Progress 01/01/16 to 12/31/17
Outputs
Target Audience:M. Alexander shared her work with peers in a presentation in the course, "Graduate Student Research Updates", where students in Cornell Plant Pathology discuss their research and practice giving scientific talks. Manuscripts for broad dissemination of results via scientific publications are being drafted. Changes/Problems:The experimental timeline for Year 2 was delayed by a seed stock error discovered in early summer, which necessitated the repetition of all screens done earlier in the year. What opportunities for training and professional development has the project provided?Researcher M. Alexander participated in a formal mentoring program through the Boyce Thompson Institute Post-Graduate Society (PGS). M. Alexander was paired with Dr. Paul Chomet, an independent consultant with extensive prior experience in industry. Dr. Chomet's mentorship was instrumental in helping M. Alexander to evaluate her skills and career goals. Through the PGS, M. Alexander also participated in a career symposium, where she learned about non-academic career paths in science. Advisor Dr. Michelle Heck provided M. Alexander with the opportunity to attend Biotech Bootcamp, an invite-only workshop in science communication in the field of agricultural biotechnology. At the workshop, M. Alexander networked with individuals working in diverse areas of science communication and learned valuable skills for effective messaging. M. Alexander was also admitted to the Cornell Broadening Experiences in Scientific Training (BEST) Program, which provides graduate students with resources and opportunities related to non-traditional science career paths. How have the results been disseminated to communities of interest?Results have been disseminated via presentations at Cornell and the NIFA project directors' meeting (see Publications and Presentations section). Draft manuscripts for formal publication of Year 2 results are in progress. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Plant pathogenic viruses are a major limiting factor in crop production in the US and around the world. Turnip yellows virus (TuYV), Potato leafroll virus (PLRV), and others in the family Luteoviridae (collectively, luteovirids) are unusual in that they localize exclusively to the phloem - the sugar-rich portion of the plant vascular system. This restriction is believed to be an adaptation facilitating acquisition of luteovirids by aphids, which feed on phloem sap and are the sole means of luteovirid plant-to-plant transmission. The mechanism underlying phloem restriction is unknown and represents a novel and attractive target for new control strategies. For this project, proteins interacting with TuYV or PLRV were identified in Year 1 and in previous studies. One of the simplest ways to investigate the role of these interactions during virus infection is to inhibit production of the host protein. Testing of plants with genetic disruptions in candidate proteins was performed by M. Alexander in Year 2. Although preliminary screens did not find a protein that appeared important for phloem restriction, a cell wall-modifying enzyme was identified which affected susceptibility to TuYV. Plants that did not produce this protein were infected at higher rates and became symptomatic sooner than wild-type plants. Follow-up studies showed that the protein was expressed in the vasculature, where it could interact with TuYV during infection.Additionally, the function of an interaction between PLRV coat protein and a host protein involved in anti-viral defense was explored. Results suggested that luteovirids can inhibit this protein to only a limited extent without severe adverse effects. A model for regulation of host protein levels was hypothesized, and further testing is underway. A great challenge in controlling plant pathogens lies in our dearth of knowledge about them. While replication and movement of many viral human pathogens can be mapped in great detail, we don't even know the function of all ten luteovirid proteins. This study provides new information about luteovirid biology, some of which may also prove informative for other plant pathogens. Additionally, this study contributes methodological knowledge broadly applicable to plant pathogenic viruses. Objective I: Identify host-virus protein-protein interactions. Objective 1 was completed in Year 1. Objective 2: Validate and characterize host protein interactors. 1) Major activities completed/experiments conducted: Select mutant lines identified and propagated in Year 1 were screened in Year 2 for evidence of altered localization of TuYV. A pectate lyase protein found interacting with TuYV was selected for further analysis. Transgenic lines to assess the subcellular localization of the pectate lyase, and to analyze which tissues the pectate lyase is expressed in, were created, propagated, and tested. The importance of a host anti-viral defense protein found interacting with PLRV coat protein in Year 1 was assessed using virus-induced gene silencing in N. benthamiana. 2) Data collected: Tissue tropism and systemic movement were assessed visually, using a TuYV mutant which causes photobleaching in infected cells. No evidence of altered phloem tropism was found in any of the A. thaliana mutant lines screened. However, a pectate lyase mutant line was observed to have a higher infection success rate, and to exhibit photobleaching sooner, than wild-type plants. TuYV titer was not significantly impacted. To further characterize this protein, transgenic plants were developed which expressed a reporter gene under control of the pectate lyase promotor. The pectate lyase promotor was found to be active specifically in vascular tissues. Initial testing by transient expression of pectate lyase fused to a fluorescent protein in N. benthamiana suggests a primarily apoplastic (cell wall) localization, although further confirmation is required. In Year 1, an Argonaute protein (AGO1) was found to directly interact with PLRV coat protein. Argonaute proteins function in post-transcriptional gene regulation and in defense against viruses. The luteovirid silencing suppressor protein, P0, has been previously shown to function by promoting degradation of AGO1, but there are no previous reports of the coat protein's involvement. To explore the function of the AGO1-coat protein interaction, AGO1 levels were further knocked down by VIGS, using Tobacco rattle virus (TRV). Plants infected with AGO1-silencing TRV exhibited stunting and leaf distortion. However, when plants were co-infected with PLRV, they quickly died. PLRV titer was significantly higher on average in AGO1-silenced plants at twelve days post inoculation, but was also more variable than in control plants, possibly due to the patchy necrosis. 3) Summary statistics and discussion of results: Current results suggest that pectate lyase mutants are more susceptible to TuYV than wild-type plants. However, it is possible that this reflects an increased susceptibility to the inoculation method (Agrobacterium infiltration) rather than the virus itself, particularly given that pectate lyases are cell wall-modifying enzymes. Testing is underway to determine whether pectate lyase mutants are also more susceptible to TuYV when aphid-inoculated. Contribution of pectate lyase to Agrobacterium susceptibility would also be an interesting result, both in terms of bacterial plant pathogens and as a factor that could be used to increase efficacy of studies utilizing Agrobacterium as a delivery method. Activity of the pectate lyase promotor in vascular tissue supports the ability of this protein to interact with phloem-restricted TuYV. Plant pectate lyases are typically annotated as expressing primarily in floral organs, so this result may indicate that this particular homolog has a unique function. The finding of an interaction between PLRV coat protein and AGO1 was surprising, as multiple studies from other labs on luteovirid P0 and AGO1 never reported involvement of a structural protein. The simplest explanation for this interaction is that the coat protein somehow assists in P0-mediated AGO1 degradation, for example by altering subcellular localization of AGO1. However, the finding of additive effects of P0 and VIGS silencing of AGO1, leading to plant death, strongly suggested that it is not advantageous for luteovirids to suppress AGO1 below certain levels. In this case, it is possible that the coat protein, which should be abundant in late stages of replication when silencing suppression may be less critical, acts to protect AGO1 from P0. Work is currently underway to test this hypothesis in vivo and in silico, by computationally modeling the AGO1-coat protein interaction. 4) Key outcomes or other accomplishments realized: Graduate student M. Alexander received training and experience in generation and testing of transgenic plants, statistical analysis of complex data sets, confocal microscopy, beta-glucaronidase activity staining, and experimental design. She also received mentorship and participated in professional development activities which greatly contributed to her knowledge of possible future career paths, and what she can do to improve her job prospects.
Publications
- Type:
Other
Status:
Other
Year Published:
2017
Citation:
Poster: M. Alexander, J.P. Mohr, J. D. Chavez, V. Ziegler-Graff, V. Brault, J. Bruce, and M. (Cilia) Heck. Turnip yellows virus: Connecting structural biology to function. NIFA Fellows Project Director Meeting. August 2017, Washington, D.C.
- Type:
Other
Status:
Other
Year Published:
2017
Citation:
Presentation: M. Alexander, J.P. Mohr, J.D. Chavez, S.L. DeBlasio, V. Ziegler-Graff, V. Brault, J. Bruce, and M. (Cilia) Heck. Probing the structural biology of viruses in the Luteoviridae. Cornell PLPPM-6820 "Graduate Student Research Updates". January 2017, Ithaca, NY.
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:Over the summer of 2016, a USDA ARS Wallace Carver Student worked as an intern on this project. Having no prior research experience, the female undergraduate student received training not only in the techniques relevant to her project, but also in experimental design, scientific problem solving, literature review, and method development. During this time, the student was mentored by M. Alexander and Dr. Cilia, and was exposed to a range of scientific projects both within and outside of Dr. Cilia's laboratory. During the reporting period, M. Alexander participated in Virology Group, a series of semi-formal research presentations given by and to scientists at Cornell University studying plant pathogenic viruses. Virology Group serves as a medium for exchange of knowledge between scientists and a forum for feedback and suggestions. Changes/Problems:Due to technical difficulties with the purification of biologically active Cereal yellow dwarf virus (CYDV) virions from N. benthamiana, we will not extend our PIR cross-linking experiments to this virus species as previously planned. Results from this and previous studies have already provided many excellent candidates for functional characterization in Objective 2. Additionally, the development of the A. thaliana high-throughput screening protocol will permit testing of more candidates than previously anticipated; thus, cross-species comparisons to prioritize candidates are no longer necessary. What opportunities for training and professional development has the project provided?In the summer of 2016, undergraduate student Laura Tucker from Iowa State University worked with M. Alexander on this project as part of a USDA Wallace-Carver Fellowship. Although L. Tucker had no prior research experience, she was able to independently carry out, analyze, and troubleshoot A. thaliana screens for Objective 2 by the end of her internship. L. Tucker also attended a seminar series for summer interns at the Boyce Thompson Institute, which exposed her to other cutting-edge research programs in plant science and permitted networking with students and professors. L. Tucker is now working in a plant virus research lab at Iowa State University and hopes to pursue a career in science. This project also provided M. Alexander with numerous professional development and training opportunities. In the summer of 2016, M. Alexander attended the International Plant Virus Epidemiology (IPVE) symposium, where she presented a poster showcasing her research and networked with eminent plant virologists from around the world. Following the conference, M. Alexander traveled to the laboratory of Dr. Veronique Brault to receive training in purification of TuYV virions and to prepare samples for PIR analysis in Objective 1. In the fall of 2016, M. Alexander participated in a biological sample preparation for electron microscopy course, to be used in Objective 2 as an additional way to assess virion localization in plant tissues. M. Alexander presented her work on this grant at the Northeast Division meeting of the American Phytopathological Society. M. Alexander has also been involved in professional development activities with the Boyce Thompson Institute Post-Graduate Society (PGS), including an alternative careers in science symposium and a new mentorship program. M. Alexander served as the 2016 Treasurer and Appropriations Committee Chair for the Cornell Graduate and Professional Student Association, where she gained valuable experience in leadership, budget management, problem solving, and communication. How have the results been disseminated to communities of interest?Results have been disseminated via submission for publication in a scientific journal, presentation at Cornell Virology Group meetings, and presentation at conferences (see Products and Target Audiences sections). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Protein interaction discovery experiments are complete. In addition to providing candidates for Objective 2, we plan to use these data to model the interaction between TuYV/PLRV and host proteins. This information may be used to design mutants or selective inhibitors that will disrupt host-virus interactions without impacting the native function of the host proteins involved. Objective 2: Over 40 mutant A. thaliana lines for proteins of interest will be subjected to high-throughput screening to discover host proteins involved in viral movement, replication, phloem restriction, and/or defense. The most promising candidates from initial screens will be subjected to further analysis, including genetic confirmation, assessment of virus localization by multiple techniques, localization of the host protein of interest, and initial tests for potential applications in disease management. Some candidates, particularly those for which there are no A. thaliana mutants available, will also be screened by virus-induced gene silencing (VIGS) in Nicotiana benthamiana to study host protein interactions with PLRV, in collaborative work with members of the Cilia lab funded by USDA-ARS and the National Science Foundation.
Impacts
What was accomplished under these goals?
Impact Statement: Viruses in the family Luteoviridae, referred to hereafter as luteovirids, are serious pests of food and fiber crops worldwide. Luteovirids are transmitted exclusively by sap-feeding insects, which inject the virions directly into host plants' vascular systems. The virus then replicates and spreads into other leaves, often causing significant yield loss. Unlike most other plant pathogens, luteovirids are found exclusively in the phloem - the portion of the vascular system used for transport of carbohydrates and other nutrients. The mechanism which prevents luteovirid diffusion out of the phloem and into other plant tissues is unknown. Studies suggest that phloem restriction is required for aphid transmission; thus, the protein or proteins responsible represent attractive targets for disease control. To examine the mechanism underlying phloem restriction, researcher Mariko Alexander, supervisor Dr. Michelle Cilia, and collaborator Dr. James Bruce used mass spectrometry and protein cross-linking to identify plant proteins directly interacting with proteins from Turnip yellows virus (TuYV), a luteovirid pathogen of importance in rapeseed production, and with Potato leafroll virus (PLRV), a luteovirid pathogen of potato. Novel direct interactions were discovered between PLRV or TuYV coat proteins and six host proteins, including one protein known to be important in viral defense and hypothesized to contribute to phloem restriction. A protocol for screening of these and other candidate host proteins as targets for new disease resistance strategies was optimized with the assistance of Laura Tucker, an undergraduate summer fellow who was part of the USDA-ARS Wallace Carver Scholars program. In addition to providing new avenues for disease management of luteovirids, results pertaining to phloem restriction may also be applicable to other plant pathogens that specifically colonize plant vasculature. Objective I: Identify host-virus protein-protein interactions. Major activities/experiments completed: Purification of virions from infected plants, a necessary first step for the cross-linking technology employed in this project, is a complex and delicate process. To receive training in purification of TuYV, M. Alexander traveled to the laboratory of collaborator Dr. Veronique Brault. Samples of virions purified during the training period were shipped back to the United States for analysis. In an expansion of previous work led by Dr. Stacy DeBlasio, M. Alexander also purified PLRV virions. Protein Interaction Reporter (PIR) technology, a protein cross-linking strategy, was applied to both TuYV and PLRV samples. Analysis of PIR data was performed by M. Alexander in conjunction with colleagues in the laboratory of collaborator Dr. James Bruce. Data collected: Direct interactions were found between TuYV virions and three different host proteins. Four additional interactions were also discovered between or within TuYV coat protein, or between coat protein and a putative viral protease. PLRV virions were found interacting with five different host proteins, including one known to be important for viral defense. Summary and Discussion of Results: Prior to this study, PIR technology had only been successfully applied to PLRV. Repeating these experiments with some modifications permitted identification of three new host-virus protein-protein interactions, and provided useful data for future optimization of the cross-linking protocol. Application of PIR technology to TuYV represents the first successful extension of this technology to a closely related species, and will help our group and others to optimize and design future experiments. The three host proteins found interacting with TuYV coat protein have never before been shown to directly interact with luteovirids and likely all represent interesting and important aspects of luteovirid biology, possibly including phloem restriction. Additionally, two of the discovered interactions between viral proteins support a new model for truncation of the read-through protein, the minor component of the virus capsid. Key Outcomes: Six new direct interactions were discovered between TuYV or PLRV coat protein and plant proteins. Several of these proteins were also found in complex with PLRV virions in previous studies by colleagues in Dr. Cilia's laboratory. These represent promising targets for understanding luteovirid biology and developing new disease control strategies. Objective 2: Validate and characterize host protein interactors. Major activities/experiments completed: Undergraduate intern Laura Tucker worked with M. Alexander to develop a protocol for high-throughput screening of host protein candidates for importance in phloem restriction, viral replication, systemic movement, and host defense. This protocol utilizes an extensive collection of mutant lines uniquely available for Arabidopsis thaliana, and a mutant form of TuYV which causes dramatic photobleaching in infected cells. A list of candidate host proteins was compiled by Dr. Stacy DeBlasio and M. Alexander, based on host-virus interaction data from this and previous studies. A. thaliana mutant lines for ~40 of these candidates were obtained and propagated by M. Alexander. Data collected: Final optimization of the screening protocol was completed in late 2016. This protocol includes changes to plant age at inoculation, concentration of inoculum, timing of post-inoculation sampling, number of plants per treatment, and plant growth conditions. Additionally, it was confirmed that TuYV localization and titer (concentration) can be further assessed by immunolocalization and enzyme-linked immunosorbant assay (ELISA). Summary and Discussion of Results: Protocol optimizations have more than doubled infection efficiency compared to initial tests in early 2016, and have significantly improved reproducibility and ease of assessment of virus localization via photobleaching. Key Outcomes: Over 40 A. thaliana mutant lines for host proteins identified in this and other interactions studies are ready for screening in 2017.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
M. Alexander, J. P. Mohr, S. L. DeBlasio, J. D. Chavez, V. Ziegler-Graff, V. Brault, J. E. Bruce, and M. Cilia. Insights in luteovirid biology guided by chemical cross-linking and high resolution mass spectrometry. Virus Research, accepted March 2017.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Poster: M. Alexander, S. L. DeBlasio, J. D. Chavez, V. Ziegler-Graff, V. Brault, J. Bruce, S. M. Gray, and M. Cilia. Exploring host-luteovirid interactions: A structural proteomic approach. International Plant Virus Epidemiology Symposium. June 2016, Avignon, France.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Presentation: M. Alexander, S. L. DeBlasio, J. D. Chavez, V. Ziegler-Graff, V. Brault, J. Bruce, S. M. Gray, and M. Cilia. Structural biology of viruses in the Luteoviridae. Northeast Division of the American Phytopathological Society Meeting. October 2016, Ithaca, NY.
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