Lee Mi-Kyung, Kwon Nak-Jung, Choi Jae Min, Lee Im-Soon, Jung Seunho, Yu Jae-Hyuk
Departments of Bacteriology and Genetics, University of Wisconsin, Madison, Wisconsin 53706.
Genetics. 2014 May;197(1):159-73. doi: 10.1534/genetics.114.161430. Epub 2014 Feb 14.
Asexual development (conidiation) of the filamentous fungus Aspergillus nidulans occurs via balanced activities of multiple positive and negative regulators. For instance, FluG (+) and SfgA (-) govern upstream regulation of the developmental switch, and BrlA (+) and VosA (-) control the progression and completion of conidiation. To identify negative regulators of conidiation downstream of FluG-SfgA, we carried out multicopy genetic screens using sfgA deletion strains. After visually screening >100,000 colonies, we isolated 61 transformants exhibiting reduced conidiation. Responsible genes were identified as AN3152 (nsdD), AN7507, AN2009, AN1652, AN5833, and AN9141. Importantly, nsdD, a key activator of sexual reproduction, was present in 10 independent transformants. Furthermore, deletion, overexpression, and double-mutant analyses of individual genes have led to the conclusion that, of the six genes, only nsdD functions in the FluG-activated conidiation pathway. The deletion of nsdD bypassed the need for fluG and flbA∼flbE, but not brlA or abaA, in conidiation, and partially restored production of the mycotoxin sterigmatocystin (ST) in the ΔfluG, ΔflbA, and ΔflbB mutants, suggesting that NsdD is positioned between FLBs and BrlA in A. nidulans. Nullifying nsdD caused formation of conidiophores in liquid submerged cultures, where wild-type strains do not develop. Moreover, the removal of both nsdD and vosA resulted in even more abundant development of conidiophores in liquid submerged cultures and high-level accumulation of brlA messenger (m)RNA even at 16 hr of vegetative growth. Collectively, NsdD is a key negative regulator of conidiation and likely exerts its repressive role via downregulating brlA.
丝状真菌构巢曲霉的无性发育(产孢)通过多种正调控因子和负调控因子的平衡活动来实现。例如,FluG(+)和SfgA(-)调控发育开关的上游调节,而BrlA(+)和VosA(-)控制产孢的进程和完成。为了鉴定FluG-SfgA下游的产孢负调控因子,我们使用sfgA缺失菌株进行了多拷贝遗传筛选。在对超过100,000个菌落进行目视筛选后,我们分离出了61个产孢减少的转化体。相关基因被鉴定为AN3152(nsdD)、AN7507、AN2009、AN1652、AN5833和AN9141。重要的是,性生殖的关键激活因子nsdD存在于10个独立的转化体中。此外,对各个基因的缺失、过表达和双突变分析得出结论,在这六个基因中,只有nsdD在FluG激活的产孢途径中起作用。nsdD的缺失在产孢过程中绕过了对fluG和flbA∼flbE的需求,但不包括brlA或abaA,并且部分恢复了ΔfluG、ΔflbA和ΔflbB突变体中霉菌毒素柄曲霉素(ST)的产生,这表明在构巢曲霉中NsdD位于FLBs和BrlA之间。使nsdD失活导致在液体深层培养中形成分生孢子梗,而野生型菌株在这种培养条件下不发育。此外,同时去除nsdD和vosA导致在液体深层培养中分生孢子梗的发育更加旺盛,并且即使在营养生长16小时时brlA信使核糖核酸(mRNA)也会高水平积累。总的来说,NsdD是产孢的关键负调控因子,可能通过下调brlA发挥其抑制作用。