於 Pseudomonas syringae pv. tomato DC3000 中染色體定點插入應用於基因表現之研究

Abstract

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), the causing agent of tomato speck disease, is a model pathogen for the studies of the molecular mechanisms underlying gene-for-gene resistance and disease susceptibility. Its genome sequence has been completed in 2003; thus, it is important to know the function of each gene in the post genomic era. In general, to explore the function of genes, we often use loss- or gain-of-function strategies. For studying bacteria, complementation of a mutant strain was usually performed using a plasmid-based method. However, some properties of the plasmid, such as multicopy and instability, may hamper the utilization of plasmid to carry the gene for complementation. In order to solve this problem, we developed a new site-specific chromosomal integration strategy for genetic expression in Pst DC3000. First, potential regions suitable for integration of genes in the genome of Pst DC3000 was identified using bioinformatic analysis. Then a sacB-based strategy was applied using a suicide vector pK18msLP to integrate gene-of-interest into the designed region. The efficacy of this method was assessed by using mutants deficient in certain T3SS and T6SS effectors. Our data revealed that the chromosome-integrated method was better than the plasmid-borne method for complementation of hcp1 and hcp2, two T6SS effectors in a growth competition assay. In addition, a plasmid carrying avrPtoB was not stable in Pst DC3000 as we observed 12-20% decrease in the plasmid retention ratio when it was inoculated into the susceptible tomato lines and N. benthamiana. In conclusion, we have successfully established a stable chromosomal integration strategy for gene expression with similar or even better effects than the plasmid-encoded strategy.