Progress 02/01/17 to 01/31/20
Outputs
Target Audience:Researchers in the area of plant-microbe interactions, graduate students, and high school students. Changes/Problems:Microbiome analysis revealed that the inner portion of crown galls from woody plant hosts do not carry large populations of bacteria, including agrobacteria. Agrobacteria (and other bacteria) are likely to be more abundant on or near the surface of crown galls. This is surprising given the energy required by these bacteria to induce gall formation and rich abundance of nutrients in galls. Further experiments on new gall samples will test different portions of the crown gall for the presence of agrobacteria and virulence genes. Issues with generating opine knockout lines for objective 2 also continued. Instead, genome sequencing and comparative population genomics of Agrobacteria isolated from the analyzed galls was undertaken, along with isolates from an extensive culture collection. This study resulted in a greater understanding of the evolution of agrobacteria and their virulence plasmids; and epidemiological studies informed on global transmission patterns of Agrobacterium in managed systems. This work was recently accepted for publication in the journal Science. What opportunities for training and professional development has the project provided?Over the course of the previous year, I have participated in several training development opportunities. Some of the most impactful have been centered around networking and promoting my research. I attended the 2019 Crown Gall Conference at U. of Missouri and presented my research, as well as presented work at the 2019 IS-MPMI conference in Glasgow Scotland. I have also engaged in developing and delivering teaching material. I was invited to teach a workshop at Virginia Tech in November 2019. This workshop covered comparative genomics analyses and making publication quality figures. This past summer I taught a bioinformatics course for a DNA biology and bioinformatics STEM camp for high school students from underrepresented groups. Last, I had opportunities to learn mentoring skills, I mentored an undergraduate student the scientific process, critical thinking, and skills in computational biology. Previous undergraduate student mentees were also co-authors on a recent publication. How have the results been disseminated to communities of interest?Publications in peer-reviewed journals. Presentations in National and International conferences. Classrooms and workshops. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The bacterial pathogen Agrobacterium tumefaciens causes crown gall disease on a diverse variety of plant host species and is a major problem in the nursery and orchard industries. It is often difficult to isolate pathogenic agrobacteria from crown galls, particularly from woody plant hosts, making fast and accurate diagnosis difficult. Microbiome analysis was performed on 92 diseased gall and healthy plant tissue samples from nurseries and orchards across Oregon and the pacific northwest. These represent 14 plant host species from nurseries in 4 states and 2 countries. Most were from woody plants such as apple, raspberry, blueberry, and rose. This work revealed that total microbial abundance inside crown galls is low, and most microbes are likely to be surface associated. This has implications for diagnostics and testing as standard protocol for diagnostic labs includes sterilization and removal of the surface of sample tissue. Despite the low overall microbial presence, gall communities were found to be rich in microbial diversity. Surprisingly, we found that Agrobacterium are not the most abundant taxa in most sampled galls and often don't even represent a plurality of bacteria in a gall. These data suggest that other bacteria are often outcompeting Agrobacterium in the gall. Future experiments will explore whether the gall itself is the true reservoir of pathogenic agrobacteria. The genome sequences of strains isolated in the course of this study, combined with genomes of strains selected from culture collections, were analyzed to determine the evolutionary history of agrobacteria and the Ti plasmids. New methods for genomic epidemiology were also developed using this analysis as a framework. These methods enable the identification of transmission patterns both within and between nurseries and grow sites. This work was recently accepted for publication and is also expected to promote advances in the optimization of Agrobacterium for genetic engineering.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Weisberg AJ, Davis II EW, Tabima J, Belcher MS, Miller M, Kuo CH, Grunwald NJ, Putnam ML, and Chang JH. 2020. Unexpected conservation and global transmission of agrobacterial virulence plasmids. Science. In press.
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Progress 02/01/18 to 01/31/19
Outputs
Target Audience:This past August I developed and taught molecular biology and bioinformatics courses as part of a multi-day DNA Biology STEM camp targeted at high school students from underrepresented groups including women and minority ethnicities. The camp was comprised of underrepresented students from an urban Portland, OR school. Students learned molecular biology techniques and analyzed sequencing data using bioinformatics tools. This past summer I also mentored another high school intern in the lab as part of a STEM academy program at Oregon State University. This student learned bioinformatics tools and programming while contributing to a project of which she will be a co-author on a future publication. Changes/Problems:Microbiome analysis revealed that the inner portion of crown galls from woody plant hosts do not carry large populations of bacteria, including agrobacteria. Agrobacteria (and other bacteria) are likely to be more abundant on or near the surface of crown galls. This is surprising given the energy required by these bacteria to induce gall formation and rich abundance of nutrients in galls. Further experiments on new gall samples will test different portions of the crown gall for the presence of agrobacteria and virulence genes. Continuing issues with generating opine knockout lines will be addressed by attempting other cloning methods. Alternatively, opine synthase insertion-mutants will be acquired from other research groups to use in experiments. Additionally, genome sequencing and comparative population genomics of Agrobacteria isolated from the analyzed galls has also been undertaken, along with isolates from an extensive culture collection. This study is nearly complete and will inform on transmission patterns of Agrobacterium in nurseries and orchards. What opportunities for training and professional development has the project provided?Over the course of the previous year, I have participated in several training development opportunities. I attended the NIFA Fellows Project Director's Meeting and presented my research, as well as learned about opportunities for grant funding. I was an invited speaker at the U. of Arizona to present my research for their department seminar, and also presented research at the Plant and Animal Genome (PAG) conference. This past summer I again taught a bioinformatics course for a DNA biology and bioinformatics STEM camp for high school students from underrepresented groups. Students attending this multi-day camp learned about transcribing and translating DNA, identifying genes with BLAST, and examined 3D protein structure using bioinformatics tools and command line programming. I also taught as part of a workshop at a Midsummer conference for high school interns as part of the ASE program; which covered the history of agriculture and the modern genetics of corn and other crops. Additionally, I also individually mentored another high school intern in our lab over the past summer. I taught this student basic programming and command line software analysis and worked with her to analyze genomics data. She contributed to an ongoing research project, and her comparative genomics analyses will be included in an upcoming publication on which she will be a co-author. The knowledge and experience in microbiome analysis that I have gained through this fellowship has also allowed me to study other microbiome datasets in collaborations with other researchers. How have the results been disseminated to communities of interest?As mentioned above, high school students from underrepresented groups, including women and ethnic minorities, participated in the DNA biology and bioinformatics STEM camp. The camp was comprised of students from an urban high school near Portland, OR. Camp participants learned bioinformatics-based approaches and analyses. They also learned molecular biology skills and introductory computer programming. Results were also presented at the NIFA Fellows Project Director's Meeting in August 2018. Once further analyses and experiments are complete, this research will be written and published as a peer-reviewed journal article describing the findings. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 is complete, and during the next reporting period, I will continue to generate constructs and knock out opine synthase genes for objective 2. Once the opine gene knockout constructs are prepared, I will proceed with knocking out those genes from the wild-type agrobacteria strains. The wild-type and opine-knockout strains (along with the gall-associated bacterial isolates) will be used to create synthetic gall communities on plants, which will be processed in a similar manner as those galls in objective 1. This will follow the established protocol described in the initial project description.
Impacts
What was accomplished under these goals?
The bacterial pathogen Agrobacterium tumefaciens causes crown gall disease on a diverse variety of plant host species and is a major problem in the nursery and orchard industries. It is often difficult to isolate pathogenic agrobacteria from crown galls, particularly from woody plant hosts, making fast and accurate diagnosis difficult. The goal of this project is to assess the microbial diversity and abundance of agrobacteria in crown galls from agriculture systems and in synthetic communities using a microbiome sequencing-based approach. As part of progress towards objective 1, I have collected 92 diseased gall and healthy plant tissue samples from nurseries and orchards across Oregon and the pacific northwest. These represent 14 plant host species from nurseries in 4 states and 2 countries. Most of the analyzed gall samples were from woody plants such as apple, raspberry, blueberry, and rose. A sequencing run on an Illumina HiSeq was performed on DNA from these samples, however microbial DNA was not abundant in the extracted samples and the estimated microbial abundance was extremely low. During the course of DNA extraction, galls were surface sterilized, and the center portion of the inner gall was sampled following standard protocol of diagnostics labs. Despite the low overall microbial presence, gall communities were found to be rich in microbial diversity. Surprisingly, we found that Agrobacterium are not the most abundant taxa in most sampled galls and often don't even represent a plurality of bacteria in a gall. These data suggest that other bacteria are often outcompeting Agrobacterium in the gall. However, agrobacteria may be primarily associated with the surface of the gall on woody plants, and are not found inside the gall tissue itself. Ongoing experiments include testing multiple regions of the gall (surface-associated, surface tissue, inner gall, etc.) for pathogenic agrobacteria, particularly on woody plant hosts. If agrobacteria are found to be primarily gall surface-associated, subsequent experiments will target this tissue for pathogen abundance. Alternatively, other experiments will explore whether the gall itself is the true reservoir of pathogenic agrobacteria. Agrobacteria also induces the plant to produce uncommon nutrients (opines) that only agrobacteria can utilize. The ability to catabolize these opines is hypothesized to provide an advantage to agrobacteria over other bacteria in the gall despite the energy required to infect a plant and induce formation of a gall (the "opine concept"). In order to test this hypothesis, I will compare the abundance of agrobacteria and the overall composition of synthetic microbial communities in galls induced by agrobacteria with and without the ability to induce the production of opines. As part of progress towards objective 2, I am generating gene knockouts for two different strains of agrobacteria that induce the production of different opines. Both strains are well characterized, and induce galls that produce either the opines nopaline or octopine. Using Gibson cloning, I am generating constructs comprised of the upstream and downstream regions of the nopaline or octopine synthesis genes, with the gene sequence itself replaced with a selectable marker. These constructs will then be cloned into the wild-type agrobacteria strains and recombined into the genome to replace the original opine synthesis gene. I have also isolated and characterized 22 bacterial isolates from gall samples. These isolates were identified based on DNA barcode sequences as belonging to diverse genera and species, including Pseudomonas, Arthrobacter, Microbacterium, Curtobacterium, Bacillus, and Agrobacterium/Rhizobium. These isolates will be used to construct synthetic communities on plants with pathogenic agrobacteria or the opine-synthesis gene knockouts described above.
Publications
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Progress 02/01/17 to 01/31/18
Outputs
Target Audience:This past August I developed and taught molecular biology and bioinformatics courses as part of (2) two week DNA Biology STEM camps targeted at high school students from underrepresented groups including women and minority ethnicities. The first camp was comprised of students local to the Corvallis, OR area, while the second was comprised of underrepresented students from an urban Portland, OR school. Students learned molecular biology techniques to do microbiome analysis of crown gall samples in the first week, and then analyzed sequencing data of their own samples using bioinformatics tools in the second week of the camp. This past summer I also mentored a high school intern in the lab as part of a STEM academy program at Oregon State University. This student, a woman from an underrepresented ethnic group, learned bioinformatics tools and programming while contributing to a project of which she will be a co-author on a publication. Changes/Problems:Following the initial sequencing run of gall microbiome DNA samples, it was determined that the inner portion of crown galls from woody plant hosts do not carry large populations of bacteria, including agrobacteria. Agrobacteria (and other bacteria) are likely to be more abundant on or near the surface of crown galls. This is surprising given the energy required by these bacteria to induce gall formation and rich abundance of nutrients in galls. Further experiments will test different portions of the crown gall for the presence of agrobacteria and virulence genes. These tissues will then be sampled for microbiome sequencing and analysis as described in the initial project description. If Agrobacterium is not abundant on the surface of galls, additional experiments with synthetic communities will test whether it is enriched in soil near plants producing or not producing opines. These opines may be secreted into soil, which could provide a selective advantage to agrobacteria despite not being physically present near the gall. What opportunities for training and professional development has the project provided?Over the course of the previous year, I have participated in several training development opportunities. I developed and taught a microbiome course for a DNA biology and bioinformatics STEM camp for high school students from underrepresented groups. Students attending this two week camp learned all of the steps for microbiome analysis, from sample preparation to bioinformatics analysis of the sequencing results. Students extracted DNA from bacteria and plant samples, amplified microbial barcode sequences using PCR, and prepared sequencing libraries. In the second week they used state-of-the-art software and tools to analyze the microbiome of different plant and soil samples that they had prepared in the previous week. Additionally, I also individually mentored a high school intern in our lab over the past summer. I taught this student basic programming and command line software analysis and worked with her to analyze genomics data. She contributed to an ongoing research project, and her work will be included in an upcoming publication on which she will be a co-author. How have the results been disseminated to communities of interest?As mentioned above, high school students from underrepresented groups, including women and ethnic minorities, participated in the DNA biology and bioinformatics STEM camp. Two camps were run, the first primarily comprised of students local to Corvallis and Albany, OR. The second camp was comprised of students from an urban high school near Portland, OR. Camp participants learned cutting-edge microbiome techniques and analyses and sequenced their own plant samples. They also learned molecular biology skills and introductory computer programming. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, I will continue to analyze the gall DNA extract samples and prepare DNA barcodes for sequencing for objective 1. These samples will be sequenced and analyzed computationally. Depending on the physical location of agrobacteria in the re-analyzed woody gall samples, additional DNA extracts will be prepared using that plant tissue and sequenced as described in the initial project description. For objective 2, once the opine gene knockout constructs are prepared, I will proceed with knocking out those genes from the wild-type agrobacteria strains. The wild-type and opine-knockout strains (along with the gall-associated bacterial isolates) will be used to create synthetic gall communities on plants, which will be processed in a similar manner as those galls in objective 1. This will follow the established protocol described in the initial project description.
Impacts
What was accomplished under these goals?
The bacterial pathogen Agrobacterium tumefaciens causes crown gall disease on a diverse variety of plant host species and is a major problem in the nursery and orchard industries. It is often difficult to isolate pathogenic agrobacteria from crown galls, particularly from woody plant hosts, making fast and accurate diagnosis difficult. The goal of this project is to assess the microbial diversity and abundance of agrobacteria in crown galls from agriculture systems and in synthetic communities using a microbiome sequencing-based approach. As part of progress towards objective 1, I have collected 92 diseased gall and healthy plant tissue samples from nurseries and orchards across Oregon and the pacific northwest. These represent 14 plant host species from nurseries in 4 states and 2 countries. A preliminary sequencing run on an Illumina HiSeq was performed on DNA from these samples, however microbial DNA was not abundant in the extracted samples and the estimated microbial abundance was extremely low. I am testing multiple DNA extraction protocols from different portions of sampled galls in order to determine if extraction can be improved, or if agrobacteria is located in different portions/regions of the gall. During the course of DNA extraction, galls were surface sterilized, and the center portion of the inner gall was sampled following standard protocol of diagnostics labs. However, agrobacteria may be primarily associated with the surface of the gall on woody plants, and are not found inside the gall tissue itself. Most of the analyzed gall samples were from woody plants such as apple, raspberry, blueberry, and rose. Ongoing experiments include testing multiple regions of the gall (surface-associated, surface tissue, inner gall, etc) for pathogenic agrobacteria, particularly on woody plant hosts. If agrobacteria is found to be primarily gall surface-associated, subsequent experiments will target this tissue for pathogen abundance. Alternatively, other experiments will explore whether the gall itself is the true reservoir of pathogenic agrobacteria. Agrobacteria also induces the plant to produce uncommon nutrients (opines) that only agrobacteria can utilize. The ability to catabolize these opines is hypothesized to provide an advantage to agrobacteria over other bacteria in the gall despite the energy required to infect a plant and induce formation of a gall (the "opine concept"). In order to test this hypothesis, I will compare the abundance of agrobacteria and the overal composition of synthetic microbial communities in galls induced by agrobacteria with and without the ability to induce the production of opines. As part of progress towards objective 2, I am generating gene knockouts for two different strains of agrobacteria that induce the production of different opines. Both strains are well characterized, and induce galls that produce either the opines nopaline or octopine. Using Gibson cloning, I am generating constructs comprised of the upstream and downstream regions of the nopaline or octopine synthesis genes, with the gene sequence itself replaced with a selectable marker. These constructs will then be cloned into the wild-type agrobacteria strains and recombined into the genome to replace the original opine synthesis gene. I have also isolated and characterized 22 bacterial isolates from gall samples. These isolates were identified based on DNA barcode sequences as belonging to diverse genera and species, including Pseudomonas, Arthrobacter, Microbacterium, Curtobacterium, Bacillus, and Agrobacterium/Rhizobium. These isolates will be used to construct synthetic communities on plants with pathogenic agrobacteria or the opine-synthesis gene knockouts described above.
Publications
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