Cloning of a large gene cluster involved in Erwinia amylovora CFBP1430 virulence

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 tomato (N.J. Panopoulos, unpublished data) or P. solanacearum (Boucher et al., 1987).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.

Molecular Characterization of Natural Erwinia pyrifoliae Strains Deficient in Hypersensitive Response

“…The disease has not been reported in countries of the Southern hemisphere other than New Zealand, except for a transient occurrence in 1997 in Australia (11). The pathogenicity of E. amylovora depends on both the synthesis of the acidic exopolysaccharide (EPS) amylovoran (2,17) and a functional type III secretion system, involved in induction of a hypersensitive response (HR) (1,5,9,17). The pear pathogen E. pyrifoliae shares substantial homologies to ams genes and also requires capsular EPS for pathogenicity (12).…”

“…Fewer than one-half of these isolates produced exudate on pear slices and the typical necrotic symptoms at the infiltrations sites on tobacco leaves ( Table 1). The ability to cause HR is a requirement for virulence of E. amylovora strains (1,17). Harpin, encoded by hrpN, is transported to the surface of the bacteria and elicits HR in the plant tissue (20).…”

Molecular Analysis of Sucrose Metabolism of Erwinia amylovora and Influence on Bacterial Virulence

“…The gene scrB of E. amylovora was amplified from plasmid pSCR100 by PCR with primers 3584 and 3523, creating SphI and HindIII sites at the ends of the 1.5-kb PCR fragment. The amplified fragment with scrB was digested and inserted into pQE31 to create pQE31scrB by fusion of scrB with the (His) 6 tag sequence of the vector. The plasmid was transformed into E. coli strain GI698.…”

“…(His) 6 tag fusions were expressed in E. coli strain GI698 after growth at 37°C to an optical density at 600 nm (OD 600 ) of 0.5. The gene was induced with 1 mM IPTG (isopropylthiogalactopyranoside) for 4 h at 28°C.…”

“…Pathogenecity depends on the ability to produce the exopolysaccharide amylovoran (10, 13), to elicit a hypersensitive response on non-host plants (6,8), and to metabolize sorbitol of the host plants (1). Rosaceous plants contain sorbitol and sucrose as storage and transport carbohydrates.…”