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Bacteriophage Mu as a genetic tool to study Erwinia amylovora pathogenicity and hypersensitive reaction on tobacco
J L Vanneste1, J P Paulin, D Expert
1Pathologie Végétale, Paris, France.
Abstract:
Erwinia amylovora 1430 was shown to be sensitive to Mu G(-) particles. Infection resulted either in lytic development or in lysogenic derivatives with insertion of the Mu genome at many sites in the bacterial chromosome. We used the Mu d1Bx::Tn9 (lac Apr Cmr) derivative, called Mu dX, to identify mutants affected in pathogenicity and in their ability to induce a hypersensitive reaction (HR) on tobacco plants. Inoculation of 1,400 lysogenic derivatives on apple root calli led to the identification of 12 mutants in three classes: (i) class 1 mutants were nonpathogenic and unable to induce an HR on tobacco plants; (ii) class 2 mutants were nonpathogenic but retained the ability to induce an HR; and (iii) class 3 mutants showed attenuated virulence. Of the 12 mutants, 8 had a single insertion of the Mu dX prophage. For class 1 and 2 mutants, reversion to pathogenicity was concomitant with the loss of the Mu dX prophage. Furthermore, revertants from the class 1 mutants also recovered the ability to induce an HR on tobacco plants. Five of the six class 3 mutants were impaired in exopolysaccharide production. No changes of the envelope structure (lipopolysaccharide and outer membrane proteins) were correlated with differences in pathogenicity. One class 3 mutant did not produce any functional siderophore, suggesting that iron uptake could be involved in pathogenicity.
Insights
Bacteriophage Mu dX insertion mutants of Erwinia amylovora were identified, revealing key genes for pathogenicity and hypersensitive response (HR). Some mutants impaired in exopolysaccharide production or iron uptake suggest these factors are crucial for virulence.
Area of Science:
- Bacteriology
- Plant Pathology
- Molecular Genetics
Background:
- Erwinia amylovora is the causative agent of fire blight, a devastating disease in apple and pear trees.
- Bacteriophage Mu, a transposable element, can be used as a genetic tool to generate insertion mutants in bacteria.
- Understanding the genetic basis of E. amylovora pathogenicity is crucial for developing effective disease control strategies.
Purpose of the Study:
- To identify genes in Erwinia amylovora essential for pathogenicity and the induction of a hypersensitive reaction (HR) in tobacco plants.
- To utilize the Mu dX transposon for generating and characterizing mutants with altered virulence.
- To investigate the role of exopolysaccharides and iron uptake in E. amylovora pathogenesis.
Main Methods:
- Generation of lysogenic Erwinia amylovora 1430 derivatives using the Mu d1Bx::Tn9 (Mu dX) transposon.
- Screening of 1,400 lysogenic derivatives for mutations affecting pathogenicity on apple root calli and HR induction on tobacco.
- Analysis of mutant phenotypes, including exopolysaccharide production, siderophore synthesis, and reversion analysis.
Main Results:
- Twelve mutants were identified and classified into three groups based on pathogenicity and HR induction: nonpathogenic with no HR, nonpathogenic with HR, and attenuated virulence.
- Eight mutants had a single Mu dX insertion; reversion to pathogenicity correlated with prophage loss, and HR recovery in class 1 revertants.
- Five of six attenuated mutants were impaired in exopolysaccharide production; one mutant showed defective siderophore production, implicating iron uptake in virulence.
Conclusions:
- The Mu dX transposon is an effective tool for identifying genes involved in E. amylovora pathogenicity and HR.
- Exopolysaccharide production and iron uptake are important factors contributing to the virulence of Erwinia amylovora.
- Further research into these identified genes will provide insights into the molecular mechanisms of fire blight disease.