炭疽病菌Nep1-like基因之選殖與功能性分析

Abstract

炭疽病菌Colletotrichum gloeosporioides (Penz.) Penz.& Sacc.為重要的植物病原真菌。其寄主範圍很廣，在熱帶果實上常造成潛伏感染，對我國水果生產及外銷影響重大。該菌感染寄主的營養型態Hemibiotroph可分為Biotrophic及Necrotrophic兩階段。病原菌在Biotrophic時期侵染寄主後，開始潛伏而不立即產生病徵，而在轉變為Necrotrophic時期後，便迅速擴張並分泌多種effectors造成寄主細胞死亡，產生黑色壞疽病徵。Nep1-like protein (NLP)為能夠迅速誘導雙子葉植物細胞死亡的effector基因。在多種微生物物種中，存在有數量不一的NLP同源性基因，形成廣大的基因家族。在先前研究中發現，NLP在病原菌侵染植物後期的表現量增加，並且其致死機制與植物細胞膜的穩定及計畫性死亡(Programmed cell death)相關。然而，NLP對於病原菌致病性的重要性，目前仍無明確的結論。因此，本研究的目標是自炭疽病菌鑑定NLP基因，並進行功能性分析。結果中，我們成功從炭疽病菌選殖出單一的NLP基因，並命名為CgNlp1。以反轉錄聚合酶鏈鎖反應分析後，發現其表現量會受環境中pH值影響，且在感染芒果的後期大量表現。將CgNlp1以農桿菌(Agrobacterium tumefaciens)表現在菸草中，發現會在36小時後造成壞疽病徵，而共表現疫病菌(Phytophthora parasitica) Suppression of necrosis 1(SNE1)與CgNlp1基因時，能夠抑制細胞死亡的發生。而我們亦在炭疽病菌上嘗試建立Polyethylene glycol (PEG)介導之轉型系統，以期能用於未來目標基因突變的研究。

Colletotrichum gloeosporioides (Penz.) Penz.& Sacc., which causes anthracnose on a wide range of crops, is regarded as a hemibiotrophic pathogen. In the biotrophic stage of infection, no obvious necrosis symptom appears on the host; however, in the necrotrophic stage, C. gloeosporioides causes rapid and wide-spread cell death of plant cells, which constitutes the lesion symptom characteristic of anthracnose. Nep1-like proteins (NLPs), which were named after the necrosis and ethylene-inducing protein (NEP1) of Fusarium oxysporum f. sp. erythroxyli, comprise a large family of secreted proteins that cause rapid cell death and general stress responses on dicotyledonous plants. In this study, we found that C. gloeosporioides contains a single copy gene encoding NLP and named it CgNlp1. Analysis by reverse transcriptase-polymerase chain reaction indicated that the CgNlp1 message was detectable in mycelia harvested from different cultures and inoculation of the mango fruit. Moreover, its expression was enhanced by alkalization and as the infection proceeding. To characterize the function, we expressed the gene by agroinfiltration, and found that the protein of CgNlp1 caused necrosis on the tobacco plants 36 hours post infiltration. These results indicated that CgNlp1 expression is related with environmental factor and infection, also its gene product is able to cause cell death on plants. We prospect to develop a PEG-mediated transformation system for functional analysis. Whether CgNlp1 plays an essential role in the pathogenesis of C. gloeosporioides awaits further investigation.