Plant Virology and Molecular Plant Pathology Laboratory, Division of Plant Pathology, SKUAST-Kashmir, Shalimar, Srinagar, 190025, India.
Research Center for Residue and Quality Control Analysis, SKUAST-Kashmir, 190025, India.
Fungal Biol. 2024 May;128(3):1771-1779. doi: 10.1016/j.funbio.2024.03.008. Epub 2024 Mar 22.
Phaseolus vulgaris L., commonly known as the common bean, is a highly nutritious crop often called the "poor man's meat". However, it is susceptible to various diseases throughout the cropping season, with anthracnose caused by Colletotrichum lindemuthianum being a significant threat that leads to substantial losses. There is still a lack of understanding about the molecular basis of C. lindemuthianum pathogenicity. The first step in understanding this is to identify pathogenicity genes that express more during infection of common beans. A reverse transcription quantitative real-time PCR (qPCR) method can be used for virulence gene expression. However, this approach requires selecting appropriate reference genes to normalize relative gene expression data. Currently, there is no reference gene available for C. lindemuthianum. In this study, we selected eight candidate reference genes from the available genome of C. lindemuthianum to bridge the gap. These genes were ACT (Actin), β-tub (β-tubulin), EF (Elongation Factor), Cyt C (Cytochrome C), His H3 (Histone H3), CHS1 (Chitin synthetase), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and abfA (Alpha-l-Arabinofuranosidase A). The primers for these candidate reference genes were able to amplify cDNA only from the pathogen, demonstrating their specificity. The qPCR efficiency of the primers ranged from 80% to 103%. We analyzed the stability of gene expression in C. lindemuthianum by exposing the mycelium to nine different stress conditions. We employed algorithms, such as GeNorm, NormFinder, BestKeeper, and RefFinder tools, to identify the most stable gene. The analysis using these tools revealed that EF, GAPDH, and β-tub most stable genes, while ACT and CHS1 showed relatively low expression stability. A large number of potential effector genes have been identified through bioinformatics analysis in C. lindemuthianum. The stable genes for qPCR (EF and GAPDH) discovered in this study will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes.
菜豆，俗称普通豆，是一种营养丰富的作物，常被称为“穷人的肉”。然而，它在整个种植季节都容易受到各种疾病的影响，炭疽病是由炭疽菌引起的，炭疽菌是一个严重的威胁，导致大量损失。目前对炭疽菌的致病机制还缺乏了解。了解这一点的第一步是鉴定在感染普通豆时表达更多的致病基因。逆转录定量实时 PCR(qPCR)方法可用于毒力基因表达。然而，这种方法需要选择合适的参考基因来归一化相对基因表达数据。目前，炭疽菌没有可用的参考基因。在这项研究中，我们从炭疽菌的可用基因组中选择了 8 个候选参考基因来填补这一空白。这些基因是 ACT(肌动蛋白)、β- tub(β-微管蛋白)、EF(伸长因子)、Cyt C(细胞色素 C)、His H3(组蛋白 H3)、CHS1(几丁质合成酶)、GAPDH(甘油醛-3-磷酸脱氢酶)和 abfA(α-L-阿拉伯呋喃糖苷酶 A)。这些候选参考基因的引物只能从病原体扩增 cDNA，证明了它们的特异性。引物的 qPCR 效率范围从 80%到 103%。我们通过将菌丝体暴露于九种不同的应激条件来分析炭疽菌中基因表达的稳定性。我们采用 GeNorm、NormFinder、BestKeeper 和 RefFinder 等算法，确定最稳定的基因。这些工具的分析表明，EF、GAPDH 和β- tub 是最稳定的基因，而 ACT 和 CHS1 表现出相对较低的表达稳定性。通过生物信息学分析，在炭疽菌中已经鉴定出大量潜在的效应基因。本研究中发现的用于 qPCR 的稳定基因(EF 和 GAPDH)将有助于科学界确定炭疽菌效应基因的相对表达。
