Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The target audience during this reporting period was fire blight researchers, apple and pear growers, researchers working on citrus greening disease, citrus breeders, and tree fruit breeders generally. Changes/Problems:Work and laboratory access restrictions made necessary by the Covid-19 pandemic slowed some of our experimental progress. However, critical experiments were resumed after a few months of shutdown, and progress towards major objectives has been continued throughout most of the reporting period. What opportunities for training and professional development has the project provided?This project has provided training opportunities for one M.S. degree student (Chad Vosburg) and one Ph.D. degree student (Jeremy Held). The M.S. student works on the most applied aspects of the citrus greening and fire blight projects, including inoculations of grapefruit trees and testing the activity of potential anti-fire blight protein activity. His work has been done both at Penn State and at the United States Horticultural Research Laboratory in Fort Pierce, FL, giving him training experiences in several locations with personnel of various specialties. Jeremy Held has been working on fire blight disease functionalities of E. amylovora, building from work of the previous Ph.D. student. This project has given Jeremy Held experience with reverse genetics, gene cloning, and gene expression in plants and in bacteria. How have the results been disseminated to communities of interest?The main mode of dissemination was one publication published about precocious blooming of our grapefruit germplasm. In addition, the results of our lab work were highlighted by the American Society for Microbiology podcast "This Week in Microbiology." What do you plan to do during the next reporting period to accomplish the goals?Objective #1: We will continue to analyze Erwinia amylovora genetics related to virulence, in particular focusing on the secreted protein HrpN and its potential novel roles in the disease process. We will also prepare auxotrophic mutants of Erwinia amylovora for blossom blight tests in a greenhouse test setting. Objective #2: Dr. McNellis will continue to help mentor the graduate student in a collaborator's lab who is studying tomato resistance genetics to Clavibacter michiganensis subsp. michiganensis. Objective #3: We will quantify and measure the level of HLB resistance, if any, of our grapefruit lines in Florida. Objective #4: We will probably not pursue the Erwinia tracheiphila project at this time in the interest of making substantial progress on fire blight management tool development.
Impacts
What was accomplished under these goals?
Objective #1: Continue analysis of Erwinia amylovora virulence genetics. This work has continued and revealed a novel extracellular polysaccharide production and transport system in Erwinia amylovora. These data were submitted as a revised manuscript and the manuscript was accepted and is in press in Journal of Bacteriology. In the process of doing this research, it became clear that a protein component of Erwinia amylovora has potential to be used as a fire blight disease management tool. An M.S. degree student has begun to assess this protein for utility and functionality, and we expect additional findings about the protein in the next reporting period. Objective # 2: Testing tomato plants potentially resistant to bacterial canker caused by Clavibacter michiganensis subsp. michiganensis. My laboratory is no longer working on this question in the interest of focusing on fire blight disease progress areas with greater potential. However, a Ph.D. student in a collaborator's lab at Penn State has begun working on this project, and Dr. McNellis is assisting and advising this student, who will be mapping resistance genes to bacterial canker disease in a tomato breeding population. Objective #3: Testing citrus germplasm for resistance to citrus greening (Huanglongbing, or HLB). Potentially HLB-resistant grapefruit germplasm is being tested for HLB resistance using three test methods: graft-inoculation; psyllid insect vectored inoculation; and transplant to the field for natural inoculation. The M.S. student working on this project has set up all of these tests and also propagated sufficient trees for repeated tests for confirmation. Inoculated trees have begun to show symptoms and will be evaluated in the next reporting period. In addition, we found that our potentially HLB-resistant grapefruit trees have a precocious blooming phenotype, which has been much sought after by citrus breeders. We prepared and submitted a manuscript describing these results, and it is now published. Objective #4: Analyze genetic information available for Erwinia tracheiphila and perform data mining and comparative genomics to understand virulence of this pathogen. Nothing to report for this objective due to focusing primarily on the citrus greening and fire blight projects.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Sinn J. P., Held J. B., Vosburg C., Klee S. M., Orbovic V., Taylor E., Gottwald T., Stover E., Moore G. A., and McNellis T. W. Flowering locus T chimeric protein induces floral precocity in edible citrus. Plant Biotechnology Journal, in press. DOI: 10.1111/PBI.13463
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience of this project during the present reporting period was apple and pear growers and grower representatives, basic researchers on fire blight and bacterial pathogenesis in plants, and citrus growers and citrus commodity representatives. Changes/Problems:We may or may not be able to pursue Goal #2 effectively due to budget limitations, so this goal is not as heavily emphasized in the plans for the upcoming reporting period. Goal #4 has become focused on obtaining information from comparisons of Erwinia tracheiphila virulence proteins with Erwinia amylovora virulence proteins, since this is more directly related to our current funding from apple grower groups. What opportunities for training and professional development has the project provided?This project has provided the opportunity to train two Ph.D. students and one M.S. student. The first Ph.D. student, Dr. Sara Klee, graduated in August, 2019, and won several awards for her dissertation, including the Penn State Alumni Association Dissertation Award and the Penn State College of Agricultural Sciences Outstanding Dissertation Award. Dr. Klee is first author on all three publications from the present reporting period. A new Ph.D. student, Mr. Jeremy Held, did a lab rotation and subsequently decided to stay for his Ph.D. work in May, 2019. He has been working on Erwinia amylovora interactions with non-host plants and passed his candidacy exam in July, 2019. New M.S. degree student Mr. Chad Vosburg was recruited in the spring of 2019 and began work on the citrus greening project, including work at the United States Horticultural Research Lab in Fort Pierce, FL, in June, 2019. His project is a combination of citrus greening disease resistance research in grapefruit and development of applications for Erwinia amylovora virulence genetics research for fire blight disease management and disease control tool development. In addition, an undergraduate began research in August, 2019, studying mechanisms of Erwinia amylovora interactions with non-host plants, including cell death defense elicitation and linking this response to genomic signatures of the bacterium. How have the results been disseminated to communities of interest?Dr. McNellis gave one presentation to the State Horticultural Society of Pennsylvania (February 21, 2019), four quarterly reports to the Citrus Research and Development Foundation, a guest lecture at the Juniata College American Society for Microbiology undergraduate club (September 19, 2019), presented a talk at the Second International Symposium on Fire Blight in Traverse City, Michigan (June 17-21, 2019), and presented a poster at the American Society for Microbiology meeting in San Francisco, CA (June 21 - 24, 2019). What do you plan to do during the next reporting period to accomplish the goals?Goal 1 - We will continue analysis of novel Erwinia amylovora genes involved in the fire blight disease development process, including new genes involved in polysaccharide production and gene regulation. One manuscript has been submitted at the end of the present reporting period and is still under review. Another manuscript is in preparation and we plan to submit it during the next reporting period. Some of this work will be continued by the new graduate students and undergraduate researcher. An NSF grant proposal to support the fire blight genetics research will be submitted. Goal 2 - As time permits, and depending on finding an undergraduate interested in working on the tomato project, we aim to create a disease rating scale for tomato canker caused by Clavibacter michiganensis subsp. michiganenesis. We will then do a partial test of some of the recombinant inbred tomato lines to determine if resistance to Clavibacter michiganensis subsp. michiganensis is segregating in that population, and whether we can reliably repeat the disease resistance level determinations in consecutive tests. Goal 3 - The multiplication of the HLB bacterium 'Candidatus Liberibacter asiaticus' in our grapefruit tree germplasm representative trees will be determined by PCR in one or more HLB infection tests transmitted by the Asian Citrus Psyllid vector (Diaphorina citrii). In addition, potentially HLB-resistant germplasm representative trees will be established in an outdoor grove in Florida and monitored for HLB disease incidence and bacterial colonization of the trees. Goal 4 - Amino acid differences between key proteins involved in bacterial wilt disease development caused by Erwinia tracheiphila and fire blight caused by Erwinia amylovora will be analyzed experimentally for impacts on host and non-host responses, focusing mainly on cell death induced in non-host plants.
Impacts
What was accomplished under these goals?
Objective 1 - Continue analysis of Erwinia amylovora virulence genetics. We published two peer-reviewed journal papers and one peer-reviewed book chapter during the reporting period that made our results with fire blight genetics available to the broader research community. These results include information about how protein translation is regulated in the pathogen cells during the infection process, how certain avirulent mutants of Erwinia amylovora have potential as biocontrol or biopesticide agents, and helped to establish immature apple fruits as a model system for fire blight infections. In addition, we began to examine fire blight in some non-host plants to better understand non-host responses to these bacteria and mutant forms of the bacteria using leaf infiltration and cell death responses measurements. Objective 2 - Testing tomato plants potentially resistant to bacterial canker caused by Clavibacter michiganensis subsp. michiganensis. Nothing to report for this objective. Objective 3 - Testing citrus germplasm for resistance to citrus greening (Huanglongbing, or HLB). We have propagated sufficient plants for multiple HLB resistance tests in a greenhouse setting. A M.S. degree student was recruited to perform work on the project, and he began work in June, 2019. Objective 4 - Analyze genetic information available for Erwinia tracheiphila and perform data mining and comparative genomics to understand virulence of this pathogen. We have slightly changed the emphasis here to doing comparisons of Erwinia tracheiphila to Erwinia amylovora, since these two strains are closely related. Similarities between proteins encoded in the Erwinia amylovora and Erwinia tracheiphia genomes were compared to reveal potential similar and divergent functions related to interaction with host plants and non-host plants as well (melon compared to apple).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Klee SM, Sinn JP, Finley M, Allman EL, Smith PB, Aimufua O, Sitther V, Lehman BL, Krawczyk T, Peter KA, and McNellis TW. Erwinia amylovora auxotrophic mutant exometabolomics and virulence on apples. Applied and Environmental Microbiology 85:e00935-19
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Klee SM, Sinn JP, Holmes AC, Lehman BL, Krawczyk T, Peter KA, and McNellis TW. Extragenic suppression of elongation factor P gene mutant phenotypes in Erwinia amylovora. Journal of Bacteriology 201:e00722-18
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
Klee SM, Sinn JP, and McNellis TW. The apple fruitlet model system for fire blight. Methods in Molecular Biology 1991:187-198
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Progress 07/01/18 to 09/30/18
Outputs
Target Audience:The main target audience of this project is researchers in the area of plant-microbe interactions, microbial genetics of virulence, and plant genetics of disease resistance and microbial recognition. In addition, the project is targeted to members of the tree fruit and tomato fruit production industry, particularly those with interest in bacterial diseases and resistance to bacterial diseases. These interested parties include apple and tomato producers in Pennsylvania and beyond, and citrus producers in Florida, California, and internationally. Changes/Problems:There have been no changes. Work is proceeding on schedule and as planned. What opportunities for training and professional development has the project provided?One undergraduate continues her work on the project. Although she had a summer job elsewhere, she returned to the lab in August to continue her work on Erwinia amylovora genetics of virulence, focusing on the metabolomics of E. amylovora interactions with their environment in host and non-host situations. A Ph.D. student continued her work under funding from a USDA Predoctoral Fellowship to analyze the genetics of E. amylovora virulence as related to metabolic interactions within and outside of the bacterial cell, analysis of protein translation and translocation alterations during the disease process, analysis of exopolysaccharide and lipopolysaccharide patterns of expression and production in the plant using labeled promoter reporters. How have the results been disseminated to communities of interest?Some of the results of our work were published or accepted for publication during the reporting period in the form of one journal article and one book chapter. This reporting period was very short, so there were relatively few opportunities to disseminate the results to communities of interest. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 - We will continue our analysis of Erwinia amylovora virulence genetics, particularly analyzing the role of the exopolysaccharide amylovoran and lipopolysaccharides in the virulence of E. amylovora. We will be doing this by analyzing several mutants, including those in the amylovoran synthesis operon and related genes, to provide more detail on the structure of amylovoran polysaccharide, how it is produced, where it is located in the E. amylovora cell, both during, prior to, and following the infection of various tissues, including flowers, shoots, and fruits. We will analyze the gene expression patterns controlled by the transcription factor RpoN and how these regulated genes are controlled during the disease and disease resistance processes in both host and non-host plants. In addition, we will assess one or more potential biocontrol bacterial strains that we have isolated from Pennsylvania for efficacy against fire blight, and for inhibition of E. amylovora growth, both in culture and inside infected plants, for the potential for systemic antagonists for the disease. Objective 2 - We plan to resume testing of potentially Clavibacter michiganensis subsp. michiganensis resistant tomato germplasm in the spring semester of 2019 and/or summer of 2019, if and undergraduate interested in the project can be identified. This will include the development of a disease rating scale for the symptoms in tomato as well as the infection of the candidate plants under several conditions (growth chamber, greenhouse, and field) in order to determine the durability and replicability of the disease resistance ratings, making sure that disease resistance differences detected in the inoculations are sufficiently robust to allow statistically significant differences to be detected and repeated. Objective 3 - We will begin testing citrus germplasm that may be resistant to citrus greening disease at quarantined greenhouse facilities in Fort Pierce, Florida, using an insect-vectored infection process, with funding obtained from the Citrus Research and Development Foundation. This will include trees that are potentially resistant, as well as control trees and untreated trees, and disease symptoms will be assessed using a standardized scale with the help of collaborators who are well-acquainted with this disase rating system and the analysis of data produced by these inoculation experiments in citrus. Objective 4 - We will analyze the genetic information available for Erwinia tracheiphila and perform data mining and comparative genomics to determine environmentally and regionally associated patterns of gene expression and gene activity and potential activity patterns of these genes under various conditions, both in the laboratory and growth chamber, that impact the progression of the bacterial wilt disease caused by E. tracheiphila. The genomes analyzed will be verified by additional targeted sequencing and also PCR-mediated gene expression analysis and profiling, to reveal correlations of gene activity and structure and pattern of expression with virulence and disease progression in pumpkins and watermelon seedlings.
Impacts
What was accomplished under these goals?
Objective 1 - We published a paper (Klee et al., 2018) detailing new findings about protein translation related to pathogenesis in the fire blight pathogen Erwinia amylovora, and submitted a book chapter detaining our protocol for infection of apple fruitlets and how this represents a viable model system for fire blight disease, which is currently lacking. This book chapter has been accepted for publication. In addition, undergraduates were training in fire blight disease biology and gained experience in plant biology and microbiology by analyzing additional E. amylovora mutants defective in the disease process.Our results about fire blight revealed bacterial protein translation patterns and responses that are associated with the disease process in apples, including anti-oxidant processes, protein secretion processes, flagellar swimming motility, and the regulation of production of exopolysaccharide, as well as the role of a poorly-understood component of the bacterial ribosome in controlling and regulating these types of proteome-level responses. The undergraduates discovered additional mutant alleles of genes involved in E. amylovora exopolysaccharide production, in particular genes in the amylovoran synthesis operon (ams), providing additional confirmation that this operon is essential for the development of fire blight disease. Objective 2 - Nothing to report, since we did not perform any additional experiments with the tomato canker project or tomato disease resistance testing for the reporting period. Objective 3 - We submitted a grant proposal to the Citrus Research and Development Foundation in July, which was selected for funding on September 26, 2018. This funding will support on graduate student to continue the analysis of the citrus trees we have developed that have potential to be resistant to citrus greening. Objective 4 - We have established a collaboration with Dr. Lori Shapiro to analyze the genomes of several Pennsylvania isoaltes of Erwinia tracheiphila, which may provide information about host adaptation and disease severity produced in different conditions, and how these traits may be regulated at the genetic level in Erwinia tracheiphila. We established an infection system using pumpkin seedlings that will provide a high-throughput way to test for virulence disease severity patterns in these strains, and allow for phenotyping of the strains for disease patterns and host-specific interactions and symptom variations related to the genome sequences.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Klee, S. M., Mostafa, I., Chen, S., Dufresne, C., Lehman, B. L., Sinn, J. P., Peter, K. A., and McNellis, T. W. (2018) An Erwinia amylovora yjeK mutant exhibits reduced virulence, increased chemical sensitivity and numerous environmentally dependent proteomic alterations. Molecular Plant Pathology 19:1667-1678
- Type:
Book Chapters
Status:
Accepted
Year Published:
2019
Citation:
Klee, S. M., Sinn, J. P., and McNellis, T. W. The apple fruitlet model system for fire blight disease. Methods in Molecular Biology, accepted for publication.
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