疫病菌Phytophthora parasitica 蛋白去磷酸酶PP2C之選殖與特性分析

Abstract

Phytophthora parasitica Dastur (疫病菌，又名P. nitcotianae Breda de Haan) 廣泛分布於全球各地。疫病菌的孢囊與游走子能靠著環境中些微水分迅速傳播，且生活史極短，能在同一植株上進行重複感染，寄主範圍又廣，經常造成重大農業損失，是世界上重要的植物病原菌之一。近年來許多研究顯示絲裂原活化蛋白激酶 (mitogen-activated protein kinases，MAPK) 訊息傳導與植物病原菌的病原性有關，例如炭疽病菌 (Colletotrichum spp.) 與稻熱病菌 (Magnaporthe grisea)。 Protein phosphatase 2C 在MAPK訊息傳遞路徑中扮演負向調控的角色，藉由去除磷酸根來抑制MAPK的活性。我們根據 Phytophthora ramorum 與 P. sojae 的 PP2C 同源性序列設計引子對，從 P. parasitica 基因體中增幅得到一長為 350 bp 之部份序列；再由 RACE (rapid amplification of cDNA ends)得到全長基因，其ORF (open reading frame)可轉譯出 344 個胺基酸序列，並將此基因命名為 ppptc1 (Phytophthora parasitica phosphatase two C 1)。ppptc1 與人類 PP2Cα的相似度高達 40%。在 P. parasitica 基因體中，ppptc1的拷貝數為一，但可能有其他同源基因存在。利用 pMAL (pMALTM Protein Fusion and Purification System) 載體於大腸桿菌中表現之 PPPTC1 重組蛋白能夠對 pNPP進行去磷酸化反應。PPPTC1 在胞外能將已磷酸化的 PPMK3 去磷酸化，但在in vivo下是否能對 PPMK3 進行逆向調控，有待進一步驗證。Ppptc1基因表現受過氧化物、鈣離子及酸性環境所抑制，但為鹼性環境而誘導。根據 ppptc1蛋白特性及逆境下之表現情形，推斷 ppptc1 極有可能參與疫病菌 MAPK 訊息傳導途徑。

Phytophthora parasitica Daster (=P. nitcotianae Breda de Haan) is an oomycete plant pathogen. Spreading easily by water, short life cycle, and wide host range contributes P. parasitica a worldwide disease. Recent studies show that MAPK pathway is related with the pathogenicity of plant pathogens, such as Colletotrichum spp and Magnaporthe grisea. PP2C negatively regulate MAPK pathway by inactive phospo-MAPKs. We designed specific primers to amplify partial sequence of PP2C from P. parasitica genomes. The entire open reading frame was gained by Rapid Amplify cDNA ends and named as ppptc1 (Phytophthora parasitica phosphatase two C 1), which encoded 344 amino acids. Ppptc1, which has 40% similarity to human PP2Cα gene, is a one-copy number gene but other homologuesin may exist in the genome of P. parasitica. To express the PPPTC1 protein, the pMAL vector was used to express heterogeneous fusion protein from E. coli. As an alkaline phosphatase, PPPTC1 can dephosphorylate pNPP (para-nitrophenyl phosphate), a non-protein substrate. PPPTC1 also seems to dephosphorylate ppmk3 in vitro, a MAP kinase in P. parasitica. ppptc1 is down-regulated by calcium ion, acidity, and oxdative stress but induced by alkalinity. Base on protein character and expression pattern under stresses, we propose that ppptc1 is involved in MAPK pathway in P. parasitica.