Progress 09/01/04 to 08/31/08
Outputs
OUTPUTS: The overall objective of this grant was to test the hypothesis that transgenic soybean plants designed to express small RNAi molecules targeted to specific soybean cyst nematode genes will have a negative impact on SCN fitness when the nematodes parasitize these plants. The specific goals were to 1) develop RNAi constructs directed against vital genes required for reproduction, viability of offspring and overall fitness of the feeding nematodes; 2) produce transgenic soybean plants containing these constructs; and 3) perform bioassays to test the efficacy of the transgenes. Fifteen genes were selected for this project: three of these are required for reproduction; three are required for fitness; and the other nine are required for embryo fertility. RNAi vectors of all fifteen genes have been completed. The first gene to be tested was the msp-1 gene coding for the major sperm protein. Experiments with this transgenic line have demonstrated about a 70% reduction in the number of cyst colonizing these plants compared to controls. The effects of plant derived dsRNA molecules appear to continue to the next generation. A non-transgenic susceptible cultivar was inoculated with eggs derived from both control plants and from eggs propagated from transgenic msp-1 plants. The msp-1 derived eggs displayed approximately 75% reduction in egg production compared to the control. An in planta hairy root transgenic system for the rapid assessment of candidate genes for SCN resistance has also been developed. The system generates chimeric plants with genetically engineered roots within one month (compared to nine to fifteen months for stable transgenics). Results from bioassays on these chimeric plants ranged from little or no effect of the number of eggs per gram of roots as in eat-3 to an 80% reduction for the Y25C1A.5. This rapid assessment of transgenes streamlines the gene selection process as stable lines are produced. Overall conclusions of this research demonstrates that 1) plant expressed dsRNA molecules targeted to specific nematode genes can be effective method in SCN resistance, 2) gene selection is critical for proper control (i.e. not all genes are effective); and 3) effects of plant derived dsRNA molecules appear to continue to the next generation and has implications on the successful deployment of RNAi technology for SCN control. PARTICIPANTS: Harold Trick, Tim Todd, Jiarui Li, Ryan Steeves, William Dall'Acqua, Juile Essig TARGET AUDIENCES: Scientists, soybean commodity commission, and soybean producers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Soybean Cyst Nematodes (SCN) is a major pest for soybean in the US causing one billion dollars in yield loss annually. Over $5 million per year in yield loss has been attributed to SCN in Kansas alone. Although there are natural sources of resistance for SCN in soybean germplasm, these traits are not resistant to all races. Our research has demonstrated the feasibility of RNA interference as a method for SCN control and has the potential to provide for significant additional protection against SCN.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We are testing the hypothesis that transgenic soybean plants designed to express small RNAi molecules, will have a negative impact on soybean cyst nematode fitness when the nematodes parasitize these plants. Our specific goals are to 1) develop RNAi constructs directed against vital genes required for reproduction, viability of offspring and overall fitness of the feeding nematodes; 2) produce transgenic soybean plants containing these constructs; and 3) perform bioassays to test the efficacy and durability of the transgenes. Of the ten separate genes initially selected for this project, six RNAi vectors have been designed. One of these genes is required for fertilization, two are required for embryo development, two are required for overall fitness of the nematode and the last is necessary for both general health and fertility. The remaining four vector constructions are under development. Of the vectors completed, two have been introduced into soybean cultures and stable
transformants are currently being identified. Bioassays of a previous transgenic line producing dsRNA of a major sperm protein (MSPi) have been ongoing and continue to show a significant reduction in cyst on transgenic plants expressing siRNAs for a male sterility gene. We are also developing an in vivo transient assay for rapidly assessing the effectiveness of candidate genes for SCN resistance. This method is based Agrobacterium rhizogenes to genetically modify the roots of whole seedlings. Although the entire plant will not be genetically modified, this method has the potential to generate genetically engineered roots as fast as two months and ready for a simple SCN assay shortly after that time. Compared to the current time of over a year, once fully developed, we should be able to greatly boost our efficiency. We have introduced three of our constructs using this method and they also have shown to show partial protection towards SCN. Currently these genes are being engineered
into soybeans for stable expression.
PARTICIPANTS: Harold Trick, Tim Todd, Jairiu Li, William Dall Acqua, Julie Essig, Shelia Stevens
TARGET AUDIENCES: Scientists, soybean commodity commission, and soybean producers
Impacts
Soybean Cyst Nematodes (SCN) is a major pest for soybean in the US causing one billion dollars in yield loss annually. Over $5 million per year in yield loss has been attributed to SCN in Kansas alone. Although there are natural sources of resistance for SCN in soybean germplasm, these traits are not resistant to all races. Our research has the potential to provide for significant additional protection against SCN.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
We are testing the hypothesis that transgenic soybean plants designed to express small RNAi molecules, will have a negative impact on soybean cyst nematode fitness when the nematodes parasitize these plants. Our specific goals are to 1) develop RNAi constructs directed against vital genes required for reproduction, viability of offspring and overall fitness of the feeding nematodes; 2) produce transgenic soybean plants containing these constructs; and 3) perform bioassays to test the efficacy and durability of the transgenes. Of the ten separate genes initially selected for this project, five RNAi vectors have been designed. One of these genes is required for fertilization, two are required for embryo development, one is required for overall fitness of the nematode and the last is necessary for both general health and fertility. The remaining five vector constructions are under development. Of the vectors completed, two have been introduced into soybean cultures and
stable transformants are currently being identified. Bioassays of a previous transgenic line producing dsRNA of a major sperm protein (MSPi) have been ongoing and continue to show a significant reduction in cyst on transgenic plants expressing siRNAs for a male sterility gene. We are also developing an in vivo transient assay for rapidly assessing the effectiveness of candidate genes for SCN resistance. This method is based Agrobacterium rhizogenes to genetically modify the roots of whole seedlings. Although the entire plant will not be genetically modified, this method has the potential to generate genetically engineered roots as fast as two months and ready for a simple SCN assay shortly after that time. Compared to the current time of over a year, once fully developed, we should be able to greatly boost our efficiency. Over the past three months we have been able to produce these engineered roots on soybean seedlings and visualize them by using the Green Fluorescent Protein from
jellyfish. In addition to this visual marker, we have also introduced our MSPi construction and two other genes which produce dsRNA to two other nematode genes. The next step in this research will be to determine expression of the MSPi gene and to inoculate this type of culture with SCN eggs.
Impacts
Soybean Cyst Nematodes (SCN) is a major pest for soybean in the US causing one billion dollars in yield loss annually. Over $5 million per year in yield loss has been attributed to SCN in Kansas alone. Although there are natural sources of resistance for SCN in soybean germplasm, these traits are not resistant to all races. Our research has the potential to provide for significant additional protection against SCN.
Publications
- Steeves, R.M., Todd, T.C., and Trick, H.N. 2006. Transgenic Soybeans Expressing siRNAs Specific to a Major Sperm Protein Gene Suppress Heterodera glycines Reproduction. Functional Plant Biology 33:991-999
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Progress 01/01/05 to 12/31/05
Outputs
We are testing the hypothesis that transgenic soybean plants designed to express small RNAi molecules, will have a negative impact on soybean cyst nematode fitness when the nematodes parasitize these plants. Our specific goals are to 1) develop RNAi constructs directed against vital genes required for reproduction, viability of offspring and overall fitness of the feeding nematodes; 2) produce transgenic soybean plants containing these constructs; and 3) perform bioassays to test the efficacy and durability of the transgenes. We are currently into our second year of the grant cycle and about six months behind our timetable due to personnel changes in the lab. We have selected ten separate genes which are vital for either reproduction or nematode fitness. Degenerate primers have been designed and five of these genes have been partially amplified. We are currently placing 250 to 300bp fragments of the 3ft end of these genes in an RNAi expression vector. Two of these
vectors are complete and were used to transform soybean cultures. We are currently in the process of selecting transgenic clones. Bioassays of a previous transgenic line have been completed demonstrating a significant reduction in cyst on transgenic plants expressing siRNAs for the major sperm protein. The long-term effect of this research project is to provide an environmentally-friendly, durable protection against soybean cyst nematodes.
Impacts
Soybean Cyst Nematodes (SCN) is a major pest for soybean in the US causing one billion dollars in yield loss annually. Over $5 million per year in yield loss has been attributed to SCN in Kansas alone. Although there are natural sources of resistance for SCN in soybean germplasm, these traits are not resistant to all races. Our research has the potential to provide for significant additional protection against SCN.
Publications
- No publications reported this period
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