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Cloning of a large gene cluster involved in Erwinia amylovora CFBP1430 virulence
M A Barny1, M H Guinebretière, B Marçais
1Laboratoire de Pathologie Végétale, I.N.R.A., Paris, France.
Abstract:
Phage MudIIPR13 insertional mutagenesis of Erwinia amylovora CFBP1430 allowed us to isolate 6900 independent CmR mutants. The frequencies of different auxotrophs in this population indicated that MudIIPR13 had inserted randomly in E. amylovora. Screening of 3500 CmR mutants on (i) apple calli and (ii) pear and apple seedlings led to the isolation of 19 non-pathogenic prototrophic single mutants, four of which expressed a LacZ+ hybrid protein. Expression of the fusion proteins was temperature sensitive. The 19 mutants could be separated into two classes according to their behaviour on tobacco: 13 were unable to elicit the hypersensitive response on tobacco (Hrp-) while six still could (Dsp-). The 19 MudIIPR13 insertions all mapped in the same virulence region. The MudIIPR13 insertions of Hrp- mutants were all clustered on the left part of this region, while the MudIIPR13 insertions of Dsp- mutants were located on the right part. All of the mutants except one, which proved to have a large deletion of the entire virulence region, could be complemented functionally by cosmids from an E. amylovora CFBP1430 genomic library. No hybridization was observed between the cosmid pPV130, which complemented 12 hrp::MudIIPR13 mutations, and the hrp genes from Pseudomonas syringae pv. phaseolicola (Lindgren et al., 1986), P. syringae pv. tomato (N.J. Panopoulos, unpublished data) or P. solanacearum (Boucher et al., 1987). Further analysis of the large virulence region will allow mapping of the border of the virulence region and facilitate the study of the function and regulation of the hrp and dsp genes.
Insights
Phage mutagenesis of Erwinia amylovora identified 19 non-pathogenic mutants, revealing distinct virulence gene clusters. This research aids in understanding Erwinia amylovora pathogenicity and developing control strategies.
Area of Science:
- Microbiology
- Plant Pathology
- Genetics
Background:
- Erwinia amylovora causes devastating fire blight disease in pome fruits.
- Understanding the genetic basis of E. amylovora pathogenicity is crucial for disease management.
Purpose of the Study:
- To identify genes involved in Erwinia amylovora virulence using insertional mutagenesis.
- To characterize mutants with altered pathogenicity and map the affected genes within the virulence region.
Main Methods:
- Insertional mutagenesis using bacteriophage MudIIPR13 in E. amylovora CFBP1430.
- Screening of chloramphenicol-resistant (CmR) mutants for non-pathogenicity on apple calli and plant seedlings.
- Categorization of mutants based on hypersensitive response (HR) on tobacco (Hrp- and Dsp-).
- Complementation analysis using a genomic library to confirm gene function.
Main Results:
- Isolated 6900 CmR mutants, with MudIIPR13 insertions showing random distribution.
- Identified 19 non-pathogenic prototrophic single mutants.
- Classified mutants into Hrp- (13) and Dsp- (6) based on tobacco HR.
- Mapped MudIIPR13 insertions to a specific virulence region, with distinct clusters for Hrp- and Dsp- mutants.
- Confirmed functional complementation of most mutations by genomic library cosmids.
Conclusions:
- The study delineated a large virulence region in E. amylovora containing distinct gene sets for pathogenicity.
- MudIIPR13 mutagenesis effectively identified key virulence factors.
- Further research on this region will elucidate the function and regulation of hrp and dsp genes.