神經肽基因flp16及flp18於草莓根腐線蟲致病力所扮演之角色

Abstract

根腐線蟲屬(Pratylenchus spp.)為內寄生濳移型的植食性線蟲，除了卵及未出卵的一齡幼蟲之外，所有齡期皆可侵入並取食寄主植物根部，廣泛分佈於全球，造成農業經濟危害。嚴重的田間線蟲問題使耕種者對廣泛性殺線蟲劑高度依賴，卻也同時危害人類健康。FMRFamide-like peptides (FLPs) 是一種廣泛存在線蟲門(Nematoda)動物神經系統內的神經肽，有潛力成為專一性殺線蟲劑的標的，然而 FLPs 於濳移型植食性線蟲行為調控所扮演的角色有待釐清。本研究希望瞭解草莓根腐線蟲 (Pratylenchus vulnus)之神經系統以及flp 基因於草莓根腐線蟲的功能為何，故透過DiI 染色法得到感覺神經元的表現圖譜，成功染出一對 amphid neurons 及 phasmid neurons。研究透過 3’快速擴增 cDNA 末端聚合酶連鎖反應及基因克隆定序，成功增幅了一個未曾被報導過的 pv-flp18 基因並獲取其序列，且同時確認了 pv-flp16 基因的存在。隨後，以 dsRNA 浸泡法建立 flp16 及 flp18 突變株、調查 flp16 及 flp18於草莓根腐線蟲於移動、侵染等致病力相關之行為所扮演的角色。最後，本研究透過原位雜合技術取得 flp16 及 flp18 轉錄體表現位置，與 C. elegans 神經元圖譜比照後，找出相對應的神經元並比較其功能。自移動行為實驗中，無論是在沙或沙壤土的環境下，pv-flp16 及 pv-flp18 突變株的移動能力都下降了 25%。而在 24 小時侵染行為實驗中，pv-flp18 突變株的侵染能力上升 73.5%，pv-flp16 突變株則是和兩個控制組都沒有顯著差異。最後，以 DIG-標定的 pv-flp 轉錄體圖譜也和行為實驗的結果相符。pv-flp16 和 pv-flp18 轉錄體圖譜於雌蟲中呈現相似的結果，表現在VC4, VC5, HSN 運動神經元中及 uv1 子宮細胞。而在幼蟲中，pv-flp16 轉錄體表現在 AVE 及 BDU 中間神經元，pv-flp18 轉錄體則是在 M2 食道運動神經元及腹部神經索的運動神經元。此論文為第一篇於濳移型植物寄生性線蟲的FLPs研究報告。此研究開啟了草莓根腐線蟲於神經解剖學的探索，不僅揭示了 flp16 及 flp18 於草莓根腐線蟲移動及侵染行為的重要性，且發現同源的 flp 基因於不同植食性線蟲會有不一樣的功能。於應用面上，此研究能作為未來應用 FLPs 為標的開發根腐線蟲屬防治藥劑的基礎。於研究面上，此研究增進了對於草莓根腐線蟲神經系統的瞭解，有助於對其行為的瞭解。

Pratylenchus spp. are migratory plant endoparasitic nematodes which can invade and feed on host roots. They distribute globally and represent a major threat to agriculture. The infective stages of Pratylenchus spp. almost include all life stages excluding egg and J1 stage. This severe nematode problem let growers rely heavily on broad spectrum nematicides for management, but often lead to human health concerns. FMRFamide-like peptides (FLPs) are neuropeptides widely expressed in nematode nervous system that could therefore serve as new specific nematicide targets, whereas the function and regulation mechanism of FLPs in migratory plant parasitic nematodes remains unknown. In this study, we aim to catch up the nervous system and identify the functions of flp neuropeptide genes in strawberry root lesion nematode P. vulnus. Through DiI staining method, one pair of amphid neurons and phasmid neurons were identified in P. vulnus. Through 3’ Rapid Amplification cDNA Ends PCR (3’ RACE PCR), cloning and sequencing, a novel pv-flp18 gene was identified. Besides, the existence of pv-flp16 was confirmed. The roles of pv-flp16 and pv-flp18 in pathogenic-related behaviors were investigated using the flp-knockdowned mutants obtained by dsRNA soaking method. Finally, compare the corresponding neuron functions in C. elegans through acquiring flp16 and flp18 transcript localization patterns with in situ hybridization. As results, the migration rate of pv-flp16 and pv-flp18 mutants were significantly decreased by 25% no matter in sands or sandy loam test column. The invasion rate of pv-flp18 mutants was significantly increased by 73.5% in 24 hr assay, while pv-flp16 mutants showed no difference to controls. Microscopic imaging of DIG-labelled pv-flp transcript patterns from in situ hybridization were consistent with behavioral assay results. Both pv-flp16 and pv-flp18 transcripts revealed similar patterns in female, with expressions in VC4, VC5, HSN motor neurons, and uv1 cells. In juveniles, pv-flp16 transcripts were found in AVE and BDU interneurons while pv-flp18 transcripts are in M2 pharyngeal motor neuron and ventral nerve cord motor neurons. This is the first FLPs research on migratory plant parasitic nematodes. and neuroanatomy study of P. vulnus starts off. To conclude, this study reveals the importance of flp16 and flp18 in migration and invasion behaviors of P. vulnus. Also, flp homologus genes among different PPNs would have different functions. For future application, this study sheds a light on using FLPs as a potential target for new Pratylenchus spp. control strategy development. For research fields, this study improves the knowledge of nervous system and nematode behaviors in P. vulnus.