鑑定秀麗隱桿線蟲促使線蟲捕捉菌捕捉網生成的信號

Abstract

自然界中，線蟲捕捉菌可以感知線蟲的存在，而 Arthrobotrys oligospora 是常見 的線蟲捕捉菌之一。當 A. oligospora 接觸到秀麗隱桿線蟲 (Caenorhabditis elegans) 時，捕捉網便會開始形成，將秀麗隱桿線蟲捕獲並分解以吸取其養分。本實驗室先前 的研究發現，秀麗隱桿線蟲散發出的費洛蒙-ascarosides ，可以使 A. oligospora 產生 捕捉網。然而，無法生成大部分之 ascarosides 的秀麗隱桿線蟲 daf-22 突變株。相較 於秀麗隱桿線蟲 N2(labstrain) 促使 A.oligospora 產生的捕捉網明顯變少，但還是能 夠促使 A.oligospora 產生捕捉網。因此，我們認為除了 ascarosides ，還有其他的因 子也會被 A.oligospora 所辨認，並促使其產生捕捉網。本研究為了鑑定出這些因子， 我們利用 ethyl methanesulfonate 誘變劑處理 daf-22 突變株，使其隨機的產生突變， 再進一步篩選出，會使 A.oligospora 產生較少捕捉網的突變株。我們總共分離出大約 27000 株 F2 子代，並測試其中大約16000 株，其促使 A. oligospora 產生捕捉網的能 力，也篩選出三株突變株，比起 daf-22 突變株會促使更少的捕捉網產生。拿到突變 株後，我們將三株中具有最明顯性狀的一株，也就是會使 A.oligospora 產生最少捕捉 網的秀麗隱桿線蟲突變株去做基因圖譜 (single nucleotide polymorphism mapping) 和 全基因體定序 (whole-genome sequencing)。根據分析結果找到了極有可能和促使 A.oligospora 產生捕捉網有關的基因。最後，利用 CRISPR-Cas9 的技術，在秀麗隱桿線 蟲的基因 cwp-5 和 npa-1 造成 gene deletion。而 cwp-5 和 npa-1 突變株，比起秀麗 隱桿線蟲野生株，促使 A. oligospora 產生更少的捕捉網。由此可知， cwp-5 和 npa- 1 可能對於促使 A. oligospora 產生捕捉網，扮演了非常重要的角色。

The nematode-trapping fungus Arthrobotrys oligospora can sense the presence of nematodes. When A. oligospora is exposed to C. elegans, trap morphogenesis is induced. Previous studies showed that ascarosides, a group of conserved nematode pheromones, are sufficient to induce trap morphogenesis in A. oligospora. However, the C. elegans daf-22 mutant which is defective in the production of most ascarosides, is still capable of triggering trap morphogenesis, suggesting that additional cues from the nematodes can be recognized. To identify the potential non-ascaroside factors from C. elegans that trigger trap morphogenesis, we used ethyl methanesulfonate to mutagenize the daf-22 mutant and screened for mutants that exhibited decreased trap induction ability in A. oligospora. Approximately 27,000 F2 mutagenized nematodes were isolated, and 16,000 of which werescreened for their ability to induce traps. Three mutants that induced fewer traps than daf-22 were identified from genetic screens. We used genetic mapping and whole-genome sequencing to identify the mutations that caused decreased trap induction phenotype in the mutant that exhibited the strongest phenotype and identified loss-of-function mutations in cwp-5 and npa-1. Clean deletion mutants of cwp-5 and npa-1 were generated via CRISPR- Cas9 and the mutants were found to induce fewer traps in A. oligospora than the wild-type N2 strain, indicating that cwp-5 and npa-1 play a role in the induction of trap formation.