存在於白紋羽病菌Rosellinia necatrix Prillieux W89 分離株中非內源dsRNA分子之研究

Abstract

由白紋羽病菌 (Rosellinia necatrix Prillieux) 所引起之白紋羽病，為常見之植物根腐病害。此病原菌 (無性世代為Dematophora necatrix Prilleux）在分類上屬子囊菌炭角菌科，Rosellinia屬。其寄主範圍廣泛，台灣地區之發病記錄有七種，包括枇杷、梨樹、桃樹、蘋果、櫻花、葡萄、茶樹等。病株受感染後由根部開始枯死進而全株凋萎，以往使用土壤灌注方法施予藥劑雖可達到一定的防治效果，但長期使用化學藥劑除成本高昂外，更對環境造成沉重負擔，故有開發其他低環境負荷防治方法的必要。約有20%之白紋羽病菌帶有雙股核醣核酸(dsRNA)，且dsRNA具使病原菌弱毒化之潛力。本研究自白紋羽病菌分離株W89中純化dsRNA及病毒顆粒，以建立cDNA library對其基因組進行完整定序。 研究結果顯示，白紋羽病菌W89分離株帶有含四個segment之dsRNA基因組(RnV-W89)，其電泳形式與Chrysoviridae之成員相似，但相對分子量較現知之Chrysovirus為大。對cDNA進行全序列解讀，四股dsRNA之分子大小分別4942、4353、4099與3685bp，依序命名為RNA1、RNA2、RNA3、RNA4，各含一較長之opne reading frame (ORF)，預測轉譯產物分別為1602、1356、1310及1061aa之蛋白質。 進一步對各序列進行分析，RNA3之轉譯產物序列中帶有RNA-dependent RNA polymerase (RdRP)之GDD motif，以BLASTp與資料庫中其他胺基酸進行比對，其與尚未確定分類地位之dsRNA病毒， 如Amasya cherry disease-associated mycovirus和Cherry chlorotic rusty spot associated totiviral-like dsRNA 4有較高相似度；其他ORF則未發現與資料庫中現存之胺基酸有顯著的相似性。 如大多數的病毒分子一般，RnV-W89中之四股dsRNA分子，在5’-與3’-非轉譯區 (untranslated region, UTR)具有序列保守性，分別為5’-(-ACGAAT) 及(TACATGAGAATATT--)-3’。其次於ORF5’上游領域亦保有(CAA)n之重複性序列；值得注意的是此(CAA)n序列與存在於植物病毒tobamoviruses 5’非轉譯區中之增強子有相似之處。純化病毒顆粒之結果，於掃描式電子顯微鏡下可觀察到直徑約48 nm之球狀病毒顆粒；SDS-PAGE分析純化之蛋白質，CBB染色後可見110、100、75及63 kDa等訊號。 綜合以上結果，於R. necatrix W89分離株所發現之dsRNA為新種之真菌病毒，目前尚未能確定其分類定位。未來需進一步對其基因組進行功能性分析，以探討此病毒在生物防治上的潛力。

White root rot is one of the most devastating, worldwide distributed diseases particularly in fruit trees. It is caused by a soilbrone ascomycetous fungus, Rosellinia necatrix Prillieux. To avoid the widespread and attack of this fungus on target plants, a control by drenching 50 to 200 L of fungicide per tree can be achieved. However, it is very labor intensive and raises environmental concerns about soil pollution. It is an attempt of developing environmentally-friendly novel management methods for controlling this phytophathogen, such as using dsRNA mycovirus to reduce the virulence of fungus. In this study, the biological and molecular properties on dsRNA isolated from R. necatrix strain, W89 (RnV-W89) were characterized. RnV-W89 contained four segments of double-stranded (ds) RNA, namely RNA1, RNA2, RNA3 and RNA4. Complementary DNA were cloned and the terminal sequences of each segments were determined by using 3’RNA ligase mediated rapid amplification of the cDNA ends (3’RLM-RACE). The full length of each segment had the size of 4942, 4353, 4099 and 3685 bp, with the deduced amino acids of 1602, 1356, 1310 and 1061 aa, respectively. blastp homology search revealed that the ORF of RNA3 encodes RNA-dependent RNA polymerase（RdRP）that had low levels of sequence identity to the unassigned viruses, such as Amasya cherry disease-associated mycovirus and Cherry chlorotic rusty spot associated totiviral-like dsRNA 4. No significant sequence similarity was found between the deduced amino acid sequences coded by the other ORFs of RnV-W89 and the known protein sequences. As most viruses in having strictly conserved region at their 5’- and 3’- terminal sequences of genome(s) for replication, the genome of RnV-W89 showed sequence similarities between the four segments at their 5’ and 3’ untranslated region (UTR). Moreover, a second region of strong sequence similarity contained a reiteration of the unique sequence “CAA”. These (CAA)n repeats were similar to the enhancer elements presented at the 5’ UTRs of tobamoviruses. The purified viral particles had a size about 48 nm in diameter. SDS-PAGE analysis of the purified virus particles showed that the main bands had the approximate sizes of 110, 100, 75 and 63 kDa. These results showed that four dsRNA segments of RnV-W89 were likely to be the genome segments of a novel mycovirus with a divided dsRNA genome. The present study provides the basic characteristics of dsRNA of RnV-W89. To ascertain the competence as a virocontrol agent of this virus, further studies are necessary.