Progress 01/01/07 to 12/14/07
Outputs
The entire sequence of the genome of Erwinia amylovora, strain Ea273, had been determined by the Sanger Institute in Cambridge, England. It can be accessed on the World Wide Web at http://www.sanger.ac.uk/Projects/E_amylovora/. Publication of the sequence is anticipated soon after the Sanger Institute completes the basic biological annotation of the genome. A preliminary annotation, developed at Cornell and UW, Madison, and the sequence can be accessed on request and registration at: https://asap.ahabs.wisc.edu/asap/logon.php. Dissemination of the news of progress in completing the sequence was done by presentation of an illustrated oral report at the 11th International Workshop on Fire Blight in Portland, Oregon, in August 2007, by word-of-mouth and by e-mail. A project aimed at determining whether DspA/E, a protein essential to the ability of E. amylovora to cause fire blight, is translocated into host (apple) plant cells was carried out. A report of the results was
prepared for submission to a peer-reviewed journal, revised and re-submitted. It is likely to be published in a learned journal soon. An innovative outreach project aimed primarily at secondary school biology students in New York State came to fruition during the reporting period. The project was undertaken in collaboration with Cornell's Institute for Biology Teaching (CIBT), which is supported by the Howard Hughes Medical Institute and the New York State Center for Advanced Technology. A DVD and a study guide were produced aimed at introducing secondary school students and their teachers to several important concepts: that bacteria can cause disease in plants; techniques for safe handling of bacterial plant pathogens; that specific genes are needed for disease-causing ability; bioinformatics strategies useful for annotation of a genome sequence, and the use of reverse genetics laboratory tests to assess gene function. The program, was presented to a group of secondary school science
teachers, who will test the materials and possibly suggest changes to the project for use in classrooms. The materials also will be made available on the internet. The secondary school outreach project is being revised and expanded for presentation to the agricultural community, especially Cooperative Extension Educators in the field and fruit growers. One of the major objectives is to foster better understanding by the target audiences of fundamental molecular biological research as applied to fruit crops and their pests and pathogens. Greater understanding of the relevance of molecular biological research, and its possible future benefit, likely will result in enhanced appreciation by the agricultural community and the general public.
Impacts
Evidence was obtained that the DspA/E protein of E. amylovora is translocated into plant cells based on use of the CyaA reporter system. In addition, translocation into plant cells requires the presence of two proteins, HrpN and HrpJ in the same cell of E. amylovora. However, two other proteins secreted by the type-three-secretion system of E. amylovora, HrpW and HrpK are not required. This finding may explain the effect of HrpN as an essential virulence determinant in E. amylovora. In addition, HrpJ was found to be essential for secretion of HrpN, thus explaining its role in virulence. Taken together the interaction of the several proteins secreted by the type-three-secretion system of E. amylovora now can be better understood by members of the scientific community. The impact of the outreach aspects of this project will require several years to become apparent. Only after the members of the target audience (secondary school students) have progressed further in life
can the effects of their exposure to the wonders of bacterial-plant pathogenesis be gauged.
Publications
- Nissinen, R.M., Ytterberg, A.J., Bogdanove, A.J., van Wijk, K. and Beer, S.V. 2007. Analyses of the secretomes of Erwinia amylovora and selected hrp mutants reveal novel type III secreted proteins and an effect of HrpJ on extracellular harpin levels. Mol. Plant Pathol. 8:55-67.
- Oh, C.-S., Martin, G., and Beer, S. V. 2007. DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death. Mol. Plant Pathol. 8: 255-265
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Progress 03/15/04 to 12/14/07
Outputs
OUTPUTS: A progress report on the sequencing of the genome of the fire blight pathogen was presented to the Eleventh International Workshop on Fire Blight. The Workshop was attended by representatives of virtually all fire blight research groups in the world. A preliminary report of progress in assessing the quality of seven published PCR primers for the identification of the fire blight pathogen was also presented to the Workshop. A full research report on the evaluation of molecular diagnostic probes for detecting and identifying Erwinia amylovora was prepared and submitted for publication in a learned journal. As part of the "Outreach" efforts for this project, a DVD presentation was conceived concerning fire blight that is aimed agricultural stakeholders and cooperative extension personnel. The 15 to 20 minute multi-media program introduces the disease and the concepts of molecular genetics and genomics. Furthermore, it provides the groundwork for understanding CSREES-funded research that is currently underway aimed at achieving better understanding of fire blight and the development of innovative control strategies. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Work continued at several locations on the analysis of the genome sequence of Erwinia amylovora. Progress was made in writing two major manuscripts reporting overall sequencing results. One manuscript deals with the discovery of several putative effectors of the fire blight pathogen, based on bioinformatic searches. The second is the actual report of the genome sequence and comparisons with known genome sequences. We envision that both manuscripts will be submitted for publication, in tandem, in a learned journal during the first half of 2009. We evaluated several published polymerase-chain-reaction (PCR) primer pairs proposed for identification of Erwinia amylovora in an attempt to find one that would differentiate all strains E. amylovora from all non-E. amylovora strains. We subjected a collection of 40 known strains of E. amylovora and 54 diverse saprophytic bacteria that had been isolated from rosaceous hosts of E. amylovora, to all published PCR-based diagnostic protocols. Several widely accepted PCR-based diagnostic probes for the detection of E. amylovora target the pEA29 plasmid, which until recently was thought to be associated with all known virulent strains of E. amylovora. However, a virulent strain of E. amylovora that lacks pEA29 and does not yield amplification products with primers based on pEA29 has put the specificity of this test into question. Our results indicated that one proposed primer pair, which amplifies a sequence of unknown function proved highly efficient and effective. No false negatives, nor false positives, were identified among our collection of 94 tested strains.
Publications
- Bocsanczy, A. M., Perna, N. T., Biehl, B., Glasner, J. D., Cartinhour, S. W., Schneider, D. J., DeClerck, G. A., Sebaihia, M., Parkhill, J., Bentley, S. and Beer, S. V. 2008. Contributions of the genome sequence of Erwinia amylovora to the fire blight community. Acta Horticulturae 793:163-170. Powney, R., Plummer, K., Luck, J., Rodoni, B. and Beer, S.V. 2008. Evaluation of PCR-based protocols for the detection of Erwinia amylovora. Acta Horticulturae 793:505-511.
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Progress 01/01/06 to 12/31/06
Outputs
Additional progress was made in understanding the genome of Erwinia amylovora, strain Ea273. The Sanger Institute completed the sequencing and closed the genome during 2006. The draft genome sequence of Ea273, as determined by the Sanger Institute, was used as an essential tool to identify 12 proteins of Ea273 that are secreted by the type three secretion system. As a result, several new proteins were identified and named and some homologs of secreted proteins from other phytopathogenic bacteria were identified. Several regions of the genome have been identified that contain homologues of genes that have been related to pathogenicty and virulence in animal and other plant pathogens. A set of genes, potentially up-regulated by sigma factors during infection, were identified by probabilistic algorithms, providing more candidate genes related to virulence and pathogenicity. Based on the described analyses and comparative genomics, several genes of Ea273 were hypothesized
to be involved in pathogenicity or virulence of E. amylovora. These genes have been mutated and their effects on pathogenicity or virulence are being assessed. Overall, the genome of E. amylovora appears to be rather more closely related to those of animal pathogenic bacteria than several other plant pathogens. Basic biological annotation of the sequence is going to be completed and uploaded on the community annotation database (ASAP) during the first quarter of 2007. An outreach aspect of the sequencing project was developed. A collection of easily adaptable multimedia lessons that introduce modern biological concepts to high school students was devised. Through these lessons, students are introduced to the concept of bacterial pathogenesis in plants, the process and potential payoff of sequencing genomes, and the reverse genetics strategies (gene mutagenesis and complementation) that can be used to uncover the genes responsible for virulence in a bacterial pathogen. Tools of
bioinformatics are introduced so that students can analyze 'new' DNA sequences to identify potential genes. Given information about homology to known genes, students develop hypotheses about the role of a series of genes in virulence and run virtual experiments to test their hypotheses. After viewing a lab-prepared video on the preparation of an assay for plant pathogenicity in immature pear fruits, which includes information on lab techniques, safety, and equipment, students run the virtual experiments, which are time-lapse videos of fire blight symptoms in immature pear fruits that had been inoculated with Ea273 and specific mutants of the pathogen. Students then can evaluate the data and revise their hypotheses about the roles of their chosen genes in the virulence of E. amylovora.
Impacts
When the outreach aspect of this project is introduced into selected high schools in New York State, we anticipate that the science teachers and students involved will gain an appreciation of plant disease, bacterial pathogenesis, the role of genes, genome sequencing and its utility in discovering fundamental biological principles and relationships.
Publications
- Wright, S.A. I., Mi, J., Clardy, J. and Beer, S.V. 2006. The biosynthetic genes of pantocin A and pantocin B of P. agglomerans Eh318. Proceedings X International Workshop on Fire Blight. Acta Horticulturae 704: 313-319.
- Asselin, J.E., Oh, C.S., Nissinen, R.M. and Beer, S.V. 2006. The secretion of EopB from Erwinia amylovora. Proceedings X International Workshop on Fire Blight. Acta Horticulturae 704: 409-415.
- Beer, S.V., Bocsanczy, A. B., Perna, N.T. and Parkhill, J. 2006. Sequencing the entire genome of Erwinia amylovora. Proceedings X International Workshop on Fire Blight. Acta Horticulturae 704: 395-396.
- Bocsanczy, A.M., Nissinen, R.M., Oh, C.S. and Beer, S.V. 2006. DspE, an effector of Erwinia amylovora, is translocated into plant cells. Proceedings X International Workshop on Fire Blight. Acta Horticulturae 704: 467-471.
- Bonasera J.M., Kim J.F. and Beer, S.V. 2006. PR genes of apple: identification and expression in response to elicitors and inoculation with Erwinia amylovora. BMC Plant Biology 6:23.
- Borejsza-Wysocka, E.E., Malnoy, M., Aldwinckle, H.S., Meng, X., Bonasera, J.M., Nissinen, R.M., Kim, J.F. and Beer, S.V. 2006. The fire blight resistance of apple clones in which the DspE-interacting proteins are silenced Proceedings X International Workshop on Fire Blight. Acta Horticulturae 704: 509-513.
- Kim, W.-S., Bonasera, J.M., Meng, X., Owens, T. and Beer, S.V. 2006. Interaction of DspE/A, a pathogenicity/avirulence protein of Erwinia amylovora, with pre-ferredoxin from apple and its relationship to photosynthetic efficiency. Proceedings X International Workshop on Fire Blight. Acta Horticulturae 704: 473-477.
- Meng, X., Bonasera, J.M., Kim, J.F., Nissinen, R.M. and Beer, S.V. 2006. Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen. Mol. Plant-Microbe Interact. 19: 53-61.
- Wang, L., and Beer, S.V. 2006. Application of signature tagged mutagenesis to the study of virulence of Erwinia amylovora. FEMS Microbiology Letters 265: 164-171.
- Wright, S.A.I. and Beer, S.V. 2006. Pantoea agglomerans, a biocontrol agent and ubiquitous microorganism - friend or foe? pp. 334-338 in W. Zeller and C. Ullrich, eds. Proceedings of the 1st Symposium of Biocontrol of Bacterial Plant Diseases. Darmstadt, Germany, October, 2005.
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Progress 01/01/05 to 12/31/05
Outputs
Sufficient sequencing of the genome of Erwinia amylovora strain Ea273 was completed in 2005 such that small numbers of contigs could be assembled into virtual artificial macromolecules. The sequencing results are available on the web site of the Sanger Institute (http://www.sanger.ac.uk/Projects/E_amylovora/). The macromolecules were constructed by concatenating the contigs at the 31, 25 and 17 contig stages. The resulting molecules were treated as genome sequences for analytical purposes. ORFs, RBS, tRNA and terminators were predicted with publicly available programs. BLAST analyses against public databases and published genomes were used to identify homologous sequences at the DNA and protein levels. Programming tools developed in-house were used to predict conserved motifs, such as putative HrpL-dependent promoters (hrp boxes) and putative iron dependent promoters (fur boxes). Candidate effectors found using these tools were analyzed further in search for
N-terminus sequences characteristics of confirmed effectors in Pseudomonas syringae. Our analyses had the following results: The genome of strain Ea273 is 3.8 Mbp, which is the smallest among the sequenced genomes of the Enterobacteriaceae. Additionally, Ea273 has two plasmids (ca. 72 kbp and 28 kbp). 76 hrp boxes, 29 fur boxes and several regions of altered G+C, including two new pathogenicity islands, were predicted. The latter contain complete sets of type-three secretion system genes and are most closely related, in sequence similarity and gene organization, to the SSR-1 island of Sodalis glossinidius, an endosymbiont of the tsetse fly, and somewhat less related to the ysa pathogenicity island of Yersinia enterocolitica, a human pathogen. Phylogenetic analysis based on the conserved homologues HrcC-YsaC-InvG protein sequences showed that HrcC in the previously known pathogenicity island (PAI-1) is closely related to other plant pathogens, while the corresponding proteins in the
novel PAI-2 and PAI-3, are closely related to those of animal pathogens. Sequence of a new plasmid was discovered that is similar in gene organization to that of pEL60 from E. amylovora strain LebB66. This indicates that the plasmid content of strains of E. amylovora differ among strains. Ten novel genes, in addition to the characterized genes of PAI-1 (Oh et al., 2005), which were shown to be expressed in vitro under hrp-inducing conditions in a proteomic analysis of MALDI-TOF MS sequences (Nissinen et al., submitted.) were also located on the sequence. Six novel putative effector proteins predicted by homology, hrp boxes and sequence characteristics at the N-terminus similar to those of P. syringae, were selected for functional testing in virulence assays. The predicted gene sequences of the PAI-1 are identical at the nucleotide level with those of E. amylovora strain Ea321 (Oh et al., 2005). The previously characterized ams operon, DFO operon and plasmid pEU29 genes and novel
potential virulence factors, including quorum-sensing, hemolysin-like proteins and regions of flagellar biosynthesis also were annotated on the sequence.
Impacts
New relationships among microorganisms have been discovered and novel candidate virulence factors have been found through the sequencing of the genome of Ea273. This information is publicly available. These results provide the Fire Blight community with new tools and information, which can impact research in disease mechanisms, disease management, quorum-sensing and bacterial survival. In addition, the extension of the sequencing findings to K-12 programs may impact science education, making available new tools in bioinformatics and biology to young students.
Publications
- Oh, C.-S. and Beer, S.V. 2005. Molecular genetics of Erwinia amylovora involved in the development of fire blight. FEMS Microbiology Letters 253:185-192.
- Oh, C.-S., Kim, J.F. and Beer, S.V. 2005. The Hrp pathogenicity island of Erwinia amylovora and identification of three novel genes required for systemic infection. Mol. Plant Pathol. 6:125-138.
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Progress 03/15/04 to 12/31/04
Outputs
Substantial progress was made during the first 9 months of this genome sequencing project. Genomic DNA was prepared from Erwinia amylovora Ea273, the strain to be sequenced, and it was shipped to the Sanger Institute near Cambridge, England. There, the DNA was incorporated into several genomic libraries for sequencing according to the standard Sanger procedures. Shotgun sequencing was carried out; according to the Sanger web site, 88,557 reads, totalling 42.322 Mb, gave a theoretical coverage of 99.99 percent of the genome. The shotgun sequencing revealed that the genome of Ea273 is approximately 3.89 Mb, about 20 percent less than estimated initially and less than that of E. carotovora (5.06 Mb), and a bit more than that of E. chrysanthemi (3.7 Mb). In November 2004 the finishing process began. Near the end of the shotgun sequencing process, the first preliminary assembly was produced and a partial preliminary annotation was made. Previously sequenced genes of the
hrp pathogenicity island and some other genes were identified that are related to pathogenicity, based on previously identified homologues in related plant pathogens. We anticipate that the sequence analysis will provide clues to genes that are potentially involved in the pathogenic or virulence capability of E. amylovora. These would be cloned and evaluated for their potential effects. The Co-PIs and principal workers on the project met to discuss and decide on efficient ways of handling the analysis of the sequence from their respective locations. Work was started to develop satisfactory procedures and materials for the outreach aspects of the project. We seek to introduce secondary school students to plant disease, critical thinking about disease causation and genome sequencing through learning about fire blight and our genome sequencing of the bacterium that causes the disease. Several potential host plant materials were evaluated from the standpoint of ease of handling in the
secondary school classroom. Apple seedlings were selected because they provide greater ease and flexibility in handling and instructional potential. Seeds were harvested, vernalized, germinated and planted in small containers suitable for the classroom. Several growing conditions, inoculation techniques, and incubation conditions are under test to determine those suitable for use in the classroom. Substantial progress was made in the development of web site providing information on the progress of the genome sequencing and other aspects of fire blight and its pathogen. The goal is a single site that will be useful to growers, their advisors, researchers and students throughout the world. It will provide information, or links to other sites, concerning diagnostics, epidemiology, control as well as the genomic aspects and scientific literature on fire blight and E. amylovora. Test versions of the site will be released early in 2005.
Impacts
Once the genome sequence of E. amylovora and its basic annotation are complete, we anticipate that there will be impacts of several sorts. New genes of the fire blight pathogen may be identified that effect pathogenic capabilities. These genes or their products may provide targets for the action of novel control techniques or materials. Comparison of the whole genome of E. amylovora with those of other pathogenic bacteria of plants and animals are likely to reveal evolutionary relationships among the bacteria and possible functional homology with better-studied genes of other pathogens. The development and utilization of instructional kits and curricula for secondary school students based on fire blight and the sequencing of the bacterium responsible is likely to broaden the understanding and appreciation of basic biology and the problems of food production for young people. The availability of all the genomic and research information on fire blight and its causal
agent in a complete and organized web page will benefit the world wide research community. It will facilitate the exchange of information among members of the research community and provide centralized information needed by concerned growers and citizens.
Publications
- Dong, H.-P., Peng, J., Bao, Z., Meng, X., Bonasera, J. M., Chen, G., Beer, S. V. and Dong, H. 2004. Downstream divergence of the ethylene signaling pathway for harpin-stimulated Arabidopsis growth and insect defense. Plant Phys. 136:3628-3638.
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