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.
This is the final report for project 8042-21220-232-00D which terminated in March 27, 2018. New NP301 approved project 8042-21220-234-00D, entitled “Biotechnology Strategies for Understanding and Improving Disease Resistance and Nutritional Traits in Soybeans and Beans” has been established. Extensive results were realized over the 5 years of the project. For Objective 1, we used mass spectrometry to identify rust fungal proteins in infected beans and soybeans. We found 24 proteins that appeared to be specifically secreted by the rust fungus that infects beans and 16 proteins that appeared to be specifically secreted by the rust fungus that infects soybeans. There was overlap between the two sets which suggests that the fungi use similar proteins and multiple mechanisms to overcome the plants’ natural resistance to cause disease. Several of these proteins appear to be involved in degrading the plant leaf cell wall, which allows the fungus to enter the plant. We also used mass spectrometry to identify proteins in the cell nucleus of soybeans resistant to soybean rust. The genes for these proteins were manipulated with a plant virus to repress the amount of RNA and protein these genes make. When the originally resistant plants produced less RNA of the target genes as a result of the virus, the plants became susceptible to soybean rust. This meant that the target 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 target 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. 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 or who want to discover new chemicals that could prevent diseases of beans and soybeans. Also for Objective 1, we prepared constructs containing full-length and signal-peptide truncated constructs for the nematode MiIDL1 gene, transformed wild-type and ida mutant Arabidopsis and obtained multiple events for each construct. Transgenic Arabidopsis plants were assayed for complementation of the inflorescence deficient in abscission (ida) mutant phenotype. Assay of multiple transgenic events and lines confirmed that expression of the full-length MiIDL1 gene in Arabidopsis rescued the delayed abscission phenotype but the truncated gene did not. The results demonstrate that the MiIDL1 gene product can function as an IDA signaling peptide and must be secreted into the apoplast to be functional. In addition, we prepared constructs containing inverted repeats (RNAi) of the MiIDL1 transcript and transformed this construct into Arabidopsis and tomato (cultivar VF36). In Arabidopsis, RNAi suppression of MiIDL1 reduced the number of galls that form on the Arabidopsis roots by 40%. In tomato, transgenic plants were genotyped to identify events that were homozygous for a single copy of the transgene. Single-copy, homozygous events and control plants were infected with juvenile M. incognita, and RNA was collected from infected and non-infected roots at 0, 7, 14 and 21 days post inoculation (dpi). Twenty-eight tomato root RNA samples have been submitted for RNA sequencing. These studies on the role of the MiIDL1 gene in nematode infection will aid other scientists and industry to develop approaches that may be used to control nematode infection of soybean and other agriculturally important crops. For Objective 2, in an effort to improve resistance in soybeans to the soybean cyst nematode, we made multiple DNA gene constructs from Arabidopsis and soybean. The genes had previously been shown to be involved in plant disease resistance and root cell wall development. The genes were inserted into the genomic DNA in soybean roots and their effects on cyst formation by the nematode were determined. One of the overexpressed genes decreased cysts formed by the soybean cyst nematode by approximately 55%. These genes may be useful to soybean breeders to broaden resistance against nematodes. This research was discontinued in 2016 as a result of a critical vacancy. For Objective 3, we used an integrated approach by applying two dimensional gel electrophoresis (2D-PAGE) and mass spectrometry (MS) and identified different classes of soybean proteins, including storage, allergen, and anti-nutritional proteins. We have developed a comprehensive database, SoyProDB, that provides a wide-range inventory of these proteins. Since soybean seeds have abundant storage proteins which hinder the extraction and identification of low abundance proteins, we developed an innovative, rapid and cost effective extraction method to deplete 80% of abundant storage proteins. This new method enriched the low abundant proteins such as allergens that are often expressed at low concentrations and produce extreme effects on the immune system causing major food allergies. We also demonstrated natural variation of storage, allergen, and anti-nutritional proteins in a wide range of soybean genotypes including wild, ancestral cultivars, landrace, and elite germplasm. We found significantly higher variation in wild genotypes compared to other genotypes. In addition, we demonstrated the impact of genetic and environmental factors on soybean storage and allergen proteins from seeds of plants grown at three different locations (Maryland, South Carolina, and South Dakota). We found statistically different amounts of proteins isolated from seeds of plants grown in different environments. Also, we demonstrated that the abundances of soybean allergen proteins were influenced by environmental and genetic factors. In addition, we analyzed various isoflavones, phenolic acids, and storage proteins in three transgenic soybean lines (transgenic with PG11: GUS) compared to its control. We illustrated that the variation of protein and phenolic compounds found in three transgenic soybean lines are within the natural range of variation observed in conventional cultivars. These data are most likely to influence the scientists from universities and breeders from the companies who assess proteins from newly developed varieties and submit such data to regulatory agencies for approval. This research project produced more than 50 scientific manuscripts published in scholarly journals, and included collaborations with more than 85 national and 15 international scientists comprising students, post-doctoral scientists, and senior scientists. Formal collaborative agreements were executed with the University of Maryland, College Park, MD, and the United States – Israel Binational Agricultural Research and Development Fund.
1. Improved nitrogen fixation in soybeans. Inefficient symbiotic nitrogen fixation is prevalent in agriculture and is a waste of natural resources. ARS scientists in Beltsville, Maryland, performed research that describes the differentially regulated proteins in Bradyrhizobium elkanii USDA 76, a rhizobium bacterium that efficiently produces fixed nitrogen symbiotically with soybean roots. The proteins include enzymes that produce signaling molecules that establish and maintain root symbiosis, that generate energy needed to fix nitrogen, and that assimilate fixed nitrogen into chemicals that can be used by both the bacterium and the soybean. The results suggest a model for the metabolism of symbiotic nitrogen fixation in soybean that is different than resolved in other plants. A better understanding of symbiotic nitrogen fixation efficiency should lead to sustainable soybean production.
2. Silencing fungi genes. Millions of indigent people in Central America and Africa depend on dry beans as a primary source of protein. Their food security is threatened by Uromyces appendiculatus, a fungus that causes rust disease on the common, dry bean plant. To better understand which fungal proteins are important for infection, ARS scientists in Beltsville, Maryland, decreased the amounts of those fungal proteins using a gene silencing mechanism. Gene silencing is a biochemical process that specifically degrades RNA as it is made from a specific DNA gene. When the RNA for four fungal proteins was reduced, the fungus did not accumulate on the leaves, and the leaves had less disease. These experiments demonstrated that four bean rust fungal genes promote fungal infection and that the inhibition of these proteins can protect bean plants from rust disease. These results will likely influence scientists who are designing new methods to fight rust diseases.
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Tabatabai, B., Arumanayagam, A.S., Enitan, O., Mani, A., Natarajan, S.S., Sitther, V. 2017. Overexpression of HlyB and Mdh genes confers high salinity tolerance (halotolerance) in Fremyella diplosiphon, a Freshwater Cyanobacterium. Enzyme and Microbial Technology. 103:12-17. https://doi.org/10.1016/jenzyme.2017.04009.
John, M., Khan, F., Luthria, D.L., Matthews, B.F., Garrett, W.M., Natarajan, S.S. 2017. proteomic and metabolomic analysis of minimax and Williams 82 soybeans grown under two different conditions. Journal of Food Biochemistry. 41:e12404. https://doi.org/10.1111/jfbc.12404.
Shukla, V., Upadhyay, R.K., Tucker, M.L., Giovannoni, J.J., Rudhrabhatla, S.V., Mattoo, A.K. 2017. Transient regulation of three clustered tomato class-I small heat-shock chaperone genes by ethylene is mediated by SIMADS-RIN transcription factor. Scientific Reports. 7:6474. https://doi.org/10.1038/s41598-017-06622-0.
Kim, J., Yang, R., Chang, C., Tucker, M.L. 2018. The root-knot nematode (Meloidogyne incognita) produces a functional mimic of the Arabidopsis inflorescence deficient in abscission (IDA) signaling peptide. Journal of Experimental Botany. 69(12):3009-3021. https://doi.org/10.1093/jxb/ery135.
Cooper, B., Campbell, K. 2017. Protection against common bean rust conferred by a gene silencing method. Phytopathology. 107(8):920-927. https://doi.org/10.1094/PHYTO-03-17-0095-R.
Krishnan, H.B., Natarajan, S.S., Oehrle, N.W., Garrett, W.M., Darwish, O. 2017. Proteomic analysis of Pigeonpea (cajanus cajan) seeds reveals the accumulation of numerous stress-related proteins. Journal of Agricultural and Food Chemistry. 65(23):4572-4581. doi: 10.1021/acs.jafc.7b00998.