- 學位論文
Pseudomonas syringae pv. tomato DC3000 第六型分泌系統表現與功能之分析
Characterization of the Expression and Function of Type VI Secretion Systems in Pseudomonas syringae pv. tomato DC3000
摘要
革蘭氏陰性菌常利用各種分泌系統外泌酵素及作用蛋白，以達到調控生理、生存或感染寄主之功能。目前已知且廣泛研究的分泌系統有第一型至第五型，除了組成與分泌機制不同之外，其外泌基質也有所差異。第六型分泌系統為2006年才被確認之新分泌系統，生物資訊分析結果顯示此分泌系統的基因叢集廣泛存在於多種細菌基因體中，並具有至少13種高度保守性基因。今已證實第六型分泌系統在多種動物與植物病原菌中受到調控並影響病原性，然而詳細作用機制及功能仍未瞭解透徹。 Pseudomonas syringae pv. tomato DC3000為造成細菌性斑點病之病原菌。分析此菌基因體資料庫得知其具兩套第六型分泌系統之基因叢集，其基因表現或功能研究才剛起步。因此，本研究的目的為瞭解此菌兩套第六型分泌系統之調控情形，以及可能扮演之角色。首先，由半定量reverse transcriptase PCR及報導系統可得知此菌的兩套分泌系統基因皆有表現，而第二套分泌系統基因表現較第一套強；其基質hcp基因則只偵測到第二套的表現。藉研究相關基因突變株之感染能力與葉表存活能力變化發現，第二套重要基因突變對番茄、阿拉伯芥等寄主的感染能力與葉表存活能力具些微影響。利用報導基因偵測第六型分泌系統啟動子在進入寄主體內後的表現，發現在感染六小時後有下降趨勢。由這些結果推論，P. s. pv. tomato DC3000的第六型分泌系統和其他動物與植物病原菌不同，可能在病原性上是不需要的或是有負面效應。
並列摘要
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.