Progress 06/15/20 to 06/14/22
Outputs
Target Audience: The objective of this project wasto determine the genetic factors required forPseudomonas syringae, a model bacterial plant pathogen, to attach to host cell surfaces during infection. The primary target audience of this project are researchers within the field of molecular plant-pathogen interactions. BecauseP. syringaeinteraction with plant cell surfaces is likely a critical stage of host infection, this research may also appeal to a broader range of researchers and agricultural scientists involved in managing and combating bacterial plant disease. I (Megan O'Malley) and my PhD advisor Jeff Anderson submitted an original research manuscript to PeerJ in September 2022 detailingP. syingaesurface attachment in response to plant chemical signals, in collaboration with Dr. Scott Peck at the University of Missouri. This manuscript is currently under review awaiting publication. During the projectfunding period, I alsoco-authored acomprehensive review on the regulation of Pseudomonas syringae virulence by host plant signals with my PhD advisor Dr. Jeff Anderson. Through research conductedduring the funding period, we have identified various plant metabolites that induceP. syringaesurface attachment. Many of these attachment-inducing metabolites have also been shown to influenceP. syringaevirulence dynamics in the host environment. By reviewing the regulation of virulence by these metabolic signals, we established necessary context for elucidating the role(s) of surface attachment inP. syringaeinfection in future publications. This work was disseminated to the public through online publication in Microorganisms, an open-access peer-reviewed scientific journal, in June 2021. A thirdmanuscript focused on our efforts to characterize the genetic basis of P. syringae surface attachment, through both targeted genetic and high-throughput screening (RB-TnSeq) approaches, is currently in preparation and will likely be submitted for publication in early 2023. Changes/Problems: A major change during the duration of the funding period was the 2020 research shutdown due to COVID-19. One of the originally stated goals of the project was to gain experiencein high-throughput sequencing data analysis. While efforts were made to conduct the data analysis while working remotely, the bioinformatics training through collaboration with other labs at OSU was unable to be carried out as originally outlined in the project goals. This setback significantly shifted the timeline of the RB-TnSeq project.To address this issue, we collaborated with the Center for Quantitative Life Sciences (formerly the Center for Genome Research and Biocomputing) at OSU for assistance in analysis of the RB-TnSeq data. Analysis of this data is currently underway. We encountered a significant technical setback in completing the initial genomic DNA sequencing of our RB-TnSeq mutant library. In order to successfully map barcoded transposon insertion sites throughout the P. syringae genome, we followed a published Illumina library preparation protocol to enrich for transposon insertion sites prior to sequencing. After sequencing the enriched library on an Illumina MiSeq platform, we found that the existing protocol was inefficient in enrichment for transposon insertion sites. As a result, we were unable to map an adequate number of the total barcoded transposon insertion sites within the mutant library. To troubleshoot the library preparation, we established personal communication with laboratories at University of California-Davis and University of California-Berkeley and obtained an updated protocol with improved transposon enrichment efficiency. After performing the updated library preparation, we re-sequenced the library on an Illumina HiSeq platform with a greater read depth in order to map a greater proportion of the mutant library. Data analysis from this re-sequencing effort is currently underway. What opportunities for training and professional development has the project provided?As project director, I (Megan O'Malley) have gained experience in both primary scientific investigation as well as overall project management. Most significantly, I have gained proficiency in conducting a high-throughput genetic screen using RB-TnSeq, a cutting-edge technique in functional genomics. Through our efforts to generate and characterize a P. syringae mutant library, I have cultivated important skills in data management and bioinformatics pipeline analysis of raw sequencing data. Through my role as project director, I have additionally gained experience in budgetary management and professional communication with project administrative faculty at Oregon State University and NIFA. During the funding period, I successfully defended my PhD dissertation and graduated from Oregon State University. Prior to graduation, I accepted a position as a postdoctoral scholar at the University of Washington. In my postdoctoral research, I am currently assessing the role of cell envelope stress in surface sensing and surface attachment in Pseudomonas aeruginosa, a research interest that directly developed from my time as a NIFA predoctoral fellow. Receipt of this funding was instrumental to my growth as a scientist and researcher, and furthered my career goals towards being a principal investigator in the microbiology field. How have the results been disseminated to communities of interest? The results of this study will be primarily disseminated to the research community through publication in peer-reviewed scientific journals. A review on the overall regulation of P. syringae virulence by plant host signals was published on Microorganisms, an open-access peer-reviewed journal, in June 2021. An original research manuscript detailing P. syringae surface attachment in response to plant chemical signals was submitted to PeerJ and is currently under review. A third manuscript detailing our efforts to establish the genetic basis of P. syringae surface attachment, including RB-TnSeq and candidate-based genetic approaches, is currently in preparation for submission to a peer-reviewed journal in 2023. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In this project, one of our primary goals is to survey for factors of P. syringae surface attachment using a genome-wide forward genetics screen. We generated a library containing an estimated ~1.2. million uniquely barcoded transposon insertion mutants. Pooled genomic DNA from this mutant library was sequenced to map unique DNA barcodes with transposon insertion sites in the P. syringae genome. Despite technical setbacks in mapping our transposon library (see future directions and Changes & Problems sections for further information), we are currently analyzing data from the sequenced library. Once the unique DNA barcodes are indexed with genomic insertion sites, barcode sequencing can be utilized to estimate fitness values for individual mutants within the P. syringae library. We have sequenced barcodes from the P. syringae mutant library after it was subjected to a battery of competitive surface attachment assays. Once library mapping is complete, we will use this barcode sequencing data to identify genes required for P. syringae surface attachment. We additionally conducted a candidate-based survey of putative attachment genes in P. syringae. In this approach, we selected a panel of six candidate attachment genes from various functional categories and generated single gene deletion mutants. These mutants were assessed for surface attachment and virulence on host plants. We identified multiple P. syringae mutants with significantly altered surface attachment, including mutants displaying both attachment-deficient and hyper-attachment phenotypes. Most significantly, we identified a gene involved in regulation of the type IV pilus, an extracellular appendage involved in surface sensing and adhesion, as required for both P. syringae surface attachment and virulence in host plants. These results suggest a role for the type IV pilus in P. syringae surface attachment, and underscore the potentially critical role of surface attachment in overall P. syringae pathogenesis. These results will be described in a manuscript currently in preparation for submission to a peer-reviewed scientific journal in 2023. In June 2021, we additionally published a comprehensive review on the regulation of P. syringae virulence within the host environment in Microorganisms, open-access, peer-reviewed online journal; an additional original research manuscript was submitted to PeerJ and is currently under review.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
OMalley, M.R.; Anderson, J.C. Regulation of the Pseudomonas syringae Type III Secretion System by Host Environment Signals. Microorganisms 2021, 9, 1227. https://doi.org/10.3390/microorganisms9061227
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
OMalley, M.R.; Kpenu, E.; Peck, S.C.; Anderson, J.C. Plant-exuded chemical signals induce surface attachment of the bacterial pathogen Pseudomonas syringae. PeerJ 2022, under review.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2021
Citation:
O'Malley, M.R. Role of GacSA Two-component System and Bacterial Surface Attachment in Pseudomonas syringae Virulence. PhD Dissertation. Oregon State University, Corvallis OR USA.
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Progress 06/15/20 to 06/14/21
Outputs
Target Audience: The objective of this project is to determine the genetic factors required for Pseudomonas syringae, a model bacterial plant pathogen, to attach to host cell surfaces during infection. The primary target audience of this project are researchers within the field of molecular plant-pathogen interactions. Because P. syringae interaction with plant cell surfaces is likely a critical stage of host infection, this research may also appeal to a broader range of researchers and agricultural scientists involved in managing and combating bacterial plant disease. During the project funding period, I (Megan O'Malley) and my PhD advisor and primary mentor Dr. Jeff Anderson co-authored a comprehensive review on the regulation of Pseudomonas syringae virulence by host plant signals. Through research conducted both preceding and during the funding period, we have identified various plant metabolites that induce P. syringae surface attachment. Many of these attachment-inducing metabolites have also been shown to influence P. syringae virulence dynamics in the host environment. By reviewing the regulation of virulence by these metabolic signals, we established necessary context for elucidating the role(s) of surface attachment in P. syringae infection in future publications. This work was disseminated to the public through online publication in Microorganisms, an open-access peer-reviewed scientific journal. Two additional manuscripts containing original research related to the project goals are currently in preparation for submission to peer-reviewed scientific journals. I additionally delivered a 50-minute talk to the students of BOT 332: Molecular Techniques in Plant Science, an upper-level undergraduate laboratory course at Oregon State University taught by Dr. Valerian Dolja. In this presentation, I detailed our laboratory's efforts to survey P. syringae genes required for surface attachment and virulence using random barcoded transposon insertion-site sequencing (RB-TnSeq), one of the central stated goals of the project. This outreach effort provided education on both molecular host-pathogen interactions and technological innovation in functional genomics to undergraduate students in plant science. Changes/Problems: Despite a research shutdown due to COVID-19 during the 2020 funding period, our overall progress towards completion of the project goals remains on track. We encountered a significant technical setback in completing the initial genomic DNA sequencing of our RB-TnSeq mutant library. In order to successfully map barcoded transposon insertion sites throughout the P. syringae genome, we followed a published Illumina library preparation protocol to enrich for transposon insertion sites prior to sequencing. After sequencing the enriched library on an Illumina MiSeq platform, we found that the existing protocol was inefficient in enrichment for transposon insertion sites. As a result, we were unable to map an adequate number of the total barcoded transposon insertion sites within the mutant library. To troubleshoot the library preparation, we established personal communication with laboratories at University of California-Davis and University of California-Berkeley and obtained an updated protocol with improved transposon enrichment efficiency. After performing the updated library preparation, we re-sequenced the library on an Illumina HiSeq platform with a greater read depth in order to map a greater proportion of the mutant library. Data analysis from this re-sequencing effort is currently underway. While troubleshooting issues with the library enrichment, we additionally conducted a candidate-based survey of potential genetic regulators of P. syringae surface attachment. Originally, we planned to limit this approach to potential attachment factors that were unlikely to be identified through competitive fitness assays, i.e. bacterial secreted products such as extracellular polymers or diffusible signals that may act as "public goods" within a pooled sample of bacterial mutants. Given our technical issues and delay with successfully mapping the RB-TnSeq mutant library, we expanded this approach to include candidate genes with predicted roles in attachment regardless of whether these genes may be detectable through our mutant screen. As a result, we identified genetic regulators across multiple functional categories that were required for full P. syringae surface attachment. In the event that we are unable to successfully map the RB-TnSeq library, this approach thus provides a limited survey of the overall biological processes that impact P. syringae attachment. What opportunities for training and professional development has the project provided? As project director, I (Megan O'Malley) have gained experience in both primary scientific investigation as well as overall project management. Most significantly, I have gained proficiency in conducting a high-throughput genetic screen using RB-TnSeq, a cutting-edge technique in functional genomics. Through our efforts to generate and characterize a P. syringae mutant library, I have cultivated important skills in data management and bioinformatics pipeline analysis of raw sequencing data. Through my role as project director, I have additionally gained experience in budgetary management and professional communication with project administrative faculty at Oregon State University and NIFA. During the funding period, I additionally presented a seminar to a prospective postdoctoral lab detailing the current goals and results of the project. I was subsequently offered and accepted a position as a postdoctoral research fellow at University of Washington following the upcoming defense of my doctoral dissertation. This is a significant milestone in my career as a researcher and aspiring principal investigator. How have the results been disseminated to communities of interest? The results of this study will be primarily disseminated to the research community through publication in peer-reviewed scientific journals. A review on the overall regulation of P. syringae virulence by plant host signals was published on Microorganisms, an open-access peer-reviewed journal, in June 2021. Two manuscripts with original research related to the project goals are currently in preparation for submission to peer-reviewed scientific journals. The first of these manuscripts, in preparation for submission to PeerJ, details the phenomenon of P. syringae surface attachment in response to virulence-inducing plant metabolites. The second manuscript, to be submitted for publication in the spring of 2022, focuses on the co-regulation of surface attachment with other key virulence factors, as well as the genetic basis of P. syringae surface attachment. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period, we intend to map the P. syringae mutant library in order to associate unique DNA barcodes with transposon insertion sites throughout the P. syringae genome. In order to achieve this goal, we have applied an updated protocol for mutant library enrichment and upgraded our sequencing efforts to the Illumina HiSeq, a platform with increased read depth. The data from the library mapping will be used to quantify mutant fitness values based on barcode abundance. DNA barcodes have already been sequenced from competitive surface attachment assays using the P. syringae mutant library, so barcode abundance data is already available for analysis once the library mapping is complete. In addition to completion of this genetic screen, we plan to complete and publish two manuscripts describing this research in peer-reviewed scientific journals before the end of the funding period.
Impacts
What was accomplished under these goals?
In this project, one of our primary goals is to survey for factors of P. syringae surface attachment using a genome-wide forward genetics screen. We generated a library containing an estimated ~1.2. million uniquely barcoded transposon insertion mutants. Pooled genomic DNA from this mutant library was sequenced to map unique DNA barcodes with transposon insertion sites in the P. syringae genome. Despite technical setbacks in mapping our transposon library (see future directions and Changes & Problems sections for further information), we are currently analyzing data from the sequenced library. Once the unique DNA barcodes are indexed with genomic insertion sites, barcode sequencing can be utilized to estimate fitness values for individual mutants within the P. syringae library. We have sequenced barcodes from the P. syringae mutant library after it was subjected to a battery of competitive surface attachment assays. Once library mapping is complete, we will use this barcode sequencing data to identify genes required for P. syringae surface attachment. We additionally conducted a candidate-based survey of putative attachment genes in P. syringae. In this approach, we selected a panel of six candidate attachment genes from various functional categories and generated single gene deletion mutants. These mutants were assessed for surface attachment and virulence on host plants. We identified multiple P. syringae mutants with significantly altered surface attachment, including mutants displaying both attachment-deficient and hyper-attachment phenotypes. Most significantly, we identified a gene involved in regulation of the type IV pilus, an extracellular appendage involved in surface sensing and adhesion, as required for both P. syringae surface attachment and virulence in host plants. These results suggest a role for the type IV pilus in P. syringae surface attachment, and underscore the potentially critical role of surface attachment in overall P. syringae pathogenesis. Data analysis from this candidate-based approach is currently in progress. In June 2021, we additionally published a comprehensive review on the regulation of P. syringae virulence within the host environment in an open-access, peer-reviewed online journal.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
OMalley, M.R.; Anderson, J.C. Regulation of the Pseudomonas syringae Type III Secretion System by Host Environment Signals. Microorganisms 2021, 9, 1227. https://doi.org/10.3390/microorganisms9061227
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