The goal of this SBIR Phase II project is to generate transgenic crops with broad-spectrum, season-long resistance to root knot nematode (RKN, Meloidogyne species) by using RNA interference to silence nematode genes in planta. Plant parasitic nematodes, of which RKN is the most significant, annually cause damage of $8 billion in the U.S. and $80 billion worldwide. Current control options including toxic nematicides have significant deficiencies. Phase I research successfully demonstrated in root culture that expression of specific double stranded RNAs (dsRNAs) from each of eight essential RKN genes limited nematode reproduction relative to untreated controls with reductions of up to 79%. In Phase II, validated dsRNA-expressing constructs will progress to tomato whole plant transformation. Transgenic plants with dsRNA expression will be tested in greenhouse large-pot assays for control of multiple RKN species, root damage, and plant vigor. In parallel, the laboratory assays validated in Phase I will be used to select next generation constructs that can further enhance the degree of nematode control. Success in Phase II, especially achieving whole plant proof-of-principle for RKN control, will justify a field trial program in Phase III and eventual commercialization of a biotechnology trait for enhancing crop yields through RKN resistance. Benefits of this approach for the grower include increased yield per acre, improved crop management and rotation options, increased tolerance of crops to stress and drought, decreased input cost for chemical application and preservation of a beneficial soil microenvironment. Benefits for consumers include enhanced food and environmental safety. OBJECTIVES: The long-term goal of this research is to commercialize a biotechnology trait for control of root knot nematode (RKN, Meloidogyne species) in important U.S. crops. Agriculture is under tremendous pressure to achieve improved yields and ensure the availability of crops for uses in food, feed, fiber, and fuel. A major limitation on crop yields are parasitic nematode worms that damage root systems and are hard to control, causing annual yield losses valued at more than $8 billion in the U.S. and $80 billion worldwide (Sasser and Freckman, 1987). Among nematodes, RKN is the most significant. RKN root damage results in plants that are less efficient in water uptake and nutrient transport and have increased vulnerability to drought, stress, and secondary infection. Examples of crops with significant yield loss of RKN include large acreage crops such as cotton, soybeans, corn, and potatoes, and high value crops such as tomatoes, carrots, pepper, and melons. The neurotoxic organophosphate nematicide fenamiphos and carbamate nematicide carbofuran were both withdrawn from the U.S. market in 2007. The fumigant nematicide methyl bromide was withdrawn in 2005 because of its role in ozone depletion (Carter, 2001). (Small scale application continues through critical use exemptions.) The fumigant 1,3-dichlorpropene is significantly restricted in its use because of toxicity and carcinogenicity. Resistant cultivars have been developed by breeding for some crops including tomato, but rapid resistance breaking has made some natural sources of resistance ineffective (Starr et al., 2002). Using RNA interference to silence essential parasite genes, we aim to create biotechnology traits that provide season-long protection of roots from RKN without deleterious effects on the plant or environment. Commercialization of this technology is likely to occur first in the major row crops such as cotton, soybeans, and corn where the vast majority of U.S. acres already employ transgenic technology for insect and weed control. Stacking an additional biotech trait, such as nematode resistance, in such crops would be widely accepted by growers following demonstrated safety and efficacy. Benefits to this approach for the grower include increased yield per acre, improved worker safety, preservation of crop management and rotation options, increased tolerance of crops to drought and stress, decreased input, labor, and fuel cost for chemical application, and preservation of a beneficial soil microenvironment. Benefits for consumers include increased food and environmental safety due to the reduction in use of hazardous chemical nematicides. APPROACH: This project uses RNAi to protect roots from root knot nematode by selectively silencing nematode genes. Success in Phase II research, especially achieving whole plant proof-of-principle for RKN control, will justify a large-scale field trial program in Phase III for yield demonstration and eventual commercialization of a safe and effective biotechnology trait for RKN control. Key factors in establishing technical feasibility include achieving consistently high levels of RKN control across multiple RKN species. In Phase II, twelve constructs driving the expression of validated dsRNAs will be progressed to whole plant transformation. For each construct, five events with low copy number (e.g. one or two) and stable expression (e.g. expression level confirmed by quantitative RT-PCR and Northern blot) will be selected for characterization in R1 and R2 generation plants. Tomato transgenic plants from Objective 1 with stable dsRNA expression will be tested in greenhouse large-pot assays to quantify reproduction of multiple root knot nematode species, root damage, and plant vigor. Divergence anticipates processing over 4,000 plants through these assays during Phase II. Our goal is to identify transgenic events that can both reduce the number of the harvested root knot nematode eggs by =50% and show a statistically significant decrease in first generation gall damage relative to control transformations. In parallel to whole plant transformation and testing, the assays validated in Phase I will be used in a supporting role to select improved constructs. Results from these experiments will affect the design and prioritization of constructs entering the whole plant transformation and testing pipeline beginning as early as midway through year one.
