運用 RNAi 技術探討褐根病菌 PnCYP51.1 的角色

Abstract

樹木褐根病 (brown root rot disease, BRRD) 由木材腐朽菌 Phellinus noxius (syn. Pyrrhoderma noxium) 引起，可危害多種樹木的根部及莖基部，造成樹勢衰弱，嚴重時會導致樹木死亡，進而造成樹木倒伏風險及經濟損失。P. noxius 具有異核性，且不易人工培養產生子實體及單核擔孢子，因此難以透過同源重組或基因編輯方式獲得同核型突變株，使基因功能分析受到限制。本研究旨在評估 RNA 干擾 (RNA interference, RNAi) 作為替代策略之可行性，並以麥角固醇生合成相關基因 CYP51 為標靶，解析其在褐根病菌中的功能。首先透過序列分析，由 P. noxius 基因體中鑑別出多個 RNAi 核心因子 (Argonaute 與 Dicer) 之同源基因，顯示其可能具備 RNAi 機制。針對 P. noxius 基因體中兩個 CYP51 同源基因 PnCYP51.1 (PNOK_0784800) 與 PnCYP51.2 (PNOK_0407000)，利用 42 株臺灣 P. noxius 菌株進行核苷酸多樣性分析，發現兩基因之序列變異可能受到純化選擇 (purifying selection) 的限制。胺基酸序列分析顯示，PnCYP51.1 及 PnCYP51.2 與其他擔子菌的 CYP51 屬於同一演化分支，其蛋白質結構與已知真菌 CYP51 高度保守，且具備與 lanosterol 等受質結合之保守結構特徵。為評估 P. noxius 對外源RNA之吸收性，以螢光標定 dsRNA 及 siRNA 處理褐根病菌節生孢子，結果顯示 siRNA 於處理後 36 小時在部分菌絲內可見螢光訊號。由於 P. noxius 侵染毛果楊 (Populus trichocarpa) 及在培養基上生長時，僅能測得 PnCYP51.1 的穩定表現，因此後續對其進行功能驗證。本研究以 Escherichia coli 生產標靶 PnCYP51.1 的 dsRNA (標靶 cDNA 1155–1653 bp處)，另以化學合成兩條標靶 PnCYP51.1 中段 (498–520 bp) 及後段 (1579–1601 bp) 之 siRNA。PnCYP51.1-siRNA 可抑制 P. noxius 之節生孢子發芽及菌絲生長，且標靶基因中段區域的 siRNA 抑制效果較佳；而 PnCYP51.1-dsRNA 僅於高濃度處理下抑制孢子發芽。於毛果楊離莖段接種試驗中，PnCYP51.1-siRNA 與 PnCYP51.1-dsRNA 處理皆略為降低褐化病斑長度，但與各自非標靶 RNA 對照組間未達顯著差異。Reverse transcription-quantitative PCR (RT-qPCR) 分析顯示，節生孢子經 dsRNA 與 siRNA 處理 72 小時後的菌絲中，PnCYP51.1 表現量並未下降，推測與褐根病菌對外源 RNA 吸收效率較低及 RNAi 介導的基因靜默作用時間性相關。本研究初步建立 P. noxius 之 RNAi 表型評估平台，藉此驗證 PnCYP51.1 為影響褐根病菌生長之關鍵功能基因，相關成果有助於對 P. noxius 生理代謝的瞭解，也為未來基因功能分析及新型防治技術研發建立基礎。

Brown root rot disease (BRRD) is caused by the wood-decaying fungus Phellinus noxius (syn. Pyrrhoderma noxium), which infects the roots and basal stems of diverse trees, leading to tree decline and, in severe instances, death, thereby increasing the risk of tree fall and causing economic losses. Due to the heterokaryotic nature of P. noxius and the difficulty in producing fruiting bodies and monokaryotic basidiospores in culture, obtaining homokaryotic mutants through homologous recombination or gene editing remains a challenge, thereby limiting functional gene analysis. This study aimed to evaluate the feasibility of RNA interference (RNAi) as an alternative strategy and to investigate the function of the ergosterol biosynthesis–related gene CYP51 in P. noxius. First, sequence analysis revealed multiple homologs of core RNAi components (Argonaute and Dicer) in the P. noxius genome, suggesting a potentially functional RNAi mechanism. Nucleotide diversity of two CYP51 homologs in the P. noxius genome, PnCYP51.1 (PNOK_0784800) and PnCYP51.2 (PNOK_0407000), was analyzed using the genomic sequences of 42 Taiwanese P. noxius isolates, revealing that sequence variation in both genes is likely restricted by purifying selection. Amino acid sequence analyses showed that PnCYP51.1 and PnCYP51.2 cluster within the same evolutionary branches as CYP51 from other basidiomycetes. Furthermore, their protein structures are highly conserved with those of known fungal CYP51, retaining conserved structural features associated with the binding of substrates such as lanosterol. To evaluate the uptake of exogenous RNA by P. noxius, fluorescently labeled dsRNA and siRNA were applied to arthrospores. Fluorescence signals were observed within a portion of the hyphae after 36 hours post siRNA treatment. Since PnCYP51.1 exhibited stable expression during infection of Populus trichocarpa and growth on culture medium, it was selected for subsequent functional validation. In this study, dsRNA targeting PnCYP51.1 (target region: 1155–1653 bp of the cDNA) was produced by Escherichia coli, while two siRNAs targeting the middle (498–520 bp) and downstream (1579–1601 bp) regions of PnCYP51.1 were chemically synthesized. PnCYP51.1-siRNA inhibited arthrospore germination and hyphal growth of P. noxius, with siRNAs targeting the middle regions showing stronger inhibitory effects, whereas PnCYP51.1-dsRNA inhibited spore germination only at high concentration. In P. trichocarpa detached stem inoculation assays, both siRNA and dsRNA treatments slightly reduced lesion length; however, no significant differences were observed relative to their respective non-targeting RNA controls. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the expression level of PnCYP51.1 in hyphae derived from arthrospores did not decrease after 72 hours of treatment with PnCYP51.1-dsRNA or PnCYP51.1-siRNA, which may be attributable to a relatively low efficiency of exogenous RNA uptake in P. noxius and the timing of RNAi-mediated gene silencing. In this study, an RNAi-based phenotypic evaluation platform for P. noxius was preliminarily established, through which PnCYP51.1 was demonstrated as a critical gene for P. noxius growth. These findings enhance our understanding of the physiology and metabolism of P. noxius and provide a foundation for future gene functional analysis and the development of novel control strategies.