Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" - Universidad de Málaga - Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Departamento de Microbiología, Universidad de Málaga, Bulevar Louis Pasteur 31 (Campus Universitario de Teatinos), 29071 Málaga, Spain.
Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" - Universidad de Málaga - Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Estación Experimental "La Mayora", 29750 Algarrobo-Costa, Málaga, Spain.
Fungal Biol. 2014 Feb;118(2):228-41. doi: 10.1016/j.funbio.2013.12.001. Epub 2013 Dec 17.
The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii) is the main causal agent of cucurbit powdery mildew and one of the most important limiting factors for cucurbit production worldwide. Despite the fungus' economic importance, very little is known about the physiological and molecular processes involved in P. fusca biology and pathogenesis. In this study, we isolated and characterised the β-tubulin-encoding gene of P. fusca (PfTUB2) to develop molecular tools with different applications in powdery mildew research. PfTUB2 is predicted to encode a protein of 447 amino acid residues. The coding region is interrupted by six introns that occur at approximately the same positions as the introns present in other fungal TUB2-like genes. Once cloned, the PfTUB2 sequence information was used in different applications. Our results showed that the TUB2 gene is a good marker for molecular phylogenetics in powdery mildew fungi but it is unsuitable for the analysis of intraspecific diversity in P. fusca. The expression of PfTUB2 was proven to be stable in different temperature conditions, supporting its use as a reference gene in quantitative gene expression studies. Furthermore, an allele-specific PCR assay for the detection of resistance to methyl-2-benzimidazole carbamate (MBC) fungicides in P. fusca was developed based on the correlation between the single amino acid change E198A in β-tubulin and the MBC resistance phenotype. Lastly, PfTUB2 was used as a target gene in the development of a high-throughput method to quantify fungal growth in plant tissues.
白粉菌真菌 Podosphaera fusca（同义 Podosphaera xanthii）是葫芦科白粉病的主要病原体，也是全球葫芦科生产的最重要限制因素之一。尽管该真菌具有重要的经济意义，但对其生物学和发病机制所涉及的生理和分子过程知之甚少。在本研究中，我们分离并鉴定了 P. fusca 的 β-微管蛋白编码基因（PfTUB2），以开发具有不同白粉病研究应用的分子工具。PfTUB2 预计编码 447 个氨基酸残基的蛋白质。编码区被六个内含子打断，这些内含子大约出现在其他真菌 TUB2 样基因中的内含子位置相同。PfTUB2 序列信息一旦被克隆，就可用于不同的应用。我们的结果表明，TUB2 基因是白粉菌真菌分子系统发育的良好标记，但不适合分析 P. fusca 的种内多样性。PfTUB2 的表达在不同的温度条件下被证明是稳定的，支持其在定量基因表达研究中作为参考基因的使用。此外，基于 β-微管蛋白中的单个氨基酸变化 E198A 与 MBC 杀菌剂抗性表型之间的相关性，开发了一种用于检测 P. fusca 对甲基-2-苯并咪唑氨基甲酸酯（MBC）杀菌剂抗性的等位基因特异性 PCR 检测方法。最后，PfTUB2 被用作目标基因，开发了一种高通量方法来量化植物组织中真菌的生长。
