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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Soybean Genomics & Improvement Laboratory » Research » Research Project #423975

Research Project: Developing Soybean and Other Legumes with Resistance to Pathogens and Assessing the Biosafety of Transgenic Soybean

Location: Soybean Genomics & Improvement Laboratory

2015 Annual Report


Objectives
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.


Approach
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.


Progress Report
Our 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. Previously we identified two genes (MiIDL1 and MiIDL2) in the genomic sequence for the root knot nematode (Meloidogyne incognita) that encode peptides (small proteins) similar to a plant IDA signaling peptide that promotes leaf abscission and the emergence of lateral roots. Last year and this year we demonstrated that the nematode IDA-like (IDL) gene could function as an IDA signaling peptide in Arabidopsis plants. When the full-length MiIDL1gene was transformed into wild-type Arabidopsis there was a greater number of lateral roots. Enhanced lateral root initiation suggests that the MiIDL peptide can alter root cell differentiation when the peptide is secreted from the nematode into the host. Most of this year’s research was directed towards a study of RNAi suppression of the MiIDL1 gene in the nematode. MiIDL-RNAi constructs were transformed into Arabidopsis and tomato and into hairy roots of soybean and tomato. Although the experiments are still in progress, first and second experimental replications indicate a reduced number of galls forming on the roots and a change in the size of the galls. The results suggest that the MiIDL-RNAi molecules made in the roots are acting negatively upon the nematode through a gene silencing mechanism, thus reducing nematode infection. 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. We also previously reported that soybean proteins resembling transcription factors (proteins that inhibit or promote gene expression) accumulate in the nucleus of soybeans harboring the Rpp1 gene that confers immunity to soybean rust. To determine if the proteins contribute in part to Rpp1 immunity, Bean pod mottle virus (BPMV) was used to attenuate or silence the expression of the transcription factors. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested transcription factors, and the plants developed rust-like symptoms after subsequent inoculation with soybean rust spores. These results helped identify some of the genes that contribute to Rpp1-mediated immunity. It is possible that these genes can be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars. To this end, we have been developing an in vitro system for over-expression of these transcription factors in soybean to test whether added amounts of the transcription factors increase resistance. Three virus systems have been investigated: The BPMV expression system also used in silencing, a Soybean mosaic virus (SMV) expression system, and a Gemini virus expression system. Each system has its advantages and drawbacks. The BPMV system produces mild-virus symptoms and does little damage to soybeans, but the BPMV icosahedron limits the size of foreign genes that can be inserted into the BPMV genome. The SMV system can express longer genes, but produces stronger symptoms and expresses lower amounts of protein. The Gemini virus system produces larger amounts of protein per cell, but the system requires Agrobacterium for infection of soybean and the virus does not move from cell to cell. Different molecular tags have been engineered onto the transcription factors so protein expression can be monitored, and the genes have been mobilized into the different expression systems for testing. 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 to develop genetically transformed plants with over-expressed and silenced genes to improve resistance to nematodes and fungi. DNA constructs were made to silence soybean genes involved in the regulation of expression of the plant defense response. Constructs overexpressing NPR1 and PAD4 show particular promise. These constructs reduced cyst formation by soybean cyst nematode approximately 55%. NPR1 and PAD4 are upstream regulators of the hormone, salicylic acid. This hormone is important in activating the defense response in plants to certain pathogens. Our 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. We previously planted early and medium maturity group soybean seeds in both field and greenhouse settings. The collected seeds were divided into 2 groups (medium and early) and prepared for protein extraction. This year we extracted proteins from 20 medium maturity groups of greenhouse and field grown soybean seeds including landraces, bred cultivars, and lines bred for seed traits. We separated the proteins using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The resulting 2D gel images were scanned. The differential expression of protein spots using analytical software is in progress.


Accomplishments
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, Maryland, made multiple DNA gene constructs designed to provide resistance to the soybean cyst nematode. 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 approximately 55%. These genes may be useful to soybean breeders to broaden resistance against nematodes.

2. Springtime for Poplar and Germinies: Poplar trees grow and produce biomass quickly, which makes them an important biofuel feedstock. Poplar trees efficiently recycle nitrogen, meaning that less nitrogen fertilizer needs to be applied to grow the trees for fuel. Consequently, this translates to less energy input and greater energy output. Thus, nitrogen recycling is an important factor in choosing biofuel feedstocks, but little is known about how poplar trees recycle nitrogen. Evidence shows that poplar stores and recycles nitrogen in its proteins. Researchers at ARS Beltsville, Maryland, and the University of Maryland, College Park, Maryland, used mass spectrometry to identify poplar proteins during stages of cell growth when nitrogen is recycled. They identified for the first time several protease proteins that degrade nitrogen storage proteins like germin. The proteases were activated during stages of new growth. It is likely that the amino acids liberated from degraded proteins are recycled to produce new proteins that support tree growth in the spring time. These results provide new insight into nitrogen recycling in poplar. These data are most likely to influence scientists at universities, government agencies and companies who are studying biofuels and energy efficiency.


Review Publications
Sundaresan, S., Philosoph-Hadas, S., Riov, J., Belausov, E., Kochanek, B., Tucker, M.L., Meir, S. 2014. A new aspect of flower abscission: involvement of a specific alkalization of the cytosol in the abscission zone cells. Journal of Experimental Botany. 66:1355-1368.
Kim, J., Tucker, M.L. 2015. To grow old: regulatory role of ethylene in senescence. Frontiers in Plant Science. 6(20):1-7.
Islam, N., Gen, L., Garrett, W.M., Lin, R., Sriram, G., Cooper, B., Coleman, G.D. 2015. The proteomics of nitrogen remobilization in poplar bark. Journal of Proteome Research. 14:1112-1126.
Natarajan, S.S., Khan, F., Luthria, D.L., Tucker, M.L., Song, Q., Garrett, W.M. 2014. A comparison of protein and phenolic compounds in seed from GMO and non-GMO soybean. Journal of Data Mining in Genomics & Proteomics. 5(3):161-169.
Tavakolan, M., Alkharouf, N.W., Matthews, B.F., Natarajan, S.S. 2014. SCNProDB: A database for the identification of soybean cyst nematode proteins. Bioinformation. 10(9):400-405.
Natarajan, S.S., Tavakolan, M., Alkharour, N.W., Matthews, B.F. 2014. SoyProLow: A protein database enriched in low abudndant soybean proteins. Bioinformation. 10(9): 598-601.
Ghosh, R., Choi, B., Cho, B., Lim, H., Park, S., Bae, H., Natarajan, S.S., Bae, H. 2014. Characterization of developmental and stress mediated expression of cinnamoyl-CoA reductase (CCR) in kenaf (Hibiscus cannabinus L.). The Scientific World. DOI: 10.1155/2014/601845.
Li, S., Darwish, O., Alkharouf, N., Matthews, B.F., Ji, P., Domier, L.L., Zhang, N., Bluhm, B.H. 2015. Draft genome sequence of Phomopsis longicolla MSPL 10-6. Genomics Data. 3:55-56.