Sordaria fimicola 光調控子實體發育及向光性分子機制之探討

Abstract

Sordaria fimicola為同宗交配型糞生子囊菌，透過細胞和形態的改變，以及多細胞組織分化，進而產生子實體，完成其有性生殖。S. fimiocla為研究遺傳重組和真菌向光性的重要生物系統，然而到目前為止，S. fimicola向光反應的分子調控機制，仍不甚清楚。在本研究中，我們以S. fimicola作為實驗系統，找到並驗證其光感受基因Sfwc-1。S. fimicola光感受蛋白質SfWC-1，含有LOV，PAS和其他保守結構區域，與模式子囊菌N. crassa 藍光感受蛋白質WC-1具有高度同源性。此外，我們亦找到並探討其光適應調控基因Sfvvd。利用農桿菌介導原生質體基因轉殖技術，經由同源重組分別成功完成Sfwc-1基因LOV保守結構區域及整個Sfvvd基因的置換，得到Sfwc-1(∆lov)及ΔSfvvd突變株。Sfwc-1(∆lov)突變株營養生長正常，但在光照條件下菌絲體類胡蘿蔔素的累積明顯減少；然而ΔSfvvd突變株在光照條件下，菌絲體類胡蘿蔔素的累積明顯增加，且菌絲生長明顯受到抑制。此外，與野生菌株相比，Sfwc-1(∆lov)突變株子囊殼的形成延遲且數量明顯減少，且子囊殼的形成失去光暗週期的帶狀特徵，此外子囊殼頸失去向光生長的特性；而ΔSfvvd突變株子囊殼頸生長亦失去向光性，但形態特徵與Sfwc-1(∆lov)突變株有所不同。突變株性狀分析結果顯示，Sfwc-1 and Sfvvd 基因共同參與S. fimicola生理及子囊殼形態分化的調控。進一步轉錄體及基因表現的分析，支持Sfwc-1光誘導調控其生理和子囊殼形態分化的重要功能，並且顯示相關分子調控網絡。此外，GFP螢光蛋白標定實驗顯示，SfWC-1蛋白質在菌絲主要集中於細胞核中，並且受光誘導短暫大量累積；然而SfVVD螢光蛋白質在黑暗中不見表現，但受光明顯誘導，並且存在於菌絲細胞質及細胞核中。本研究探討S. fimicola藍光感受及調控蛋白質，藉由本研究的發現期望有助於闡明真菌藍光調控基因功能及其網絡。

Sordaria fimicola, a coprophilous ascomycete, is a homothallic fungus that can undergo sexual differentiation with cellular and morphological changes followed by multicellular tissue development to complete its sexual cycle. S. fimicola is an important model to study genetic recombination and phototropism. However, its molecular mechanism related to phototropic response is largely unknown. In this study, we identified and characterized the blue light photoreceptor gene Sfwc-1 in S. fimicola. SfWC-1 photoreceptor contains light-oxygen-voltage (LOV)-sensing, Per-Arnt-Sim (PAS) and other conserved domains and is highly homologous to the WC-1 blue-light photoreceptor of Neurospora crassa. Moreover, we also identified and characterized vivid orthologue (Sfvvd) in S. fimicola. The LOV domain of Sfwc-1 and entire Sfvvd gene were deleted by homologous recombination using Agrobacterium-mediated protoplast transformation. The Sfwc-1(∆lov) mutant showed normal vegetative growth but produced less carotenoid pigment, whereas ΔSfvvd mutants displayed enhanced accumulation of carotenoid in hyphae and dramatic reduction of mycelial growth under constant light illumination. The Sfwc-1(∆lov) mutant showing delayed and less pronounced perithecial formation, was defective in phototropism of perithecial beaks, and lacked the fruiting-body zonation pattern compared with the wild type under illumination condition. Interestingly, ΔSfvvd mutants were also defective in photoresponsive bending of perithecial beaks but morphological feature was different from that found in Sfwc-1(∆lov). These results suggest Sfwc-1 and Sfvvd genes coordinately mediate not only physiology but also the development of sexual fruiting body in S. fimicola. Transcriptome and gene expression analyses further supported the importance of light-induced functions of Sfwc-1 and photoresponsive networks in the physiology and developmental process of perithecial formation. Moreover, GFP-tagged SfWC-1 fluorescent signals were transiently strong upon light induction and prominently located inside the nuclei of living hyphae. In contrast, SfVVD fluorescent signals were hardly seen in the dark but apparently observed both in cytosol and nuclei under illumination condition. Our studies focus on the putative blue light photoreceptor and regulator in a model ascomycete and contribute to better understandings of their photoregulatory functions and networks across diverse fungal phyla.