The long-term objective is to develop soybean with resistance to pests and pathogens. Soybean is the second largest crop in the United States with a farm value of $30 billion in 2009. A number of diseases affect soybean yield, but by far the greatest loss is due to soybean cyst nematode(SCN), Heterodera glycines. While nematodes are a long-standing problem, the greatest emerging threat to the US soybean crop is soybean rust (SBR), a disease caused by the fungus Phakopsora pachyrhizi. Gene expression will be examined in the SCN-induced syncytium (multinucleated cell) to determine if gene expression and protein targeting is asymmetric across the length of the syncytium (i.e., polarized). New candidate genes responsible for susceptibility and resistance to SCN and SBR will be identified and their functions investigated. Transgenic and mutagenized soybean plants will be examined to determine if there are unintended changes in the proteome. The specific objectives of the proposal are: Objective 1: Discover and characterize plant and pathogen genes and molecular signals important for resistance or pathogenicity at the molecular level with emphasis on soybean interactions with soybean rust and soybean cyst nematode. Objective 2: Identify genes expressed during interactions of soybean with nematodes and fungi at various intervals on resistant and susceptible plants, and develop transformed plants with over-expressed and silenced genes to improve resistance to nematodes and fungi. Sub-Objective 2A. Identify genes expressed by the host and pathogen during a resistant and susceptible interaction of soybean with SCN and SBR. This will provide insights into host-pathogen interactions and will identify candidate genes for testing. Sub-Objective 2B. Overexpress and silence candidate genes in transformed soybean plants and soybean roots to determine their effect on pathogen growth and development. Objective 3: Determine the collateral variation in seed composition between crop plants developed using genetic engineering, mutagenesis and classical breeding.
Plant hormonal signal pathways (e.g. ethylene, auxin, IDA), will be examined to determine how they contribute to growth of the feeding structure (syncytium) for SCN. Fluorescent markers will be used to identify expression patterns for genes and proteins involved in auxin, ethylene and IDA synthesis, transport and signaling to determine their interactive roles in the asymmetric growth of the syncytium. A novel IDA-like gene discovered in root-knot nematodes will be assayed for its role in nematode growth within the plant by overexpression of the nematode IDA in the plant roots and suppression of the IDA in the nematode by RNAi gene silencing. Fifteen proteins that accumulate in the nucleus at a higher level and 52 proteins that accumulate at a lower level in Rpp1 plants resistant to SBR will be examined. Using virus-induced gene silencing and virus-induced over-expression, it will be tested whether altered levels of these proteins contribute to Rpp1 resistance and whether these genes can be used to improve resistance. Roots infected with SCN will be examined using Illumina RNA-seq. DNA constructs representing genes of interest will be transformed into soybean roots and challenged with SCN to determine if they contribute to resistance or susceptibility. The seed proteins and their abundance in soybean lines derived via conventional plant breeding, mutagenesis, and genetic engineering and soybean landraces and wild soybean from which they are derived will be compared using 2-dimensional electrophoresis, mass spectrometry, and other techniques.
The first objective is to discover and characterize plant and pathogen genes and molecular signals important for resistance or pathogenicity at the molecular level with emphasis on soybean interactions with soybean rust and soybean cyst nematode. Two genes (MiIDL1 and MiIDL2) were identified in the genomic sequence of the root knot nematode (Meloidogyne incognita), which encode peptides (small proteins) similar to the plant IDA signaling peptide which promotes leaf abscission and the emergence of lateral roots. Attempts to localize the expression of the MiIDL genes in nematode cells were not successful, possibly due to the very low level of expression of these genes early in nematode development. Nonetheless, application of synthetic nematode IDA-like (IDL) peptide on Arabidopsis plants that carry the ida mutant promoted leaf abscission and lateral root emergence was sucessfully demonstrated. This indicated that the nematode peptide may have a similar mode of action to the plant IDA signaling peptide. Moreover, when the full-length root knot nematode MiIDL1 gene was transformed into Arabidopsis plants with the ida mutant, the transformed plants with the full-length MiIDL1 gene also had normal leaf abscission and lateral root emergence. In addition, when the full-length MiIDL1 gene was transformed into non-mutant (wild type) Arabidopsis plants, the plants displayed altered root phenotypes that may help in understanding the role of the MiIDL peptide in nematode infection. RNAi (gene suppression) experiments are in progress. Nematode and plant signaling molecules that regulate the nematode infection process are of special interest because they are useful targets for controlling nematode infection of agriculturally important plants. Soybean proteins resembling transcription factors (proteins that inhibit or promote gene expression) that accumulate in the nucleus of soybeans harboring the Rpp1 gene that confers immunity to soybean rust were examined. To determine if the proteins contribute in part to Rpp1 immunity, Bean pod mottle virus was used to attenuate or silence the expression of genes associated with Rpp1-conditioned immunity to rust. Rpp1 plants subjected to the virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with soybean rust spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help identify some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean disease defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars. The second objective is to identify genes and proteins expressed during interactions of soybean with nematodes and fungi at various intervals on resistant and susceptible plants, and develop genetically transformed plants with over-expressed and silenced genes to improve resistance to nematodes and fungi. DNA constructs were made to silence genes of soybean cyst nematode and root-knot nematode. Initial trials indicate that several constructs show promise and reduce gall formation by the root knot nematode or cyst formation by the soybean cyst nematode more than 60%. These studies will aid public and private researchers who are trying to develop nematode and rust resistant soybeans. The third objective is to determine the collateral variation in seed composition between crop plants developed using genetic engineering, mutagenesis and classical plant breeding. Seeds were collected from a diverse set of soybeans and extracted proteins from the seeds. The extracted seed proteins from medium Maturity Group soybean genotypes were separated using a procedure called 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The identification of separated proteins using mass spectrometry is in progress.
1. Soybean genes that retard cyst nematode development. Soybean cyst nematodes attack the roots of soybean plants and cause approximately $1-2 billion in damage each year to U.S. soybeans. In an effort to improve resistance in soybeans to the soybean cyst nematode, ARS researchers at Beltsville, MD made multiple DNA constructs designed to provide resistance to both soybean cyst and root-knot nematodes. The constructs were inserted into soybean roots and their effects on cyst formation by the soybean cyst nematode and gall formation by the root-knot nematode were determined. Three of the DNA constructs with overexpressed genes decreased the galls formed by the root-knot nematode and cysts formed by the soybean cyst nematode by approximately 70%. These gene constructs may be useful to soybean breeders to broaden resistance against nematodes.
2. Soybean genes contributing to soybean rust immunity. Soybean rust is a fungus that causes disease on soybeans. The discovery of soybean genes and proteins that are important for disease resistance to soybean rust may help improve soybean cultivars through breeding or transgenic technology. Proteins previously discovered in the cell nucleus of soybeans resistant to soybean rust were thought to help activate resistance. The genes for these proteins were manipulated with a plant virus by ARS researchers at Beltsville, MD to repress the amount of RNA and protein these genes make. When the originally resistant plants produced less RNA of the test genes as a result of the virus, the plants became susceptible to soybean rust. This meant that the test genes were important for disease resistance. An analysis of other genes also known to be important for disease resistance was performed, and it was found that when the virus reduced the amounts of RNA for the test genes, the RNA for some of the other disease resistance genes was reduced as well. This meant that these genes participate in a network of genes important for disease resistance, and this study showed that the disruption of one gene disrupts others in the network leading to loss of resistance. Two of the known genes that were disrupted in the network control the production of molecules that are toxic to fungi. Thus, soybean disease resistance is attributed in part to the production of antimicrobial molecules, and this research discovered several genes that control the production of these antimicrobial molecules. These data are most likely to influence scientists at universities, government agencies and companies who are searching for the soybean genes needed to fight rust diseases.
3. The first fully synthetic plant virus. The ribonucleic acid sequence of Tobacco mosaic virus (TMV) was discovered in 1982. However, no known DNA clone of the sequenced strain exists. Therefore, it is unknown if the sequence reported in 1982 is for an infectious virus. In this study, synthetic DNA of the TMV sequence was constructed by ARS researchers at Beltsville, MD using small pieces of DNA. The DNA was converted to synthetic RNA and then encapsided in a test tube using free viral capsid protein. Synthetic virions formed in the test tube. The synthetic virus was used to inoculate tobacco plants, but the plants did not develop symptoms. Mutations were discovered in the original TMV DNA sequence. When the mutations were corrected, new versions of the synthetic virus became infectious. The corrected TMV DNA was then combined with DNA from Tomato mosaic virus (ToMV) and this hybrid-synthetic virus elicited a resistant reaction on Nicotiana sylvestris (tobacco) whereas TMV did not. These experiments show that a gene from N. sylvestris causes resistance to ToMV but not TMV. TMV DNA was also combined with DNA from Barley stripe mosaic virus. The synthetic virus did not infect barley, but it did produce unique symptoms on N. benthamiana, a close relative of tobacco. This report corrects a long-standing error in the DNA sequence of TMV, shows which virus genes elicit disease resistance in N. sylvestris, and shows that unexpected symptoms can be produced by synthetic viruses. Synthetic biology is a discipline that includes making life forms artificially from chemicals. Therefore, these data are most likely to influence scientists at universities, government agencies and companies who are interested in making new molecules and genes using this technology.
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