Progress 03/15/12 to 03/14/16
Outputs
Target Audience:This project has provided training for students recruited from an NIH Minority Biomedical Research Support-Research Initiative in Science Enhancement (MBRS-RISE) training program. This enhanced our ability to provide training for students from historically underrepresented groups. The University of Texas at San Antonio is a Hispanic serving Institution with >55% minority enrollment, and this project provides significant opportunities for continued recruitment of undergraduate and graduate scholars to conduct research in plant molecular virology. This will help to increase the number of graduates trained in agricultural-related sciences. Of the participants in this project, four are women, one is African American and two are hispanic. Our work has also targeted the general plant science community, including plant virologists, through dissemination of results at local, national and international meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the course of this project, we have provided training opportunities for a number of people: One post-doctoral fellow: This person advanced expoeriments concerning ERF13 and WRKY33. Two technicians: Both have provided support for all facets of the proposal. This included performing experiments to examine interactions between host factors that regulate the TGMV AL1629 promoter and to help in the maintenance of plants, generation of transgenic lines and microscopy. This proposal has provided training opportunities for several students, including students from historically underrepresented groups. A total of three undergraduates: One has continued his work in my lab on this project as a Masters student. One has entered a PhD program at Boston University School of Medicine. The third student is currently employed at the Texas Biomedical research Institute in San Antonio, TX working on HIV. Two Masters students: One student is currently completing a thesis and is expected to graduate in May 2016. The second finished her thesis and was admitted to the Cell and Molecular Biology PhD program at UTSA and is currently completing her dissertation work in my lab. Two PhD students: One carried out some of the experiments on analyzing regions of the TGMV genome that confer silencing of the AL1629 promoter. As mentioned above, one is completing her dissertation work in my lab. An outcome of this recruitment effort was the expansion of training opportunities in current topics related to plant molecular virology, and an increase the number of students graduating with experience in the agricultural sciences. This support has increased our capacity to provide and provided important training for the next generation of students engaging in research in plant pathology and has provided an opportunity to develop the future leaders in agriculture, and to increase the number of scientists that pursue careers in the critical area of food security and safety. How have the results been disseminated to communities of interest?We have presented results from the experiments conducted during this time period at several scientific meetings: 31st Annual Meeting American Society for Virology (July, 2012. Madison, WI); Society for Advancement of Chicanos and Native Americans in Science (October 2012. Seattle WA). University of Stuttgart (August, 2012. Stuttgart, Germany) 10th EMBL Conference on Transcription and Chromatin (August 2012. Heidelberg, Germany) Advances in Plant Virology Conference (April, 2012. Dublin, Ireland). 32nd Annual Meeting American Society for Virology (July 2014. Boulder CO) IS-MPMI meeting (July 2014. Rhodes, Greece) Zhejiang University, China (May, 2014), Each year of the project students presented their work at the UTSA College of Sciences Research Symposium. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Work under this proposal sought to increase our understanding of how viruses manipulate the host plant to facilitate infection. We have successfully developed the tools to examine the role of the host in regulating expression of viral genes important for pathogenesis. Using constructs that express a reporter gene from a viral promoter, we have identified sequences necessary for expression of AL2 and AL3 in two distinct yet related viruses, Tomato golden mosaic virus (TGMV) and Spinach curly top virus (SCTV). The fact that TGMV has a bipartite genome organization and SCTV has a monopartite genome indicates that regulation of the AL2/3 promoter could be conserved in both sub groups of geminiviruses. This has important ramifications for developing resistance as a strategy based on interfering with AL1629 promoter activity could provide broad-based resistance. We have also identified two host factors that may be critical for virus transcription. This work carried out in this project has changed our knowledge of how plant viruses interact with the host. Aim #1: Characterize interactions between geminivirus AL1629 promoters and chromatin. For this aim we developed the assays to examine the state of chromatin and DNA when the TGMV AL1629 promoter is active and inactive. During the course of the project we expanded this aim due to concerns raised by the Dissertation committee of the PhD student. It was suggested that replication of the virus could impact the status of the promoter. We therefore have been generating additional promoter-reporter constructs that can produce replicons. These constructs have been examined in transient assays and due to technical problems generating the replicons we have just recently introduced these into Nicotiana benthamiana plants to study the chromatin state. The experiments carried out under this aim have determined the following: a) Methylation pattern of geminivirus AL1629 promoters: Our data has demonstrated that there are differences in the DNA methylation pattern of cytosine residues between active and silenced AL1629 promoters. The majority of cytosine residues are contained within the CHH context with very few CG or CNG sites. There are however some slight differences between the sites. We found methylation at CG and CNG sites was high, although this could be reflective of the limited number of these sites. However, there was a lower level of methylation at CHH sites within the AL1629 promoter. Whether this is reflective of regulation of the viral promoter waits to be confirmed, but this does support our hypothesis that the promoter is subject to methylation. Although we did not observe wholesale changes to methylation of the DNA, two specific cytosine residues were always methylated in silenced promoters and non-methylated in active promoters. We are currently undergoing a mutational analysis of these two residues to determine their role in AL1629 promoter activity. b) Analysis of histone methylation in a chromatin context: As mentioned above these experiments have been delayed due to technical issues in constructing transgenic plants containing the replicons needed. However, we have now generated the transgenic plants necessary for the analysis. c) Analysis of constructs capable of replication has revealed that replication appears to suppress promoter activity. From these results we are hypothesizing that there could be two different pools of viral templates. One pool of viral DNA provides a template for transcription, while a second pool is template for replication. The mechanism for how these templates are formed is under investigation. Aim #2:Characterize the mechanism for activation of geminivirus AL1629 promoters: a) Using transient assays we have identified sequences necessary for expression of the SCTV AL2/3 promoter. Within this region are sequences 100% conserved between both SCTV and TGMV. Although, we had generated transgenic N. benthamiana plants containing SCTV promoter-reporter constructs, which exhibited loss of activity, additional constructs were generated that were active in transgenic plants. Interestingly, the SCTV AL1629 promoter is significantly stronger than the equivalent TGMV promoter. As SCTV is a significantly more virulent virus than TGMV, it is possible that the increased promoter activity allows SCTV to escape the methylation defense of the plant. This has yet to be definitively demonstrated. b) In previous work we had shown that Introducing mutations that prevent TGMV AL1 protein from associating with its cognate binding site, result in increased AL62 transcription, and decreased transcription from the downstream AL1629 initiation site present within the AL1 coding region. Work in this project has shown that this also appears to be the case for SCTV. We have generated transgenic plants containing mutations in the AL1 coding region and the AL1 binding site, in both TGMV and SCTV. Analysis of these plants indicates that the presence of AL1 appears to suppress activity of the AL1629 promoter, in contrast to the results observed in transient assays. When AL1 is removed the AL1629 promoter becomes active. This paradoxical discovery is currently being investigated. c) Analysis of histone methylation in a chromatin context: These experiments have only just been initiated and we anticipate analysis to begin within the next month. Aim #3: Characterize interactions between geminivirus AL1629 promoters and ERF13. a) In this project we have identified a host ethylene response factor (ERF13) that interacts with the TGMV AL1629 promoter. Using yeast genetic analysis we have demonstrated that ERF13 behaves as a typical transcription factor. Using deletions and a bioinformatics analysis of the amino acid sequence suggests that ERF13 contains a typical acidic type activation domain. b) This work has also shown that ERF13 interacts with a second host factor, WRKY33. Both proteins have been implicated in host defense against pathogen infection. Thus, geminiviruses may highjack the host response to provide transcription factors for expression of two viral genes critical for infection. c) We have tested the effect of infection on ERF13 expression. Using quantitative PCR, our data indicates that expression is induced upon infection with different geminiviruses, indicating that these viruses stimulate expression of ERF13. In addition we have shown that many of the genes involved in both the ethylene signaling and biosynthetic pathway are up-regulated during infection. This is consistent with our hypothesis that geminiviruses may manipulate the ethylene pathway during infection. As ethylene is synthesized from S-adenosyl methionine this provides a possible link between the methyl cycle, an important defense pathway targeting DNA viruses. This would be a novel and exciting new avenue of research concerning plant defenses against geminiviruses. d) Effect of over-expression of ERF13. Attempts to assess the effects of over-expressing ERF13 on virus infectivity have proven unsuccessful, but revealed some interesting information. When ERF13 was over-expressed, both from the constitutive Cauliflower mosaic virus 35S promoter and from a TMV-based replicon, plants generated an HR-like response. This was evident in N.benthamiana plants, which lack the N resistance gene. This strongly suggests that ERF13 may be involved in this defense response downstream of R-avr gene interaction.
Publications
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Progress 03/15/14 to 03/14/15
Outputs
Target Audience: This project has provided training for two undergraduate student recruited from an NIH Minority Biomedical Research Support-Research Initiative in Science Enhancement (MBRS-RISE) training program. This enhanced our ability to provide training for students from historically underrepresented groups. The University of Texas at San Antonio is a Hispanic serving institution with >55% minority enrollment, and this project provides significant opportunities for continued recruitment of undergraduate and graduate scholars to conduct research in plant molecular virology. This will help to increase the number of graduates trained in agricultural-related sciences. One additional student was also supported during this time. Two gradaute students, one Masters and one PhD were supported. A post-doctoral scientist and two technicians, one an African American, were supported in part by this project. Of the participants in this project, three were women. Our work has also targeted the general plant science community, including plant virologists, through dissemintation of results at local, national and international meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A technician has provided support for all facets of the proposal, and performed experiments to examine interactions between host factors that regulate the TGMV AL1629 promoter. An additional technician was also hired to help in the maintenance of plants, generation of transgenic lines and microscopy. This year has provided training opportunities for several students. Two undergraduates were recruited from an NIH MBRS-RISE training program. This support has increased our capacity to provide training for students from historically underrepresented groups. One Masters student was involved in evaluating interactions between different host factors and the AL1629 promoter. I have successfully recruited two PhD students, one of which is continuing the experiments on DNA and histone methylation. Of the people recruited, four are women, one is African American and two are hispanic. An outcome of this recruitment effort is the expansion of training opportunities in current topics related to plant molecular virology, and to increase the number of students graduating with experience in the agricultural sciences. How have the results been disseminated to communities of interest? We presented results from the experiments conducted during this time period at several scientific meetings: 32nd Annual Meeting American Society for Virology (July 2014. Boulder CO) IS-MPMI meeting (July 2014. Rhodes, Greece) Local meetings included: UTSA College of Sciences Research Symposium (October 2014. San Antonio TX) I was also invited to present results of our research at Zhejiang University, China (May, 2014), What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Work under this proposal seeks to address fundamental issues concerning plant health. Specifically, we are conducting experiments to increase our understanding of how viruses manipulate the host plant to facilitate infection. Students at the PhD, Masters and undergraduate levels have been developing the tools to examine the role of the host in regulating expression of viral genes important for pathogenesis. Using constructs that express a reporter gene from a viral promoter, they have determined the sequences necessary for that expression and identified two host factors that may be critical for the virus. This work has changed our knowledge of how plant viruses interact with the host and provided important training for the next generation of students engaging in research in plant pathology. This increases the options for these students to pursue a productive career in the agricultural sciences. Increasing our understanding how plant viruses interact with a host to cause disease will allow us to develop new strategies to interfere with these processes and provide resistance to these viruses. This new information will allow the use of methods that will reduce the use of pesticides, that are the primary strategy for removing insects that transmit these particular plant viruses. The training of students in this field provides an opportunity to develop the future leaders in agriculture, and to increase the number of scientists that pursue careers in the critical area of food security and safety. Aim #1: Characterize interactions between geminivirus AL1629 promoters and chromatin. For this aim we have continued to develop the assays to examine the state of chromatin and DNA when the Tomato golden mosaic virus (TGMV) AL1629 promoter is active and inactive. We have also expanded this aim due to concerns raised by the Dissertation committee of the PhD student that replication of the virus could impact the staus of the promoter. The student has therefore been generating additional promoter-reporter constructs that can produce replicons. This change will expand our understanding of regulation of the AL1629 promoter, by providing additional data on how replication of the virus can play a role in regulation. We have also been continuing our optimization of the chromatin purification scheme. Aim #2:Characterize the mechanism for activation of geminivirus AL1629 promoters. In this aim, we have generated all the constructs necessary for analysis. Using transient assays we have identified sequences necessary for expression of the Spinach curly top virus (SCTV) AL2/3 promoter. These sequences contain regions that are 100% conserved between both SCTV and TGMV. Transgenic N. benthamiana plants containing SCTV promoter-reporter constructs also show loss of activity, comparable to that observed for equivalent TGMV constructs. This suggests a common mechanism for regulation and indicates that any future strategy for interfering with this promoter could provide broad-based resistance. Additional transgenic lines have been generated, that contain mutations in the AL1 coding region and the AL1 binding site, in both TGMV and SCTV. These plants are now ready for analysis. Aim #3: Characterize interactions between geminivirus AL1629 promoters and ERF13. We have conducted experiments in this aim that have demonstrated that ERF13 can act as a transcription factor and interacts with a second host factor, WRKY33. We have been developing the assays to demonstrate that this interaction occurs in vivo, using transgenic plants containing the AL1629 promoter. We have generated constructs expressing a FLAG-tagged version of ERF13 to use in Chromatin immunoprecipitation experiments. A second set of experiments that was performed was to test the effect of infection on ERF13 expression. Our data indicates that expression is induced upon infection with different geminiviruses, indicating that these viruses stimulate expression of the gene needed for expression of a viral pthogenciity determinant. Additional experiments usinf quantitative real time PCR indicate that these viruses may manipulate the ethylene pathway during infection. As ethylene is synthesized from S-adenosyl methionine. this provides a possible link between the methyl cycle, an important defense pathway targeting DNA viruses. This would be a novel and exciting new avenue of research concerning plant defenses againts geminiviruses.
Publications
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Progress 03/15/13 to 03/14/14
Outputs
Target Audience: This project has provided training for an undergraduate student recruited from an NIH Minority Biomedical Research Support-Research Initiative in Science Enhancement (MBRS-RISE) training program. This enhanced our ability to provide training for students from historically underrepresented groups. The University of Texas at San Antonio is a Hispanic serving institution with >55% minority enrollment, and this project provides significant opportunities for continued recruitment of undergraduate and graduate scholars to conduct research in plant molecular virology. This will help to increase the number of graduates trained in agricultural-related sciences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A technician was hired to provide support for all facets of the proposal, and to aid in examination of interactions between host factors that regulate the TGMV AL1629 promoter. We have provided training opportunities for several students. One undergraduate was recruited from an NIH MBRS-RISE training program. This support has increased our capacity to provide training for students from historically underrepresented groups. One Masters student was involved in evaluating interactions between different host factors and the AL1629 promoter. I have successfully recruited two PhD students, one of which will be supported by this grant and is going to be continuing the experiments on DNA and histone methylation. An outcome of this recruitment effort is the expansion of training opportunities in current topics related to plant molecular virology, and to increase the number of students graduating with experience in agricultural sciences. How have the results been disseminated to communities of interest? We presented results from the experiments conducted during this time period at several scientific meetings: 31st Annual Meeting American Society for Virology (July, 2012. Madison, WI); Society for Advancement of Chicanos and Native Americans in Science (October, 2012. Seattle WA). I was invited to present results of our research at University of Stuttgart (August, 2012. Stuttgart, Germany) as well as at the 10th EMBL Conference on Transcription and Chromatin (August, 2012. Heidelberg, Germany) and the Advances in Plant Virology Conference (April, 2012. Dublin, Ireland). What do you plan to do during the next reporting period to accomplish the goals? Our efforts for the next year are to confirm the interaction of ERF13 and WRKY33 and to test the ability of ERF13 to bind the AL1629 promoter. Our efforts will also be focused on determining the histone modifications that are associated with the AL1629 promoter when repressed and active. We will also focus on testing whether the region of TGMV DNA responsible for silencing of the AL1629 promoter can be used for disrupting viral infection.
Impacts
What was accomplished under these goals?
For the objectives outlined in Aim 1 we have performed experiments to optimize conditions for the chromatin immunoprecipitation of viral DNA/histone complexes using antibodoies to markers for both active and repressed chromatin. A new PhD student is continuing these experiments where we will be comparing the levels of markers associated with active or repressed chromatin under conditions where the TGMV AL1629 promoter is silenced or active. For experiments outlined in Aim 2 we have begun to generate the constructs necessray for analysis of role of AL1 in remodeling the viral chromatin architechture to regulate expression from the AL1629 promoter. In Aim 3 a host ERF factor binds to the AL1629 promoter and it is possible that this occurs in a complex with a second host factor, WRKY33. This hypothesis stems from information that ERF13 and WRKY33 are predicted to interact with each other in an interactome study. A Masters level student has therefore been performing yeast two hybrid experiments to test our hypothesis. Preliminary data seems to indicate that these two proteins do in fact interact. However, we are investigating wether this actually occurs at the viral promoter.
Publications
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Progress 03/15/12 to 03/14/13
Outputs
OUTPUTS: Conducting and analyzing experiments: Experiments for this project were performed primarily for aim 1 and 3 of the project. Outcomes of those experiments have contributed to the overall objective of the proposal: to characterize the role of the viral AL1 protein, chromatin and a host ethylene response factor (ERF) in regulating viral transcription. In Aim 1 the outcome of our experiments is an increased understanding of the role of DNA and histone methylation in regulation of the promoter for the Tomato golden mosaic virus (TGMV) AL1629 transcription unit. The mRNA produced expresses AL2 and AL3 proteins, which are critical for viral infection. The outcome of experiments performed for Aim 3 is expansion of our understanding of the interaction between the ERF host factor and the TGMV AL1629 promoter. The overall outcome of these experiments is increased understanding of the role of the host in regulating expression of highly conserved genes that suppress antiviral defenses and recondition the cellular environment. Specifically, the role of host methylation and host transcription factors in this process. With respect to our long-term goals, we are using this information to develop methods for disruption of these interactions. Training and mentoring: A technician was hired to provide support for all facets of the proposal, and to aid in examination of interactions between host factors that regulate the TGMV AL1629 promoter. We have provided training opportunities for several students. One undergraduate was recruited from an NIH MBRS-RISE training program. This support has increased our capacity to provide training for students from historically underrepresented groups. One Masters student was involved in evaluating interactions between different host factors and the AL1629 promoter. I have successfully recruited two PhD students, one of which will be supported by this grant and is going to be continuing the experiments on DNA and histone methylation. An outcome of this recruitment effort is the expansion of training opportunities in current topics related to plant molecular virology, and to increase the number of students graduating with experience in agricultural sciences. Dissemination: We presented results from the experiments conducted during this time period at several scientific meetings: 31st Annual Meeting American Society for Virology (July, 2012. Madison, WI); Society for Advancement of Chicanos and Native Americans in Science (October, 2012. Seattle WA). I was invited to present results of our research at University of Stuttgart (August, 2012. Stuttgart, Germany) as well as at the 10th EMBL Conference on Transcription and Chromatin (August, 2012. Heidelberg, Germany) and the Advances in Plant Virology Conference (April, 2012. Dublin, Ireland). Collaborations. One outcome of these presentions is a potential collaboration with Drs. H. Jeske and C. Wege in Stuttgart. This will involve exchange of information regarding chromatin state of viral minichromosomes during infection. We have also initiated proteomics work with Dr. William Haskins at UTSA to investigate the proteins associated with minichromosomes during infection. PARTICIPANTS: Garry Sunter: Project Director. I have been responsible for overseeing progress on all aims of the proposal, and directed the efforts of the technician and students who are participating on the project. I hold weekly meetings with all personnel involved in the project to evaluate progress for each of the aims outlined in the proposal. I have also been responsible for dissemination of results at National and International scientific meetings. A technician has been hired with the main responsibility for maintaining cell cultures and plants essential to the success of the project. To date this project has provided training or professional development opportunities for undergraduate and graduate students An undergraduate student has been in the lab for the past year learning fundamental molecular biology techniques and the generation of transgenic plant lines important for this project. A Masters level student successfully defended her thesis based on the work outlined in this proposal and we are preparing to submit a manuscript describing this work. TARGET AUDIENCES: This project has provided training for an undergraduate student recruited from an NIH Minority Biomedical Research Support-Research Initiative in Science Enhancement (MBRS-RISE) training program. This enhanced our ability to provide training for students from historically underrepresented groups. The University of Texas at San Antonio is a Hispanic serving institution with >55% minority enrollment, and this project provides significant opportunities for continued recruitment of undergraduate and graduate scholars to conduct research in plant molecular virology. This will help to increase the number of graduates trained in agricultural-related sciences. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
For the objectives outlined in Aim 1 we have performed experiments to optimize conditions for the chromatin immunoprecipitation of viral DNA/histone complexes using antibodoies to markers for both active and repressed chromatin. A new PhD student is continuing these experiments where we will be comparing the levels of markers associated with active or repressed chromatin under conditions where the TGMV AL1629 promoter is silenced or active. For experiments outlined in Aim 2 we have begun to generate the constructs necessray for analysis of role of AL1 in remodeling the viral chromatin architechture to regulate expression from the AL1629 promoter. In Aim 3 a host ERF factor binds to the AL1629 promoter and it is possible that this occurs in a complex with a second host factor, WRKY33. This hypothesis stems from information that ERF13 and WRKY33 are predicted to interact with each other in an interactome study. A Masters level student has therefore been performing yeast two hybrid experiments to test our hypothesis. Preliminary data seems to indicate that these two proteins doe in fact interact. However, we are investigating wether this actually occurs at the viral promoter.
Publications
- No publications reported this period
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