Bugeja Hayley E, Hynes Michael J, Andrianopoulos Alex
Department of Genetics, University of Melbourne, Victoria, Australia.
Eukaryot Cell. 2010 Apr;9(4):578-91. doi: 10.1128/EC.00226-09. Epub 2010 Jan 29.
Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. The opportunistic human pathogen Penicillium marneffei is a dimorphic fungus, displaying vegetative (proliferative) multicellular hyphal growth at 25 degrees C and unicellular yeast growth at 37 degrees C. P. marneffei also undergoes asexual development into differentiated multicellular conidiophores bearing uninucleate spores. These morphogenetic processes require regulated changes in cell polarity establishment, cell cycle dynamics, and nuclear migration. The RFX (regulatory factor X) proteins are a family of transcriptional regulators in eukaryotes. We sought to determine how the sole P. marneffei RFX protein, RfxA, contributes to the regulation of morphogenesis. Attempts to generate a haploid rfxA deletion strain were unsuccessful, but we did isolate an rfxA(+)/rfxADelta heterozygous diploid strain. The role of RfxA was assessed using conditional overexpression, RNA interference (RNAi), and the production of dominant interfering alleles. Reduced RfxA function resulted in defective mitoses during growth at 25 degrees C and 37 degrees C. This was also observed for the heterozygous diploid strain during growth at 37 degrees C. In contrast, overexpression of rfxA caused growth arrest during conidial germination. The data show that rfxA must be precisely regulated for appropriate nuclear division and to maintain genome integrity. Perturbations in rfxA expression also caused defects in cellular proliferation and differentiation. The data suggest a role for RfxA in linking cellular division with morphogenesis, particularly during conidiation and yeast growth, where the uninucleate state of these cell types necessitates coupling of nuclear and cellular division tighter than that observed during multinucleate hyphal growth.
真菌是能够经历多种复杂发育程序的小型真核生物。机会性人类病原体马尔尼菲青霉菌是一种二态真菌，在25摄氏度时呈现营养型（增殖性）多细胞菌丝生长，在37摄氏度时呈现单细胞酵母生长。马尔尼菲青霉菌还会经历无性发育，形成带有单核孢子的分化多细胞分生孢子梗。这些形态发生过程需要在细胞极性建立、细胞周期动态和核迁移方面进行调控变化。RFX（调节因子X）蛋白是真核生物中的一类转录调节因子。我们试图确定马尔尼菲青霉菌唯一的RFX蛋白RfxA如何促进形态发生的调控。生成单倍体rfxA缺失菌株的尝试未成功，但我们确实分离出了一个rfxA(+)/rfxADelta杂合二倍体菌株。使用条件性过表达、RNA干扰（RNAi）和显性干扰等位基因的产生来评估RfxA的作用。RfxA功能降低导致在25摄氏度和37摄氏度生长期间有丝分裂缺陷。在37摄氏度生长期间，杂合二倍体菌株也观察到了这种情况。相比之下，rfxA的过表达导致分生孢子萌发期间生长停滞。数据表明，rfxA必须受到精确调控以进行适当的核分裂并维持基因组完整性。rfxA表达的扰动也导致细胞增殖和分化缺陷。数据表明RfxA在将细胞分裂与形态发生联系起来方面发挥作用，特别是在分生孢子形成和酵母生长期间，这些细胞类型的单核状态需要核分裂和细胞分裂的耦合比多核菌丝生长期间观察到的更紧密。
