Articles
A SIMPLE GENETIC TOOL FOR STUDYING GENE FUNCTION AND SIGNAL TRANSDUCTION IN ERWINIA AMYLOVORA
Article number
793_22
Pages
171 – 178
Language
English
Abstract
Using an in vivo expression technology (IVET) system, we have previously identified several hundred E. amylovora genes that are induced during infection of immature pear fruit.
In order to further determine the function of the genes identified by IVET, we have adopted an easy gene knockout technique PCR-based one step inactivation of chromosomal genes, also referred to as red-cloning.
This technique is not only easy and fast (capable of mutant generation within a week without any cloning steps), but can also generate a single gene, an operon or a genomic island deletion mutant.
So far, we have constructed several dozen single, double, triple and island deletion mutants in E. amylovora using this technique.
Early studies in elucidating the molecular basis for pathogenesis have identified an essential virulence system the hrp type III secretion system (hrp-T3SS) in E. amylovora which delivers effector proteins into host plants.
Subtractive hybridization followed by analysis of the E. amylovora genome sequence has revealed two novel T3SS pathogenicity islands (PAIs) (Erwinia-pathogenicity islands, EPI1 and EPI2). Deletion mutants for these three PAIs, type II secretion system (T2SS) and the amylovoran (ams) operon have been generated.
Pathogenicity assays with immature pear fruit and apple seedlings showed that only the hrp-T3SS and ams operons are essential pathogenicity factors, whereas T2SS, EPI1 and EPI2 are not involved in virulence in plants.
In addition, using genomic and bioinformatics techniques, we have also identified about 45 signal transduction regulatory genes in the genome of E. amylovora. Knock-out mutants for all of these genes have been constructed and phenotypic and genetic characterizations of these mutants are now underway.
In the future, transcriptome analysis using microarrays for these signal transduction systems will be performed.
In order to further determine the function of the genes identified by IVET, we have adopted an easy gene knockout technique PCR-based one step inactivation of chromosomal genes, also referred to as red-cloning.
This technique is not only easy and fast (capable of mutant generation within a week without any cloning steps), but can also generate a single gene, an operon or a genomic island deletion mutant.
So far, we have constructed several dozen single, double, triple and island deletion mutants in E. amylovora using this technique.
Early studies in elucidating the molecular basis for pathogenesis have identified an essential virulence system the hrp type III secretion system (hrp-T3SS) in E. amylovora which delivers effector proteins into host plants.
Subtractive hybridization followed by analysis of the E. amylovora genome sequence has revealed two novel T3SS pathogenicity islands (PAIs) (Erwinia-pathogenicity islands, EPI1 and EPI2). Deletion mutants for these three PAIs, type II secretion system (T2SS) and the amylovoran (ams) operon have been generated.
Pathogenicity assays with immature pear fruit and apple seedlings showed that only the hrp-T3SS and ams operons are essential pathogenicity factors, whereas T2SS, EPI1 and EPI2 are not involved in virulence in plants.
In addition, using genomic and bioinformatics techniques, we have also identified about 45 signal transduction regulatory genes in the genome of E. amylovora. Knock-out mutants for all of these genes have been constructed and phenotypic and genetic characterizations of these mutants are now underway.
In the future, transcriptome analysis using microarrays for these signal transduction systems will be performed.
Publication
Authors
Y.F. Zhao, G.W. Sundin
Keywords
recombineering, λ red recombination, pathogenicity islands, type III secretion, two-component systems, gene regulation
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