疫病菌鈣離子通道次單元基因Mid1之功能性分析

Abstract

疫病菌為重要的植物病原卵菌，其寄主範圍廣泛，可於多種重要經濟作物造成危害。病原卵菌外觀形態和真菌相當類似，但進一步的核酸類源關係分析顯示兩者明顯不同，使得分類學將其獨立於真菌界之外。 疫病菌孢囊會進行細胞分裂以產生游走子，是其感染寄主時的初級感染原；在潮濕環境下，游走子會泳動至適合的寄主表面，形成靜止子、發芽並入侵寄主，故孢囊的形成及游走子的釋放在病原傳播過程中扮演了重要的角色，但其中詳細的分子機制目前卻尚未完全明瞭。過去的研究發現，添加鈣離子抑制劑會影響孢囊細胞分裂與游走子釋放，認為鈣離子為此過程的關鍵因子。本研究利用序列比對尋找鈣離子訊息傳遞過程的相關蛋白，獲得一真菌高親和性鈣離子通道次體之同源基因—Ppmid1，並以基因靜默 (gene silencing) 之形式取得Ppmid1基因靜默轉殖株。以即時定量聚合酶連鎖反應偵測Ppmid1，發現其在孢囊中的表現量明顯較高。實驗中觀察發現Ppmid1基因靜默株無法生成正常的孢囊，並且無游走子釋出，所形成的孢囊也常直接發芽，但添加離子至誘導孢囊產生的溶液中，有助於Ppmid1基因靜默株所形成的異常孢囊恢復至正常型態，並且釋放游走子。另外於接種試驗中也發現Ppmid1基因靜默株有致病力下降的情形，在煙草所造成的病斑明顯較野生型P. parasitica (94069) 為小，很可能是因為Ppmid1基因靜默株無法產生正常孢囊所致。這些結果顯示Ppmid1極有可能在疫病菌孢囊形成及游走子釋放過程中扮演重要的角色。

Phytophthora parasitica is a notorious oomycete pathogen that causes severe disease in a wide variety of plant species. Plant infection by this pathogen involves mainly its asexual development, whereby papillate sporangia are formed, followed by differentiation and release of biflagellate zoospores. Calcium signaling has been proposed to play a key role in these processes. This study deals with the functional characterization of Ppmid1, which encodes a component of a putative calcium channel. Analysis by quantitative reverse transcriptase-PCR indicated that the expression of Ppmid1 is induced in the stage of sporangia. Moreover, we generated Ppmid1-silenced mutants of P. parasitica, which showed reduced expression of Ppmid1 and exhibited severe defects in its asexual development. Sporangia formed by the mutants lacked papilla and failed to release zoospores in response to cold shock. Instead, they tended to germinate directly. Further analysis revealed that, in response to cold shock for induction of zoospore formation, no sign of cytoplasmic cleavage was detected in the sporangia of Ppmid1-silenced mutants. Interestingly, when the sporangia induction solution was amended with CaCl2, the mutants became able to form papillate sporangia, which released zoospores upon cold shock. Inoculation experiments indicated that Ppmid1-silenced mutants showed reduced virulence toward Nicotiana benthamiana. These results indicated that Ppmid1 plays an important role in the asexual development of P. parasitica.