Pseudomonas syringae pv. tomato DC3000 第六型分泌系統表現與功能之分析

Abstract

For the proposes of survival, regulation of physiology and infection, Gram negative bacteria may utilize specialized apparatus to secrete enzymes, toxin or effector proteins. To date, five types of secretion systems (T1SS to T5SS) have been well studied. Type VI secretion system (T6SS) was formally named in 2006. In silico analysis suggested that the highly conserved gene clusters encoding T6SS are widespread in a broad range of microbes, and at least 13 conserved genes have been identified so far. Accumulating evidences connected T6SS to virulence in many animal and plant pathogens; however, the secretory mechanisms and functions remain largely unclear so far. Pseudomonas syringae pv. tomato DC3000 is the causing agent of tomato speck disease. Previous in silico analysis identified two copies of type VI gene clusters in PstDC3000 genome from Pseudomonas genome database, and studies on the gene expression, regulatory mechanisms and functions of T6SSs in PstDC3000 were just initiated two years ago. Therefore, the objectives of this study were to investigate gene expression and functions of T6SSs in PstDC3000. Based on the results of semi-quantitative RT-PCR, expression levels of the t6ss2 cluster and its putative substrate hcp-2 were higher than that of the t6ss1 cluster in all growth conditions we tested, while expression of hcp-1 was barely detectable. Furthermore, in the pathogenicity assay and epiphytic fitness analysis on tomato and Arabidopsis, the single or double mutants of t6ss2-related genes showed slightly redunction in their ability to colonize inside or on host plants. The in planta promoter activity assay showed decreased levels of the promoters of the t6ss2 cluster and the hcp-2 suggested that, unlike animal pathogens or certain plant pathogens, T6SS may play a minor or a negative role in virulence.