The APSES Transcription factor Efg1 regulates a novel phenotype switch in Candida parapsilosis [RNA-seq].

In Candida albicans the Efg1 transcription factor (a member of the APSES family) is an important regulator of hyphal growth, and of the white-to-opaque transition. In contrast, we show that the Efg1 ortholog in Candida parapsilosis is a major regulator of a different morphological switch at the colony level, from a concentric to smooth morphology. The rate of switching is at least 100-fold increased in an efg1 knockout relative to wild type. Deleting efg1 also reduces biofilm formation, and results in increased sensitivity to SDS, Congo red, caspofungin and calcofluor white in cells of both morphologies. Biofilm reduction is more dramatic in in vitro than in in vivo models. We use ChIP-seq to show that Efg1 binds to 502 promoter regions, including 70 potential transcription factors or regulatory proteins. Several of the transcription factors belong to networks that regulate biofilm development and white-opaque switching in C. albicans. Efg1 also binds to its own promoter. The binding site for C. parapsilosis Efg1 resembles that of orthologs in other fungi. Many Efg1 targets are probably also regulated by the Ndt80 transcription factor. We show that a paralog of Efg1 (Efh1) is restricted to Candida species. Efh1 does not regulate concentric-smooth phenotype switching, biofilm formation or stress response in C. parapsilosis. Our analysis supports the hypothesis that Efg1 has an ancient role as regulator of development in fungi, but we have identified a new role in C. parapsilosis as a regulator of colony switching that is distinct from the white-opaque switch in C. albicans. Overall design: RNA was isolated from C. parapsilosis wild type (three biological replicates, concentric phenotype), and from efg1 deletion strains (three biological replicates from both concentric and smooth phenotype). Gene expression was determined using strand-specific RNA-seq.在白色念珠菌（Candida albicans）中，Efg1转录因子（Efg1 transcription factor，属于APSES家族（APSES family））是菌丝生长与白色-不透明形态转换的重要调控因子。与之形成鲜明对比的是，本研究证实副念珠菌（Candida parapsilosis）中的Efg1直系同源蛋白是调控一类全新菌落水平形态转换的核心调控因子，该转换为菌落从同心态至光滑态的形态转变。相较于野生型菌株，efg1基因敲除菌株的形态转换速率提升至少100倍。敲除efg1还会削弱生物膜形成能力，并使两种形态的细胞对十二烷基硫酸钠（SDS）、刚果红、卡泊芬净及荧光增白剂的敏感性显著升高。体外模型中生物膜形成能力的削弱程度较体内模型更为突出。本研究通过染色质免疫共沉淀测序（ChIP-seq）证实，Efg1可结合502个启动子区域，其中包含70个潜在转录因子或调控蛋白。其中部分转录因子属于调控白色念珠菌生物膜发育及白色-不透明形态转换的调控网络。Efg1还可结合自身启动子。副念珠菌Efg1的DNA结合位点与其他真菌中的同源蛋白结合位点高度相似。多数Efg1靶基因可能同时受Ndt80转录因子（Ndt80 transcription factor）的调控。本研究还发现，Efg1的旁系同源蛋白Efh1仅存在于念珠菌属物种中。在副念珠菌内，Efh1并不调控同心态-光滑态表型转换、生物膜形成及应激响应。本研究分析结果支持以下假说：Efg1作为真菌发育调控因子具有古老的进化起源，但本研究鉴定出其在副念珠菌中存在全新的功能——作为不同于白色念珠菌白色-不透明转换的菌落形态转换调控因子。整体实验设计：从副念珠菌野生型菌株（3个生物学重复，均为同心态表型）以及efg1基因敲除菌株（3个生物学重复，分别来自同心态与光滑态表型）中提取总RNA，采用链特异性RNA测序（strand-specific RNA-seq）技术检测基因表达水平。