SBIR Phase I: Intracellular delivery of nematicidal proteins

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.