Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici.
Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici.
复制标题
DOI：
10.1016/j.fgb.2015.03.023
复制
发表时间：
2015-06
期刊：
影响因子：
--
通讯作者：
Steinberg G
中科院分区：
文献类型：
--
作者：
Ma W;Kilaru S;Collins C;Courbot M;Steinberg G
我们提供了三个酵母和两个杂交文库用于小麦纹枯病菌。质量控制实验证实存在长达2000个碱基对的cDNA插入片段。使用Atg4和Atg8，我们表明这些文库对识别蛋白质相互作用是有用的。病原菌对抗真菌治疗不断产生耐药性。因此，寻找新的杀菌剂靶标具有重要意义。很好的候选者是必不可少的真菌蛋白及其调节器。寻找相互作用的因素是揭示必需蛋白质分子环境的有效方法。在这里，我们建立了三个酵母双杂交文库，涵盖了小麦病原菌ZymosepVictoria tritici的酵母期和菌丝期。3个文库均未发现可检测到的基因组DNA。随机扩增结果显示，文库包含长达2000个碱基对的cDNA片段，表明文库中含有中小型蛋白质。事实上，在所有文库中都发现了五种蛋白质的全长cDNA。大片段甲壳素合成酶基因mcs1(含内含子5742个碱基，不含内含子5568个碱基)全长cDNA未能扩增，但其5‘和3’区均有表达，表明文库中含有更大的基因。最后，我们测试了自噬蛋白ZtAtg4和ZtAtg8在Z.tritici中预期的相互作用，然后使用ZtAtg4筛选其中一个双杂交文库。事实上，我们发现ZtAtg8是一个积极的相互作用伙伴，证实了可以识别相互作用的蛋白质。因此，这些分子工具有望在识别新的杀菌剂靶蛋白方面发挥作用。
We provide three yeast two hybrid libraries for use in Z. tritici. Quality control experiments confirm the presence of cDNA insert of up to 2000 bp. Using Atg4 and Atg8 we show that the libraries are useful to identify protein interactions. Pathogenic fungi are constantly emerging resistance to anti-fungal treatments. Therefore, identification of new fungicide targets is important. Good candidates are essential fungal proteins and their regulators. An efficient way to reveal the molecular environment of an essential protein is the search for interacting factors. Here, we establish three yeast two-hybrid libraries, covering yeast and hyphal stages of the wheat pathogen Zymoseptoria tritici. No detectable genomic DNA was present in any of the 3 libraries. Random amplification revealed that the libraries include cDNA fragments of up to 2000 bp, suggesting that small-to-medium sized proteins are represented therein. Indeed, full-length cDNAs of five proteins were found in all libraries. The full-length cDNA of large chitin synthase gene mcs1 (5742 bp with introns; 5568 bp without introns) could not be amplified, but its 5′ and 3′ regions were represented, suggesting that even larger genes are covered in all libraries. Finally, we tested for the expected interaction of the autophagy proteins ZtAtg4 and ZtAtg8 in Z. tritici, and then used ZtAtg4 to screen one of the two-hybrid libraries. Indeed, we found ZtAtg8 as a positive interaction partner, confirming that interacting proteins can be identified. Thus, these molecular tools promise to be useful in identifying novel fungicide target proteins.