Centro de Investigação das Ferrugens do Cafeeiro/Instituto de Investigação Científica Tropical, Oeiras, Portugal.
Fungal Biol. 2011 Sep;115(9):891-901. doi: 10.1016/j.funbio.2011.07.002. Epub 2011 Jul 18.
Hemileia vastatrix is a biotrophic fungus, causing coffee leaf rust in all coffee growing countries, leading to serious social and economic problems. Gene expression studies may have a key role unravelling the transcriptomics of this pathogen during interaction with the plant host. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the golden standard for gene expression analysis, although an accurate normalisation is essential for adequate conclusions. Reference genes are often used for this purpose, but the stability of their expression levels requires validation under experimental conditions. Moreover, pathogenic fungi undergo important biomass variations along their infection process in planta, which raises the need for an adequate method to further normalise the proportion of fungal cDNA in the total plant and fungus cDNA pool. In this work, the expression profiles of seven reference genes [glyceraldehyde-3-phosphate dehydrogenase (GADPH), elongation factor (EF-1), Beta tubulin (β-tubulin), cytochrome c oxidase subunit III (Cyt III), cytochrome b (Cyt b), Hv00099, and 40S ribosomal protein (40S_Rib)] were analysed across 28 samples, obtained in vitro (germinated uredospores and appressoria) and in planta (post-penetration fungal growth phases). Gene stability was assessed using the statistical algorithms incorporated in geNorm and NormFinder tools. Cyt b, 40S_Rib, and Hv00099 were the most stable genes for the in vitro dataset, while 40S_Rib, GADPH, and Cyt III were the most stable in planta. For the combined datasets (in vitro and in planta), 40S_Rib, GADPH, and Hv00099 were selected as the most stable. Subsequent expression analysis for a gene encoding an alpha subunit of a heterotrimeric G-protein showed that the reference genes selected for the combined dataset do not differ significantly from those selected specifically for the in vitro and in planta datasets. Our study provides tools for correct validation of reference genes in obligate biotrophic plant pathogens, as well as the basis for RT-qPCR studies in H. vastatrix.
咖啡驼孢锈菌是一种活体营养型真菌，会导致所有咖啡种植国家的咖啡叶锈病，从而造成严重的社会和经济问题。基因表达研究可能在揭示该病原体与植物宿主相互作用时的转录组学方面发挥关键作用。逆转录定量实时聚合酶链反应（RT-qPCR）是目前进行基因表达分析的金标准，尽管为了得出充分的结论，准确的归一化是必不可少的。参考基因通常用于此目的，但它们的表达水平稳定性需要在实验条件下进行验证。此外，病原真菌在其感染植物过程中会发生重要的生物量变化，这就需要有一种适当的方法来进一步归一化总植物和真菌 cDNA 库中真菌 cDNA 的比例。在这项工作中，分析了 7 个参考基因[甘油醛-3-磷酸脱氢酶（GADPH）、延伸因子（EF-1）、β-微管蛋白（β-tubulin）、细胞色素 c 氧化酶亚基 III（Cyt III）、细胞色素 b（Cyt b）、Hv00099 和 40S 核糖体蛋白（40S_Rib）]在 28 个样本中的表达谱，这些样本分别来自体外（萌发的夏孢子和附着胞）和体内（穿透后真菌生长阶段）。使用 geNorm 和 NormFinder 工具中包含的统计算法评估基因稳定性。Cyt b、40S_Rib 和 Hv00099 是体外数据集最稳定的基因，而 40S_Rib、GADPH 和 Cyt III 是体内最稳定的基因。对于组合数据集（体外和体内），选择 40S_Rib、GADPH 和 Hv00099 作为最稳定的基因。随后对编码异三聚体 G 蛋白α亚基的基因进行表达分析表明，组合数据集选择的参考基因与专门为体外和体内数据集选择的参考基因没有显著差异。我们的研究为正确验证专性活体营养型植物病原体中的参考基因提供了工具，也为咖啡驼孢锈菌的 RT-qPCR 研究奠定了基础。
