褐根病菌之基因體與轉錄體分析

Abstract

由褐根病菌(Phellinus noxius)所引起的樹木褐根腐病(Brown root rot)是熱帶及亞熱帶地區樹木常見的樹木病害,主要危害果樹及木 本園藝樹木,常造成根部組織的壞死,使其容易倒伏而造成危害與經 濟損失。褐根病在東南亞國家如臺灣、日本逐漸受到重視,但目前對 其病原菌基因體的了解甚少。本研究針對來自宜蘭大溪的擔孢子分離 株 A42 進行基因體及轉錄體之定序與組裝。Illumina 定序總共建立一 組 paired-end library,與四組 insert size 為 1k 到 9k 不等的 mate-paired libraries,組裝得到大小約 31.8 Mb、N50 為 2.7 Mb 的基因體序列;透過 ab initio 基因預測,共得到 9,684 筆可轉譯成蛋白的核酸序列,其中有 8,574 筆可在 NCBI non-redundant(nr)蛋白資料庫中找到同源性序列。 為了增進基因預測與了解病原在逆境時之基因表現,本研究共針對 5 種條件下之轉錄體進行分析,包含對照組(麥芽萃取物培養基)、溫 度逆境與三組不同的營養逆境(基本培養基、缺乏氮源、缺乏碳源), 結果顯示參與營養代謝、次級代謝物生合成與疏水蛋白生合成途徑上 的基因在不同逆境中均有被誘導的現象;而三組營養逆境中,草酸生 合成的關鍵基因 glyoxylate dehydrogenase (GLOXDH) 與 oxaloacetase チ(OXA) 亦有特別顯著的表現量上升。由於草酸在前人研究中發現可 以增進木材腐朽菌細胞壁分解酵素之活性,為了瞭解草酸代謝、碳源 使用與營養逆境的關係,挑選 18 個參與在三羧酸循環(TCA cycle)、 乙醛酸循環(glyoxylate cycle)、草酸生合成與調控相關的候選基因, 進一步以 qRT-PCR 做驗證。在三次獨立重複試驗及一個從病樹上取 得的菌絲面樣本中,GLOXDH 及 OXA 之表現量,在所有的營養逆境 處理與 in planta 的樣本中均有一致且明顯的上升,顯示草酸在褐根病 菌營養缺乏及侵染寄主時的角色,值得未來深入探討。在與資料庫比 對的結果中,找到兩個控制真菌交配型的基因座:A 與 B 基因座。其 中 A 基因座含有兩對距離 50-kb 的 HD1/HD2 基因組,在 mitochondrial intermediate peptidase(MIP)基因旁邊的一對 HD1/HD2 基因其轉譯方 向相同,此種組合方式可能為刺革菌目之特徵。分析 10 株來自單一子 實體之擔孢子分離,發現 A 基因座明顯分為 2 個序列變異度高之等位 基因,B 基因座則帶有一個具 7 個跨膜結構域的完整費洛蒙受體基因 STE3,但鄰近序列中卻沒有費洛蒙基因。檢視 24 個擔孢子分離株與 チ37 株來自臺灣與日本菌株的 STE3 序列,發現其多樣性低(與保守性 基因 MIP 與 ELF1α 相似),故應與交配型之決定無關。綜合以上結果 可推斷褐根病菌可能是二級異宗交配型(bipolar mating system)。

Phellinus noxius causes brown root rot disease in more than 200 tree species in tropical and subtropical regions. Although the potential risk of toppling and economic losses caused by this disease have gained much attention in East Asia, little is known about the genome of P. noxius. In this study, one paired-end and four mate-paired libraries of a P. noxius basidiospore isolate A42 were sequenced by using Illumina platform, which yielded a total of 31 Gb data. Genome assembly and ab intio gene prediction resulted in a draft genome of ~31.8 Mb (N50 = 2.7 Mb) consisting of 149 scaffolds and 9,684 coding sequences. 8,574 out of the 9,684 predicted genes were annotated according to NCBI non-redundant (nr) database. To facilitate gene annotation and to better understand the expression profiles of genes involved in stress responses, transcriptomes under various stress conditions were examined, encompassing temperature shift, nutrient deficiency (minimal medium, carbon deficiency, nitrogen deficiency), and a control (liquid complete medium). Genes involved in nutrient metabolism, secondary metabolites biosynthesis, and hydrophobin production were induced under all the stress conditions. A particularly up-regulated pattern was found in two oxalate production genes, glyoxylate dehydrogenase (GLOXDH) and oxaloacetase (OXA). Oxalic acid is known to facilitate the activity of cell-wall degrading enzymes in wood-decay fungi. To understand the association of oxalate metabolism, carbon utilization, and nutrient deficiencies, 18 candidate genes involved in tricarboxylic acid (TCA) cycle, glyoxylate (GLOX) cycle, and oxalate metabolism were selected for qRT-PCR validation. A total of 3 replicates from 3 independent trials and an in planta mycelial mat sample from an infected tree were tested. GLOXDH and OXA were consistently highly induced in all nutrient deficiency treatments and the in planta sample. The role of oxalate in P. noxius under nutrient stress and during pathogenesis is worth further investigation. Two unlinked MAT loci, A and B locus, were identified through genomic screening. The A locus of P. noxius encoded two pairs of HD1/HD2 genes separated by a 50-kb insertion. The HD1/HD2 pair flanking to MIP are in head-to-tail arrangement, which may be a characteristic feature of Hymenochaetales. Sequence analysis of the A locus of 10 single-basidiospore isolates from a single basidiocarp showed a clear segregation into two distinct alleles with highly polymorphic sequences. Only one functional STE3- like pheromone receptor with 7 transmembrane domains is identified in P. noxius B locus, but no pheromone gene is found nearby. The STE3 sequences of 24 basidiospore isolates and 37 isolates from Taiwan and Japan revealed no specifically elevated polymorphism compared to the conserved genes MIP and ELF1α. Taken together, a bipolar heterothallic mating system in P. noxius is supported.