A new fungus-mediated RNAi method established and used to study the fatty acid and retinol binding protein function of the plant-parasitic nematode <i>Aphelenchoides besseyi</i>

RNA interference (RNAi) is a powerful tool for gene functional analysis of plant-parasitic nematodes (PPNs). RNAi involving soaking in a dsRNA solution and <i>in planta</i> methods is commonly applied in the study of gene function in PPNs. However, certain problems restrict the application of these methods. Therefore, more convenient and effective RNAi methods need to be established for different PPNs according to their biological characteristics. In this study, the fatty acid and retinoid binding protein genes (<i>Ab-far-1, Ab-far-4</i>, and combinatorial <i>Ab-far-1</i> and <i>Ab-far-4</i>) of the rice white tip nematode (RWTN), <i>Aphelenchoides besseyi</i>, were used as target genes to construct a fungal RNAi vector, and the <i>Ab-far-n</i> dsRNA transgenic <i>Botrytis cinerea</i> (ARTB<i>n</i>) were generated using <i>Agrobacterium</i>-mediated transformation technology. After RWTN feeding on ARTB<i>n</i>, the expression of <i>Ab-far-1</i> and <i>Ab-far-4</i> in the nematodes was efficiently silenced, and the reproduction and pathogenicity of the nematodes were clearly inhibited. The <i>Ab-far-1</i> and <i>Ab-far-4</i> co-RNAi effects were better than the effects when each gene was individually targeted with RNAi. Additionally, the RNAi induced when RWTNs fed on ARTB<i>1</i> were persistent and heritable. Thus, a new method of fungus-mediated RNAi was established for fungivorous PPNs and was verified as effective and applicable to the study of nematode gene function. This technique will remove the technological bottlenecks and provide a new method to studying the multiple genes with polygene co-RNAi in fungivorous PPNs. This study also provides a theoretical basis and new thought for further study of the gene function in PPNs. <b>Abbreviations:</b> FAR(Fatty acid and retinol-binding proteins); RWTN (The rice white tip nematode, <i>Aphelenchoides besseyi); Ab-far-n</i> (Fatty acid and retinol binding protein gene of <i>A. besseyi)</i>; ARTB<i>1</i> (<i>Ab-far-1</i> hpRNA transgenic <i>Botrytis cinerea</i>); ARTB<i>4</i> (<i>Ab-far-4</i> hpRNA transgenic <i>Botrytis cinerea</i>); ARTB<i>1</i>/<i>4</i> (combinatorial <i>Ab-far-1</i> and <i>Ab-far-4</i> hpRNA transgenic <i>B. cinerea</i>); EVTB (Empty vector transgenic <i>B. cinerea</i>); GRTB (eGFP hpRNA transgenic <i>B. cinerea</i>); WTB (Wild-type <i>B. cinerea</i>).

RNA干扰（RNA interference, RNAi）是开展植物寄生线虫（plant-parasitic nematodes, PPNs）基因功能研究的强有力工具。当前植物寄生线虫的基因功能研究中，常用双链RNA（double-stranded RNA, dsRNA）溶液浸泡法与植株体内（in planta）递送法开展RNA干扰实验，但上述方法存在一定局限，制约了其推广应用。因此，需针对不同植物寄生线虫的生物学特性，开发更为便捷高效的RNA干扰技术体系。本研究以水稻白尖线虫（rice white tip nematode, RWTN），其学名为*Aphelenchoides besseyi*，的脂肪酸与视黄醇结合蛋白基因（*Ab-far-1*、*Ab-far-4* 及二者联合靶标组合）为靶标基因，构建真菌RNAi载体，通过农杆菌介导转化（Agrobacterium-mediated transformation）技术获得表达*Ab-far-n*双链RNA的转基因灰葡萄孢（*Botrytis cinerea*，简称ARTB*n*）。将水稻白尖线虫饲喂ARTB*n*后，线虫体内*Ab-far-1*与*Ab-far-4*的表达量被高效沉默，线虫的繁殖能力与致病力均受到显著抑制；其中*Ab-far-1*与*Ab-far-4*的联合RNA干扰效果优于单基因RNA干扰的效果。此外，水稻白尖线虫取食ARTB*1*后诱导产生的RNA干扰效应具有持久性与可遗传性。本研究成功建立了一种针对食真菌性植物寄生线虫的真菌介导RNA干扰新方法，经验证该方法高效且适用于线虫基因功能研究，可突破现有技术瓶颈，为食真菌性植物寄生线虫的多基因联合RNA干扰研究提供全新技术手段。本研究同时为植物寄生线虫的基因功能后续研究提供了理论基础与新思路。**缩写说明：** FAR（脂肪酸与视黄醇结合蛋白，Fatty acid and retinol-binding proteins）；RWTN（水稻白尖线虫，*Aphelenchoides besseyi*）；*Ab-far-n*（贝西滑刃线虫的脂肪酸与视黄醇结合蛋白基因，Fatty acid and retinol binding protein gene of *A. besseyi*）；ARTB*1*（表达*Ab-far-1*发夹RNA（hairpin RNA, hpRNA）的转基因灰葡萄孢，*Ab-far-1* hpRNA transgenic *Botrytis cinerea*）；ARTB*4*（表达*Ab-far-4* hpRNA的转基因灰葡萄孢，*Ab-far-4* hpRNA transgenic *Botrytis cinerea*）；ARTB*1*/4（共表达*Ab-far-1*与*Ab-far-4* hpRNA的转基因灰葡萄孢，combinatorial *Ab-far-1* and *Ab-far-4* hpRNA transgenic *B. cinerea*）；EVTB（空载质粒转化的灰葡萄孢，Empty vector transgenic *B. cinerea*）；GRTB（表达增强型绿色荧光蛋白（enhanced green fluorescent protein, eGFP）hpRNA的转基因灰葡萄孢，eGFP hpRNA transgenic *B. cinerea*）；WTB（野生型灰葡萄孢，Wild-type *B. cinerea*）。