ABSTRACT Macrophomina phaseolina, causative agent of the charcoal rot disease, impacts over 500 plant species, causing devastating crop failures worldwide. Due to the lack of effective management approach, charcoal rot can result in serious loss, especially during years with hot and dry summers. This study aims to evaluate the effectiveness of using the RNA interference (RNAi) mechanism to suppress the growth of M. phaseolina, through exogenous application of small interfering RNA (siRNA) targeting one of the chitin synthase (MpCHS) genes in the M. phaseolina genome. Since chitin is one of the main components in the fungal cell wall, we hypothesize that suppressing the expression of MpCHS through RNAi will negatively affect the growth of the fungus, which may result in reduced virulence toward the host. Of the seven candidate MpCHS genes assessed, MpCHS6 showed consistent expression in plate-grown fungus and in infected plant tissue and was chosen as the target for RNAi knockdown. Two siRNAs were designed to target MpCHS6 and tested through in vitro application in M. phaseolina culture. Both siRNAs suppressed growth of the fungus at multiple time points and under varied environmental conditions. More importantly, the growth suppression was well correlated with down-regulation of the MpCHS6 gene. These results indicate that exogenous siRNAs can suppress gene expression in M. phaseolina even when applied in vitro. In addition, targeting a MpCHS gene may provide an effective way to control the fungal growth. Our study provides firsthand knowledge on RNAi in M. phaseolina, which benefits not only basic research that focuses on understanding gene function in this species but also application of RNAi-based technology in disease control in the future.

中文翻译：

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针对几丁质合成酶基因的外源siRNA抑制病原真菌Macrophomina phaseolina的生长

摘要 木炭腐烂病的病原体 Macophhomina phaseolina 影响 500 多种植物物种，在世界范围内造成毁灭性的作物歉收。由于缺乏有效的管理方法，木炭腐烂会导致严重的损失，尤其是在夏季炎热干燥的年份。本研究旨在评估使用 RNA 干扰 (RNAi) 机制抑制小菜豆生长的有效性，通过外源应用小干扰 RNA (siRNA)，靶向小菜豆中的一种几丁质合酶 (MpCHS) 基因基因组。由于几丁质是真菌细胞壁的主要成分之一，我们假设通过 RNAi 抑制 MpCHS 的表达会对真菌的生长产生负面影响，这可能会导致对宿主的毒力降低。在评估的七个候选 MpCHS 基因中，MpCHS6 在平板生长的真菌和受感染的植物组织中表现出一致的表达，并被选为 RNAi 敲低的目标。设计了两种 siRNA 以靶向 MpCHS6，并通过在菜豆培养物中的体外应用进行测试。两种 siRNA 在多个时间点和不同的环境条件下都抑制了真菌的生长。更重要的是，生长抑制与 MpCHS6 基因的下调密切相关。这些结果表明，即使在体外应用时，外源 siRNA 也可以抑制小菜豆中的基因表达。此外，靶向 MpCHS 基因可能提供一种控制真菌生长的有效方法。我们的研究提供了关于菜豆中 RNAi 的第一手知识，