Ramos John E, Jain Ritesh G, Powell Charles A, Dawson William O, Gowda Siddarame, Borovsky Dov, Shatters Robert G
U.S. Horticultural Research Laboratory (USDA-ARS), Fort Pierce, FL, United States.
Indian River Research and Education Center, UF/IFAS, Fort Pierce, FL, United States.
Front Physiol. 2021 Apr 9;12:571826. doi: 10.3389/fphys.2021.571826. eCollection 2021.
Citrus Greening or Huanglongbing (HLB) is a disease of citrus, causing high reduction in citrus production and is transmitted by the Asian citrus psyllid Kuwayama vectoring a phloem-limited bacterium sp. We report research results using crowdsourcing challenge strategy identifying potential gene targets in to control the insect using RNA interference (RNAi). From 63 submitted sequences, 43 were selected and tested by feeding them to using artificial diet assays. After feeding on artificial diet, the three most effective dsRNAs causing 30% mortality above control silenced genes expressing iron-sulfur cluster subunit of the mitochondrial electron transport chain complex (Rieske), heme iron-binding terminal oxidase enzyme (Cytochrome P450) and tetrahydrobiopterin (BH) pathway enzyme (Pterin 4α-Carbinolamine Dehydratase). These sequences were cloned into a citrus phloem-limited virus (, CTV T36) expressing dsRNA against these target genes in citrus. The use of a viral mediated "para-transgenic" citrus plant system caused higher mortality to adult than what was observed using artificial diet, reaching 100% when detached citrus leaves with the engineered CTV expressing dsRNA were fed to adult . Using this approach, a virus-induced gene silencing (VIGS) can be used to test future transgenic cultivars before genetically engineering citrus. RNA Seq analysis after feeding CTV-RIE on infected leaves identified transcriptionally modified genes located upstream and downstream of the targeted RIE gene. These genes were annotated showing that many are associated with the primary function of the Rieske gene that was targeted by VIGS.
柑橘黄龙病（HLB）是一种柑橘病害，会导致柑橘产量大幅下降，由亚洲柑橘木虱传播，该木虱携带一种韧皮部限制性细菌——韧皮部杆菌属。我们报告了利用众包挑战策略的研究结果，该策略用于识别韧皮部杆菌属中潜在的基因靶点，以便使用RNA干扰（RNAi）来控制这种昆虫。从提交的63个序列中，选择了43个，并通过人工饲料试验将它们喂给亚洲柑橘木虱进行测试。在以人工饲料喂养后，导致死亡率比对照组高出30%的三种最有效的双链RNA（dsRNA）使线粒体电子传递链复合物（铁硫蛋白）的铁硫簇亚基、血红素铁结合末端氧化酶（细胞色素P450）和四氢生物蝶呤（BH）途径酶（蝶呤4α-甲醇胺脱水酶）的表达基因沉默。这些序列被克隆到一种柑橘韧皮部限制性病毒（柑橘衰退病毒，CTV T36）中，该病毒在柑橘中表达针对这些靶基因的dsRNA。使用病毒介导的“准转基因”柑橘植物系统导致成年亚洲柑橘木虱的死亡率高于使用人工饲料时观察到的死亡率，当将表达dsRNA的工程化CTV的离体柑橘叶喂给成年亚洲柑橘木虱时，死亡率达到100%。使用这种方法，在对柑橘进行基因工程之前，可以利用病毒诱导的基因沉默（VIGS）来测试未来的转基因品种。在用CTV-RIE感染叶片后进行的RNA测序分析确定了位于靶向RIE基因上游和下游的转录修饰基因。对这些基因进行注释表明，许多基因与VIGS靶向的铁硫蛋白基因的主要功能相关。
