Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, Himachal Pradesh, 173234, India.
ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, 171001, India.
Mol Biol Rep. 2023 Oct;50(10):8421-8429. doi: 10.1007/s11033-023-08736-3. Epub 2023 Aug 24.
Venturia inaequalis is an apple scab causing fungal pathogen. It is a highly contagious and destructive pathogen which rapidly spreads infection in the surrounding orchards if not managed. The management and control of disease require multiple fungicides to be sprayed at different development stages of the apple. Persistent applications of fungicides also raises environmental concerns. Here, we demonstrate the potential of using spray induced gene silencing (SIGS) by developing target specific gene constructs for the synthesis of corresponding double-stranded RNA (dsRNA).
The exogenous application of dsRNAs was found to reduce mycelial growth and spore formation of V. inaequalis on culture plates. Four genes of V. inaequalis viz. CIN1, CE5, VICE12 and VICE16 which get upregulated during infection, were selected as targets for the development of gene construct expressing the corresponding dsRNA. The effect of exogenously supplied in vitro synthesized dsRNA on V. inaequalis was assessed in culture bioassay experiments with respect to growth, and spore formation. The expression level of the target genes in treated and control fungus was evaluated using quantitative PCR. Fungus treated with VICE12 targeted dsRNA showed maximum reduction in colony size (~ 55%), conidia formation (~ 93%) and expression level of the corresponding gene (2.2 fold), which was followed by CIN1-dsRNA. VICE16-dsRNA treatment was least effective with 32% reduction in growth, the non-significant effect of conidial spore formation and 1.13 fold down regulation of corresponding target gene expression level.
The result of this investigation validates the hypothesis that RNAi is evoked in V. inaequalis by exogenously supplied dsRNA and spray induced gene silencing (SIGS) based solutions may reduce burden of fungicide usage on apple crop against apple scab disease in future.
梨火疫病菌是一种引起苹果黑星病的真菌病原体。如果不加以管理，它是一种高度传染性和破坏性的病原体，在周围果园中迅速传播感染。该病害的防治需要在苹果不同的发育阶段喷洒多种杀菌剂。持续使用杀菌剂也引起了环境问题。在这里，我们通过开发用于合成相应双链 RNA（dsRNA）的靶标特异性基因构建体来展示使用喷雾诱导基因沉默（SIGS）的潜力。
在培养板上发现外源性 dsRNA 的应用可降低梨火疫病菌的菌丝生长和孢子形成。选择了在感染过程中上调的梨火疫病菌的四个基因 CIN1、CE5、VICE12 和 VICE16 作为开发表达相应 dsRNA 的基因构建体的靶标。在培养生物测定实验中，评估了外源合成 dsRNA 对梨火疫病菌的影响，涉及生长和孢子形成。用定量 PCR 评估处理和对照真菌中靶基因的表达水平。用 VICE12 靶向 dsRNA 处理的真菌显示出最大的菌落大小 (55%)、分生孢子形成 (93%)和相应基因表达水平的降低（2.2 倍），其次是 CIN1-dsRNA。VICE16-dsRNA 处理的效果最差，生长减少 32%，分生孢子形成无显著影响，相应靶基因表达水平降低 1.13 倍。
该研究结果验证了外源性 dsRNA 可在梨火疫病菌中引发 RNAi 的假设，并且基于喷雾诱导基因沉默（SIGS）的解决方案可能会减少未来苹果黑星病防治中对苹果作物杀菌剂使用的负担。
