疫病菌 Phytophthora parasitica mitogen-activated protein kinases 之功能性分析

Abstract

Phytophthora parasitica Dastur (=P. nitcotianae Breda de Haan) 是一種卵菌綱的植物病原菌，分布於全球，可感染多種植物，包括蔬菜類、果樹類、觀賞植物及樹木等，包含多種重要的經濟作物。在台灣，根據「台灣植物病害名彙」的記載，至少有49種植物受到P. parasitica 危害的紀錄，且因為P. parasitica的病害史非常短暫，於氣候條件適合的情況下在短時間內即會造成嚴重病害。因為P. parasitica 是如此重要的植物病原菌，所以我們開始探討其細胞內的訊息傳導途徑。mitogen-activated protein kinase (MAP kinase)訊息傳導途徑在真核細胞扮演著重要的角色，其傳導途徑中的成員，在演化過程中具有高度的保守性。根據植物病原真菌MAPKs的保守性序列，我們在P. parasitica找到四個MAPK基因，分別命名為ppmk1a、 ppmk1b、 ppmk2及ppmk3。為了探討其在P. parasitica所扮演的角色，我利用real-time quantitative reverse transcriptase-PCR來探討其RNA表現情形，結果發現在無性世代中只有ppmk2 在 sporangia 時期會被誘導表現，此外在酸性環境及高滲透壓下都會被誘導表現，ppmk3則會受到高滲透壓、特定碳素源及10 mM cAMP等誘導表現，此結果顯示ppmk2及ppmk3所參與的訊息傳導途徑與外界環境的刺激有相關，且ppmk2及ppmk3 RNA的表現會受到相同訊息的誘導，所以這兩個MAPK所參與的訊息傳導途徑可能是相關的。另外，我們利用E. coli表現ppmk1a、 ppmk1b、 ppmk2及ppmk3重組蛋白，以in vitro kinase assay 分析其磷酸化活性，發現ppmk3重組蛋白具有對Elk1磷酸化的活性。我亦利用yeast two-hybrid system釣取與ppmk1a、 ppmk1b、 ppmk2及ppmk3具有交互作用的蛋白，篩選出來的結果待進一步的分析。最後，我也嘗試了在P. parasitica建立有效的轉殖方法。

Phytophthora parasitica Dastur (=P. nitcotianae Breda de Haan) is an Oomycete plant pathogen which causes severe disease in many economically improtant crops. According to “List of plant diseases in Taiwan”, there were at least 49 records of the plant diseases caused by P. parasitica. Since P. parasitica is an important plant pathogen, we are interested in the signal transduction mechanism of P. parasitica. Mitogen-activated protein kinase (MAP kinase) pathways operate at the core of eukaryotic signal transduction networks, and their component kinases have been highly conserved through evolution. Four MAP kinases that are homologous to fungal MAP kinases were cloned from P. parasitica and designated as ppmk1a, ppmk1b, ppmk2, and ppmk3, respectively. In order to investigate their roles, expression of these genes in different life stages of P. parasitica was analyzed by real-time quantitative reverse transcriptase-PCR. The results indicated expressions of all four genes were induced in the process of plant infection, while ppmk2 were expressed only in the stage of sporangia. Moreover, while expression of ppmk2 was induced in response to acidic pH and osmotic stress, ppmk3 was induced in response to osmotic stress, high concentration of cAMP and specific carbon sources including sorbitol, glycerol and pectin. It was thus suggested that ppmk2 and ppmk3 were involved in the signaling pathways of P. parasitica in response to different environmental signals. To analyze the kinase activity of these MAPKs, in vitro kinase assay was performed using recombinant proteins obtained from E. coli. The result indicated that Ppmk3 exhibited kinase activity toward an exogenous substrate. Furthermore, yeast two-hybrid system was employed to screen genes which might interact with ppmk1a, ppmk1b, ppmk2 or ppmk3. Putative functions of proteins encoded by these genes will be discussed. In order to know the function and regulatory mechanism of the MAP kinase signaling pathways, I have also tried to establish a transformation method for P. parasitica.