SBIR Phase I: Intracellular delivery of nematicidal proteins

这个小型企业创新研究(SBIR)第一阶段研究项目将开发一种方法，使用细胞穿透肽(CPPs)在植物寄生线虫中携带生物活性蛋白穿过细胞膜。多个CPP将被开发出来，当连接到15 kDa的蛋白质支架上时，它们将穿过线虫细胞膜。这种支架是未来新型文库(称为STEM)的开始结构，它将涉及到选择抑制线虫基本细胞内靶标的蛋白质。CPP-STEM融合蛋白将在细菌中产生并纯化。大豆胞囊线虫(SCN)和根结线虫(RKN)J2幼虫将被这些蛋白质浸泡，条件是促进分子进入寄生虫的肠道。为了证实CPP-STEM支架可以在植物中以线虫可接近的形式表达，将使用毛状根中融合蛋白的转基因表达以及SCN和RKN的摄取作为生物检测。CPP-STEM蛋白也将被定位在线虫中，这将提供关于这种方法可以针对哪些组织和细胞隔间的信息。在商业上，该应用程序是一种控制寄生性线虫感染的转基因解决方案。寄生线虫每年给美国的农业造成超过80亿美元的损失，在全球范围内每年造成780亿美元的损失。杀线虫剂的使用受到严格限制。以安全的方式使用它们也很麻烦，而且使用起来也很昂贵。许多化学品，如甲基溴，对环境有害，将被取消注册。该项目的更广泛影响集中在控制线虫的新方法的潜力上，以减少在主要农作物上使用有毒和对环境不友好的化学品的需要。这种方法是以靶标为基础的，在转基因植物中表达穿过线虫细胞膜的杀线虫蛋白。这一策略可以用来控制广泛的寄生线虫，从大豆上的孢囊线虫，蔬菜、水果和棉花上的根结线虫，到玉米上的病损线虫。进一步应用于植物的昆虫和真菌病原体可能会扩大这项技术的影响。

This Small Business Innovation Research (SBIR) Phase I research project will develop a method to use cell-penetrating peptides (CPPs) to carry bioactive proteins across cell membranes in plant parasitic nematodes. Multiple CPPs will be developed which cross nematode cell membranes when attached to a 15 kDa protein scaffold. This scaffold is the starting structure for future novel libraries (called STEM), which will involve the selection of proteins that inhibit essential intracellular targets in the nematode. CPP-STEM fusion proteins will be generated in bacteria and purified. Soybean cyst nematode (SCN) and root knot nematode (RKN) J2 larvae will be soaked in these proteins under conditions that facilitate molecular uptake into the parasite intestine. To confirm that the CPP-STEM scaffolds can be expressed in plants in a form that is accessible to the nematode, transgenic expression of the fusion proteins within hairy roots as well as uptake by SCN and RKN will be used as the bioassay. The CPP-STEM proteins will also be localized in the nematode, which will provide information on what tissues and cellular compartments can be targeted by this approach. Commercially, the application is a transgenic solution for the control of parasitic nematode infections. Parasitic nematodes cost the agricultural industries in excess of $8 billion annually in the United States and $78 billion annually worldwide. The use of nematicides is tightly restricted. They are also cumbersome to apply in a safe manner and are expensive to use. Many chemicals, such as methyl bromide, are hazardous to the environment and are slated for deregistration. The broader impact of this project centers on the potential for novel means of nematode control to lessen the need for the application of toxic and environmentally unfriendly chemicals on major crops. This approach is target based, expressing nematicidal proteins that cross the nematode cell membrane in transgenic plants. This strategy can be used to develop control of a broad array of parasitic nematodes from cyst nematodes on soybean, root knot on vegetables, fruits and cotton, to lesion nematode on corn. Further application to insects and fungal pathogens of plants could broaden the impact of this technology.