Amnuaykanjanasin Alongkorn, Daub Margaret E
Department of Plant Biology, North Carolina State University, Raleigh, North Carolina 27695, USA.
Fungal Genet Biol. 2009 Feb;46(2):146-58. doi: 10.1016/j.fgb.2008.11.007. Epub 2008 Dec 6.
The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.
缺乏CRG1转录因子的烟草尾孢菌突变体在毒素尾孢菌素的抗性和生物合成方面均有显著降低。我们克隆并测序了之前从野生型（WT）和crg1突变体之间的消减文库中鉴定出的两个基因ATR1和CnCFP的全长拷贝。ATR1是一个ABC转运蛋白基因，有一个4368bp的开放阅读框（ORF），含一个内含子。CnCFP编码一种与菊池尾孢菌CFP具有同源性的MFS转运蛋白，之前认为其与尾孢菌素的输出有关，有一个1975bp的ORF，含三个内含子。ATR1的破坏表明atr1缺失突变体在固体和液体培养中尾孢菌素的产量分别大幅降低（为WT水平的25%和20%）。ATR1破坏突变体对尾孢菌素也表现出适度更高的敏感性。在crg1突变体中组成型表达ATR1可恢复尾孢菌素的生物合成并适度提高抗性。相比之下，在突变体中过表达CnCFP并不能恢复毒素的产生，然而，它适度增强了毒素抗性。这些结果共同表明ATR1在这种真菌中作为尾孢菌素的外排泵发挥作用，并在抗性中起部分作用。
