ICAAM-Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Instituto de Investigação e Formação Avançada, Universidade de Évora, Pólo da Mitra, Ap.94, 7006-554 Évora, Portugal.
Departamento de Fitotecnia, ICAAM-Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Escola de Ciências e Tecnologia, Universidade de Évora, Pólo da Mitra, Ap.94, 7006-554 Évora, Portugal.
Viruses. 2018 Aug 9;10(8):416. doi: 10.3390/v10080416.
RNA silencing is an important defense mechanism in plants, yet several plant viruses encode proteins that suppress this mechanism. In this study, the genome of the (OMMV) was screened for silencing suppressors. The full OMMV cDNA and 5 OMMV open reading frames (ORFs) were cloned into the Gateway binary vector pK7WG2, transformed into , and agroinfiltrated into 16C plants. and showed suppressor activity, with showing significantly higher activity than , yet activity that was lower than the full OMMV, suggesting a complementary action of and . These viral suppressors were then used to induce OMMV resistance in plants based on RNA silencing. Two hairpin constructs targeting each suppressor were agroinfiltrated in plants, which were then inoculated with OMMV RNA. When silencing of both suppressors was achieved, a significant reduction in viral accumulation and symptom attenuation was observed as compared to those of the controls, as well as to when each construct was used alone, proving them to be effective against OMMV infection. This is the first time that a silencing suppressor was found in a necrovirus, and that two independent proteins act as silencing suppressors in a virus member of the family.
RNA 沉默是植物的一种重要防御机制，但有几种植物病毒编码的蛋白质能够抑制这种机制。在本研究中，对 （OMMV）的基因组进行了筛选，以寻找沉默抑制剂。完整的 OMMV cDNA 和 5 个 OMMV 开放阅读框（ORFs）被克隆到 Gateway 双元载体 pK7WG2 中，转化为 和 ，并用农杆菌浸润 16C 植物。 和 表现出抑制活性， 表现出比 更高的活性，但比完整的 OMMV 活性低，这表明 和 的作用是互补的。然后，利用这些病毒抑制剂基于 RNA 沉默诱导植物对 OMMV 的抗性。针对每个抑制剂的两个发夹结构被农杆菌浸润到 植物中，然后用 OMMV RNA 接种。当两个抑制剂的沉默都实现时，与对照相比，病毒积累和症状减弱显著减少，与单独使用每个构建体相比也是如此，证明它们对 OMMV 感染有效。这是首次在坏死病毒中发现沉默抑制剂，并且两个独立的蛋白在 家族的病毒成员中作为沉默抑制剂发挥作用。
