SBIR Phase I: Intracellular delivery of nematicidal proteins

这项小型企业创新研究（SBIR）一期研究项目将开发一种方法，利用细胞穿透肽（CPPs）在植物寄生线虫的细胞膜上携带生物活性蛋白。将开发多个CPPs，当附着在15 kDa的蛋白质支架上时，它们可以穿过线虫细胞膜。这个支架是未来新文库（称为STEM）的起始结构，这将涉及到选择抑制线虫细胞内必需靶点的蛋白质。CPP-STEM融合蛋白将在细菌中生成并纯化。大豆囊线虫（SCN）和根结线虫（RKN） J2幼虫将在这些蛋白质中浸泡，以促进分子进入寄生虫肠道的吸收。为了证实CPP-STEM支架能够以线虫可及的形式在植物中表达，将使用毛状根内融合蛋白的转基因表达以及SCN和RKN的吸收作为生物测定。CPP-STEM蛋白也将定位在线虫中，这将提供关于这种方法可以靶向哪些组织和细胞区室的信息。在商业上，该应用是一种控制寄生线虫感染的转基因解决方案。在美国，寄生线虫每年给农业造成的损失超过80亿美元，在全球范围内则超过780亿美元。杀线虫剂的使用受到严格限制。它们在安全的情况下使用起来也很麻烦，而且使用起来很昂贵。许多化学品，如甲基溴，对环境是有害的，计划取消登记。这个项目更广泛的影响集中在线虫控制新手段的潜力上，以减少在主要作物上使用有毒和对环境不友好的化学品的需要。这种方法是基于靶标的，在转基因植物中表达穿过线虫细胞膜的杀线虫蛋白。该策略可用于防治大豆上的囊线虫、蔬菜、水果和棉花上的根结线虫、玉米上的病变线虫等多种寄生性线虫。进一步应用于昆虫和植物真菌病原体，可以扩大该技术的影响。

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.