Ralstonia solanacearum UW551 Project Description



Brief Summary

This collaborative project is to determine the genomic DNA sequence of Ralstonia solanacearum Race 3 Biovar 2, a USDA Select Agent. This Select Agent has has been introduced repeatedly into the U.S. but has not become established. Race 3 Biovar 2 is of particular concern because it is more cold tolerant than Race 1 strains, which are already present in the U.S., and because of unknown pathogenicity factors that may reside in the Race 3 Biovar 2 genome.

The project is organized under the direction of Dean Gabriel. Strain UW551 was chosen because it was introduced into the U.S. on latently infected geranium and its taxonomic status and pathogenicity were extensively characterized independently by Caitilyn Allen and John Elphinstone. Initial DNA sequencing was performed by SeqWright (Houston, TX) and initial annotation by Integrated Genomics (Chicago, IL). Bioinformatics tools, website design and additional DNA sequencing were performed by the ICBR Genomics Core, directed by William Farmerie

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About the Organism

Phylum: Proteobacteria;
Class: Betaproteobacteria;
Order: Burkholderiales;
Family: Burkholderiaceae.

Ralstonia solanacearum (Smith 1896) Yabuuchi et al. 1996 is a soil-borne bacterial plant pathogen that causes systemic vascular wilting of many economically important crops, including tomato, tobacco, pepper, peanut, ginger, banana, eucalyptus, and potato. It infects a very wide range of plant species in over 50 different families, and can be carried asymptomatically in some species.

Ralstonia solanacearum Race 3 Biovar 2 (R3B2) strains are a highly clonal group that are of particular concern because they are cold-tolerant and capable of surviving and infecting crops in temperate environments. The economic threat to plant industry is such that R3B2 strains are listed as Select Agents in the United States and are quarantined pathogens in Europe and Canada.

Ralstonia solanacearum Race 3 Biovar 2 strain UW551 was isolated in Wisconsin from a wilted geranium imported from Kenya, and it is pathogenic on tomato and potato.

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Sequencing Strategies

High molecular weight total DNA from UW551 was sheared into 1-3 kb fragments, subcloned into pUC18 and 38,000 randomly selected clones were single pass sequenced using M13F and M13R primers. Sequencing was performed by SeqWright (Houston, TX).

High molecular weight Sau3A1 digested DNA was extracted and ligated to cosmid vector pUFJ10. Seven hundred and sixty eight cosmid clones were end sequenced using vector based primers (T7 and M13R).

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Annotation Strategies

Automated annotation was performed by Integrated Genomics (IG; Chicago, IL) to identify open reading frames (ORFs), rRNA and tRNA encoding regions and, where possible, assign function.

All predicted ORFs of R. solanacearum UW551 were compared to all predicted ORFs of R. solanacearum GMI1000 [Genbank accession NC_003295.1] using BLASTP (low complexity filter, off; E-value cutoff, 100; -b, 1; -v, 1) and TBLASTN (low complexity filter, on; E-value cutoff, 10-11, -v, 1; -b, 1).

A secure web-based interface (http://vision.biotech.ufl.edu) was set up and maintained by the UF ICBR Genomics Core, which also curated all original DNA sequencing reads, assembly edits, ORF calls and ORF edits. All ORFs that appeared to be found in UW551 but missing in GMI1000 were manually annotated. These ORFs were compared against the GenBank nonredundant (nr) database using BLAST, PFAM and also run individually against 171 publically available completely sequenced microbial genomes, including 9 eukaryotes, 144 eubacteria and 18 archaebacteria.

In addition to the UW551-unique ORFs, particular attention was given to ORFs that were likely involved in pathogenicity or cold tolerance, including: 1) the hypersensitive response and pathogenicity (hrp) or Type III secretion gene region; 2) ORFs with a plant inducible promoter consensus sequence (PIP Box); 3) ORFs encoding predicted hydrolytic enzymes; 4) ORFs encoding type II secretion genes; 5) ORFs corresponding to genes known by IVET analysis to be up-regulated in planta; 6) flagellar genes; 7) type IV and tight adherence pilus genes; 8) efflux pump genes; 9) twin-arginine protein translocation (TAT) substrate genes, and 10) extracellular polysaccharide (EPS) genes.

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Funding Agencies

This work was funded by a grant to Dean Gabriel by the U.S. Department of Agriculture, Animal and Plant Health Inspection Service (USDA-APHIS).

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Personnel

Participants are listed in alphabetical order.

Plant Pathology Group

Caitilyn Allen, Ph.D.
Department of Plant Pathology
University of Wisconsin, Madison
U.S.A.

Anne M. Alvarez, Ph.D.
Department of Plant and Environmental Protection Sciences
University of Hawaii, Honolulu
U.S.A.

Timothy Denny, Ph.D.
Department of Plant Pathology
University of Georgia, Athens
U.S.A.

Yong Ping Duan, Ph.D.
Plant Pathology Department
University of Florida, Gainesville
U.S.A.

John Elphinstone, Ph.D.
Plant and Environmental Bacteriology
Central Science Laboratory, Sand Hutton, York
U.K.

Enid Gonzalez.
Department of Plant Pathology
University of Wisconsin, Madison
U.S.A.

Dean Gabriel, Ph.D.
Plant Pathology Department
University of Florida, Gainesville
U.S.A.

Qi Huang, Ph.D.
USDA/ARS, US National Arboretum
Beltsville, MD
U.S.A.

Joseph D. Reddy, Ph.D.
Plant Pathology Department
University of Florida, Gainesville
U.S.A.

Gerry Saddler, Ph.D.
Scottish Agricultural Science Agency
East Craigs, Edinburgh
U.K.

 		Bioinformatics Group

William G. Farmerie, Ph.D.
Interdisciplinary Center for Biotechnology Research (ICBR)
University of Florida, Gainesville
U.S.A.

Li Liu, M.D.
Interdisciplinary Center for Biotechnology Research (ICBR)
University of Florida, Gainesville
U.S.A.

Vincent Mulholland, Ph.D.
Scottish Agricultural Science Agency
East Craigs, Edinburgh
U.K.

Manjeera Patnaikuni
Department of Computer and Information Science and Engineering
University of Florida, Gainesville
U.S.A.

Gernot Presting, Ph.D.
Department of Molecular Biosciences and Bioengineering
University of Hawaii, Honolulu
U.S.A.

Mark A. Schell, Ph.D.
Department of Microbiology
University of Georgia, Athens
U.S.A.

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