Department of Agriculture Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy.
Interdepartmental Research Center Nutrafood "Nutraceuticals and Food for Health", University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy.
Int J Mol Sci. 2023 Mar 2;24(5):4869. doi: 10.3390/ijms24054869.
is a pathogen of wide agronomic and scientific importance partly due to its tendency to develop fungicide resistance. Recently, there has been great interest in the use of RNA interference as a control strategy against . In order to reduce the possible effects on non-target species, the sequence-dependent nature of RNAi can be used as an advantage to customize the design of dsRNA molecules. We selected two genes related to virulence: (a MAP kinase essential for fungal pathogenesis) and (a tetraspanin related to appressorium penetration). After performing a prediction analysis of small interfering RNAs, dsRNAs of 344 () and 413 () nucleotides were synthesized in vitro. We tested the effect of topical applications of dsRNAs, both in vitro by a fungal growth assay in microtiter plates and in vivo on artificially inoculated detached lettuce leaves. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination for , an evident growth retardation for , and a strong reduction in necrotic lesions on lettuce leaves for both genes. Furthermore, a strongly reduced expression of the and genes was observed in both in vitro and in vivo experiments, suggesting that these genes could be promising targets for the development of RNAi-based fungicides against .
是一种具有广泛农业和科学重要性的病原体，部分原因是其倾向于产生抗真菌剂的抗性。最近，人们对利用 RNA 干扰作为防治 的控制策略产生了极大的兴趣。为了减少对非目标物种的可能影响，可以利用 RNAi 的序列依赖性将 dsRNA 分子的设计定制化。我们选择了两个与毒力相关的基因：（一种对真菌发病机制至关重要的 MAP 激酶）和 （一种与附着胞穿透有关的四跨膜蛋白）。在进行小干扰 RNA 的预测分析后，体外合成了 344 个核苷酸（）和 413 个核苷酸（）的 dsRNA。我们测试了 dsRNA 的局部应用的效果，无论是在微滴定板中的真菌生长测定中体外进行，还是在人工接种的离体生菜叶片上体内进行。在这两种情况下，dsRNA 的局部应用均导致基因敲低， 导致分生孢子萌发延迟， 导致明显的生长迟缓， 导致两种基因在生菜叶片上的坏死病变强烈减少。此外，在体外和体内实验中均观察到 和 基因的表达强烈降低，表明这些基因可能是针对 的基于 RNAi 的杀菌剂开发的有前途的靶标。
