探討活性氧及鈣離子在白絹病菌菌核形成之功能

Abstract

菌核 (sclerotia) 為真菌菌絲發育形成的特殊構造，對環境逆境的耐受性高，可於田間殘存並作為初次感染源。菌核的形成受到養分、光、酸鹼值、溫度及活性氧 (reactive oxygen species, ROS) 等多種因子影響，目前多數研究支持ROS在誘導菌核數量、大小及色素累積具重要性。除了ROS外，作為細胞訊息傳遞信使的鈣離子(calcium ion, Ca2+) 也會影響菌核形成，雖有諸多研究指出ROS及Ca2+在細胞內調節路徑相互影響，但兩者在真菌菌核形成過程中的角色及其交互作用仍有待探討。本研究以白絹病菌 (Athelia rolfsii) 作為材料，針對菌核形成的四個時期 (包含菌絲期、初始菌核期、菌核發展期、及菌核成熟期) 進行轉錄體分析，分析結果顯示ROS解毒基因大量表現於菌核發展期和成熟期，且後續實驗的結果亦顯示氧化壓力的變動不會誘導菌核的形成，僅有添加抗氧化劑二硫蘇糖醇 (DTT) 和穀胱氨酸 (glutathione) 會導致菌核色素沈澱的能力下降，故認為ROS主要影響在A. rolfsii菌核形成後期。ROS抑制試驗中，僅在添加glutathione的處理下會抑制A. rolfsii菌核形成數量，因文獻指出glutathione亦會誘導Ca2+釋放，故後續實驗藉由調整培養基中CaCl2濃度以測試Ca2+對菌核數量的影響，並於結果中發現CaCl2添加濃度提高會導致A. rolfsii、Macrophomina phaseolina、和Sclerotinia sclerotiorum菌核或微菌核的數量下降。此外，在探討ROS及Ca2+交互影響的實驗中，發現僅有Ca2+會降低A. rolfsii的菌核形成數量，反觀ROS則對後期發育和成熟階段影響較大；而在M. phaseolina之微菌核和S. sclerotiorum之菌核形成，ROS及Ca2+皆參與其中，且ROS及Ca2+交互影響模式在兩種真菌之間有所差異。總結上述，本研究針對A. rolfsii之菌核形成階段進行轉體學分析，另藉由改變氧化壓力的實驗驗證ROS參與在菌核形成後期，且以Ca2+作為抑制A. rolfsii菌絲發育成菌核之主要因子，並發現雖然ROS及Ca2+皆參與在菌核形成路徑，但其功能及機制因真菌種類而異。

Sclerotia are specialized structures formed by various fungi, and they serve as the long-term survival structures and primary inoculum in the subsequent season. Sclerotia formation is influenced by several factors, including nutrients, light, pH, temperature, and reactive oxygen species (ROS). Among these factors, ROS is one of the most studied factors and its importance in affecting the number, size, and melanin accumulation of sclerotia has been supported. In addition to ROS, calcium ion (Ca2+) acts as the second messenger and participates in sclerotia formation. Several studies revealed that ROS and Ca2+ interact with each other in many pathways, but the individual effects and crosstalk between ROS and Ca2+ during sclerotia formation remain to be explored. In this study, RNA-Sequencing was performed on four stages of sclerotia formation, including the mycelial stage, initiation stage, developmental stage, and maturation stage of Athelia rolfsii. Transcriptome analysis revealed that ROS detoxification genes are highly expressed in the developmental and mature stages. Consistently, changing the ROS stress in growth media did not affect the sclerotia number, and the addition of antioxidants DTT and glutathione affected only pigmentation of sclerotia. The observations suggested that ROS are involved in the later stages of A. rolfsii sclerotia formation. However, in the treatment of glutathione, which is known to induce Ca2+ influx, resulted in the reduction of sclerotia number. The subsequent experiments with amended CaCl2 in culture media revealed a significant decrease in the sclerotia number of not only A. rolfsii, but also Macrophomina phaseolina and Sclerotinia sclerotiorum. Investigating the individual effect and the crosstalk between ROS and Ca2+ revealed that only Ca2+ is involved in the sclerotia formation of A. rolfsii, acting as the suppression signal for hyphal differentiation into sclerotia, while ROS may play roles in the latter developmental and mature stages. On the other hand, both ROS and Ca2+ are participated in the sclerotia formation of M. phaseolina and S. sclerotiorum. However, the crosstalk between ROS and Ca2+ cause different results on these two fungi. Collectively, this study completed the transcriptomic analysis to investigate the sclerotia formation of A. rolfsii. It validated the involvement of ROS in the later stages of sclerotia formation and confirmed that Ca2+ acts as a negative stimulus for hyphal differentiation into sclerotia. This study also pointed out that ROS and Ca2+ both play roles in sclerotia formation pathway, with unique mechanisms depending on the fungal species.