Conditional promoters for analysis of essential genes in Zymoseptoria tritici.
Conditional promoters for analysis of essential genes in Zymoseptoria tritici.
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DOI：
10.1016/j.fgb.2015.03.024
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发表时间：
2015-06
期刊：
影响因子：
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通讯作者：
Steinberg G
中科院分区：
文献类型：
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作者：
Kilaru S;Ma W;Schuster M;Courbot M;Steinberg G
我们建立了5个用于小麦的诱导/抑制启动子。在培养基补充诱导下，所有启动子均表达细胞质GFP。测试条件启动子的表达范围从弱到强。5个启动子中有3个在抑制条件下紧密表达，2个启动子呈现背景表达。条件α-微管蛋白突变体在抑制条件下死亡，证明了这一原理。为了可持续地控制植物真菌病原体，开发新的杀菌剂需要鉴定新的抗真菌靶点。必需的真菌特异性蛋白质是很好的候选者，但由于它们的重要性，基因缺失突变体是不可行的。因此，它们在细胞中的作用往往是难以捉摸的。这种障碍可以通过使用条件突变体来克服，条件突变体的表达由诱导/抑制启动子控制。本研究引入5个诱导/抑制启动子系统，对小麦酵母病病原菌的关键基因进行了研究。我们将增强绿色荧光蛋白（egfp）基因融合到小麦硝酸还原酶（Pnar1，氮诱导，铵抑制）、1,4-β-内木聚糖酶A （Pex1A，木糖诱导，麦芽糊精抑制）、l-阿拉伯糖醛酸苷酶B （PlaraB，阿拉伯糖诱导，葡萄糖抑制）、半乳糖-1-磷酸尿苷转移酶7 （Pgal7，半乳糖诱导，葡萄糖抑制）和异柠檬酸裂解酶（Picl1，葡萄糖抑制）的启动子区域。醋酸钠诱导，葡萄糖抑制)。然后在诱导和抑制条件下定量分析细胞质报告荧光。我们发现Pnar1， PlaraB和Pex1A在被抑制时很少或不驱动egfp表达，但在被诱导时诱导适度的蛋白产生。相比之下，Pgal7和Picl1在受到抑制时表现出相当大的egfp表达，并且在它们的诱导剂存在下被强烈诱导。将所有启动子的表达水平正常化为α-微管蛋白启动子Ptub2的表达水平，发现PlaraB是最弱的启动子（约占Ptub2的20%），而Picl1则强烈表达报告子（约占Ptub2的250%）。这些工具的使用有望更好地了解必需基因，这将有助于开发新的控制策略，保护小麦免受小麦小麦黑穗病的侵害。
We establish five inducible/repressible promoters for use in Z. tritici. All promoters express cytoplasmic GFP when induced by media supplements. The tested conditional promoter range from weak to strong expression. 3 of 5 promoters are tight under repressed conditions, 2 promoters show background expression. A conditional α-tubulin mutant dies under repressed conditions, showing proof of principle. Development of new fungicides, needed for sustainable control of fungal plant pathogens, requires identification of novel anti-fungal targets. Essential fungal-specific proteins are good candidates, but due to their importance, gene deletion mutants are not viable. Consequently, their cellular role often remains elusive. This hindrance can be overcome by the use of conditional mutants, where expression is controlled by an inducible/repressible promoter. Here, we introduce 5 inducible/repressible promoter systems to study essential genes in the wheat pathogen Zymoseptoria tritici. We fused the gene for enhanced green-fluorescent protein (egfp) to the promoter region of Z. tritici nitrate reductase (Pnar1; induced by nitrogen and repressed by ammonium), 1,4-β-endoxylanase A (Pex1A; induced by xylose and repressed by maltodextrin), l-arabinofuranosidase B (PlaraB; induced by arabinose and repressed by glucose), galactose-1-phosphate uridylyltransferase 7 (Pgal7; induced by galactose and repressed by glucose) and isocitrate lyase (Picl1; induced by sodium acetate and repressed by glucose). This was followed by quantitative analysis of cytoplasmic reporter fluorescence under induced and repressed conditions. We show that Pnar1, PlaraB and Pex1A drive very little or no egfp expression when repressed, but induce moderate protein production when induced. In contrast, Pgal7 and Picl1 show considerable egfp expression when repressed, and were strongly induced in the presence of their inducers. Normalising the expression levels of all promoters to that of the α-tubulin promoter Ptub2 revealed that PlaraB was the weakest promoter (∼20% of Ptub2), whereas Picl1 strongly expressed the reporter (∼250% of Ptub2). The use of these tools promises a better understanding of essential genes, which will help developing novel control strategies that protect wheat from Z. tritici.