The cereal cyst nematode, Heterodera avenae is distributed worldwide and causes substantial damage in bread wheat, Triticum aestivum. This nematode is extremely difficult to manage because of its prolonged persistence as unhatched eggs encased in cysts. Due to its sustainable and target-specific nature, RNA interference (RNAi)-based strategy has gained unprecedented importance for pest control. To date, RNAi strategy has not been exploited to manage H. avenae in wheat. In the present study, 40 H. avenae target genes with different molecular function were rationally selected for in vitro soaking analysis in order to assess their susceptibility to RNAi. In contrast to target-specific downregulation of 18 genes, 7 genes were upregulated and 15 genes showed unaltered expression (although combinatorial soaking showed some of these genes are RNAi susceptible), suggesting that a few of the target genes were refractory or recalcitrant to RNAi. However, RNAi of 37 of these genes negatively altered nematode behavior in terms of reduced penetration, development and reproduction in wheat. Subsequently, wheat plants were transformed with seven H. avenae target genes (that showed greatest abrogation of nematode parasitic success) for host-induced gene silencing (HIGS) analysis. Transformed plants were molecularly characterized by PCR, RT-qPCR and Southern hybridization. Production of target gene-specific double- and single-stranded RNA (dsRNA/siRNA) was detected in transformed plants. Transgenic expression of galectin, cathepsin L, vap1, serpin, flp12, RanBPM and chitinase genes conferred 33.24-72.4 % reduction in H. avenae multiplication in T1 events with single copy ones exhibiting greatest reduction. A similar degree of resistance observed in T2 plants indicated the consistent HIGS effect in the subsequent generations. Intriguingly, cysts isolated from RNAi plants were of smaller size with translucent cuticle compared to normal size, dark brown control cysts, suggesting H. avenae developmental retardation due to HIGS. Our study reinforces the potential of HIGS to manage nematode problems in crop plant.

中文翻译：

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若干Heterodera avenae保守基因的表达干扰扰乱小麦线虫寄生成功

谷物胞囊线虫 Heterodera avenae 分布于世界各地，对面包小麦和普通小麦造成严重损害。这种线虫极难控制，因为它的未孵化卵被包裹在包囊中的时间很长。由于其可持续和特定目标的性质，基于 RNA 干扰 (RNAi) 的策略在害虫控制中获得了前所未有的重要性。迄今为止，尚未利用 RNAi 策略来管理小麦中的 H. avenae。在本研究中，合理选择了40个具有不同分子功能的H. avenae靶基因进行体外浸泡分析，以评估它们对RNAi的敏感性。与 18 个基因的靶向特异性下调相反，7 个基因被上调，15 个基因表现出未改变的表达（尽管组合浸泡显示其中一些基因对 RNAi 敏感），表明一些靶基因对 RNAi 是难治的或顽固的。然而，这些基因中 37 个的 RNAi 对线虫行为产生了负面影响，降低了小麦的渗透、发育和繁殖。随后，小麦植株被转化为七个 H. avenae 目标基因（显示最大程度地消除线虫寄生成功）用于宿主诱导的基因沉默 (HIGS) 分析。通过PCR、RT-qPCR和Southern杂交对转化植物进行分子表征。在转化植物中检测到目标基因特异性双链和单链 RNA (dsRNA/siRNA) 的产生。半乳糖凝集素、组织蛋白酶 L、vap1、serpin、flp12、RanBPM 和几丁质酶基因在 T1 事件中使 H. avenae 繁殖减少 33.24-72.4%，单拷贝的减少最大。在 T2 植物中观察到的相似程度的抗性表明在后续世代中具有一致的 HIGS 效应。有趣的是，与正常大小的深棕色对照包囊相比，从 RNAi 植物中分离的包囊尺寸较小，具有半透明的角质层，表明 H. avenae 由于 HIGS 发育迟缓。我们的研究加强了 HIGS 在管理作物植物线虫问题方面的潜力。与正常大小的深棕色对照包囊相比，从 RNAi 植物中分离的包囊尺寸较小，具有半透明的角质层，表明 H. avenae 由于 HIGS 发育迟缓。我们的研究加强了 HIGS 在管理作物植物线虫问题方面的潜力。与正常大小的深棕色对照包囊相比，从 RNAi 植物中分离的包囊尺寸较小，具有半透明的角质层，表明 H. avenae 由于 HIGS 发育迟缓。我们的研究加强了 HIGS 在管理作物植物线虫问题方面的潜力。