RNA isolated from developing cotton plants and cottonseed will be hybridized with PCR-generated probes for selected cotton plastid genes using standard Northern hybridization technology. The promoters from those genes that demonstrate high levels of expression in green and/or non-green cotton tissues based on Northern hybridization results will be cloned and characterized using standard molecular biological methods. Promoter fragments of select plastid genes will be fused to reporter genes (GUS, GFP, etc.) and transformed into tobacco and cotton plastids in order to identify the minimal functional promoter sequences. While tobacco plastid transformation protocols have been developed, the same cannot be said of cotton, and protocols will have to be optimized for this plant. Once an efficient cotton plastid transformation system has been developed, cotton plastid will be transformed with transformation vectors in which reporter, antifungal, and selectable marker genes are placed under control of selected cotton plastid gene promoters. Transplastomic cotton plants will be analyzed for expression and production of reporter, antifungal, and selectable marker genes by standard molecular biological techniques (PCR, Northern and Western blotting). In planta antifungal bioassays will be performed to determine levels of resistance to A. flavus, as well as other cotton fungal pathogens. Transformation of the chloroplast (a compartment within a plant cell that contains chlorophyll and deoxyribonucleic acid (DNA)) genome of cotton with antifungal genes is being studied as a means to reduce Aspergillus (A.) flavus invasion and subsequent aflatoxin contamination of cottonseed. Aflatoxin is a toxic and carcinogenic compound produced by A. flavus during growth on some plants. Successful production of the antifungal protein/peptide will depend in large part on the ability of the promoter structure controlling expression of the antifungal gene to generate high levels of gene expression in the chloroplast genomes present in cells of both green and non-green tissues. Based on northern hybridization (a method to measure how much a gene is being expressed) studies using 20 different plastid DNA probes and total ribonucleic acid (RNA) extracted from cotton tissues grown in the light or dark, 6 promoters were selected for real time quantitative polymerase chain reaction (PCR) gene expression analysis (qPCR). Two gene promoters directed levels of expression more than 200 fold higher than that of the RNA polymerase gene that was used as a control: the psbA and rrn16 gene promoters. Since rrn16 is expressed in all tissues at higher levels than psbA (at least ~10 fold higher than that of psbA in all tissues except light-grown cotyledons and leaves), its promoter is our best candidate for generation of a plastid transformation vector for high level expression in a wide variety of tissues. Both nuclear- and chloroplast-transformed transgenic tobacco plants have been developed that express an antifungal peptide capable of disrupting the cell membrane of the fungus. These plants are currently being studied using microscopic techniques to determine the sites and levels of expression of the antifungal peptide. We have completed a preliminary study of tobacco tissues to determine which wavelengths of light are appropriate for these microscopic studies and will be using a commercially available antibody to directly visualize the peptide. These studies should be completed by the end of August 2010. Thirty-one cotton cultivars are currently being grown in the greenhouse to generate sufficient seeds for a cottonseed screening assay to determine if any of the seed of these cultivars demonstrate increased resistance to infection by the fungus. Once sufficient seed is obtained from these and any additional varieties that we obtain as well as ancestral strains, we will infect the cottonseed with a strain of Aspergillus flavus that produces a fluorescent protein that will allow us to quantitatively determine its growth in the cottonseed screening assay. Development of transgenic cottons expressing antifungal genes will be used to control aflatoxin contamination in cottonseed. Research progress is monitored by phone, email, and site visits.
Impacts
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Publications
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Identify cotton plastid promoters that demonstrate high expression levels in both green and non-green plant tissues for use in development of cotton plastid transformation vectors. Determine levels of expression of reporter genes in both green (leaf and outer boll) and non-green (cottonseed and root) cotton tissues under control of select, engineered plastid promoters. Generate cotton plastid transformation vectors that place antifungal genes and selectable marker genes under control of selected cotton plastid promoters and transform cotton. Test transformed tissues for expression of antifungal genes and selectable marker genes under both light and dark growth conditions. Perform in planta bioassays for antifungal activity in transplastomic cotton plants. Approach (from AD-416) Total RNA isolated from developing cotton plants and cottonseed will be hybridized with PCR-generated probes for selected cotton plastid genes using standard Northern hybridization technology. The promoters from those genes that demonstrate high levels of expression in green and/or non-green cotton tissues based on Northern hybridization results will be cloned and characterized using standard molecular biological methods. Promoter fragments of select plastid genes will be fused to reporter genes (GUS, GFP, etc.) and transformed into tobacco and cotton plastids in order to identify the minimal functional promoter sequences. While tobacco plastid transformation protocols have been developed, the same cannot be said of cotton, and protocols will have to be optimized for this plant. Once an efficient cotton plastid transformation system has been developed, cotton plastid will be transformed with transformation vectors in which reporter, antifungal, and selectable marker genes are placed under control of selected cotton plastid gene promoters. Transplastomic cotton plants will be analyzed for expression and production of reporter, antifungal, and selectable marker genes by standard molecular biological techniques (PCR, Northern and Western blotting). In planta antifungal bioassays will be performed to determine levels of resistance to A. flavus, as well as other cotton fungal pathogens. Significant Activities that Support Special Target Populations Efforts are underway to add specific antifungal genes into the deoxyribonucleic acid (DNA) of certain organelles (plastids) of cotton as a means to reduce A. flavus invasion and subsequent aflatoxin contamination of cottonseed. Successful production of the antifungal protein/peptide will depend in large part on the ability of the machinery controlling expression of the antifungal gene to generate high levels of gene expression in the plastid genomes present in cells of both green and non-green tissues. A total of 18 genes from the cotton plastid genome were assayed for their levels of expression in a number of green (e.g. leaf and stem) and non-green (e.g. seed) tissues at different growth stages using standard techniques. Two genes in particular, matK and rrn16, demonstrated high level expression at almost all growth stages and tissues. Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technology was used to confirm the data, and though both matK and rrn16 were expressed in all tissues and times tested, the rrn16 gene expression was found to be much higher than matK. Therefore, when specific antifungal piece of DNA or specific gene needs to be added to cotton plastids, the transfer mechinery (vectors) will utilize the relevant-part (promoter) of the rrn16 gene to drive high level expression of the antifungal genes in both green and non-green cotton tissues. Development of transgenic cottons expressing antifungal genes will be used to control aflatoxin contamination in cottonseed. Research progress is monitored by phone, email, and site visits.
Impacts
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