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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

2016 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. We identified a short sequence motif (SCNbox1 - WGCATGTG) common to several promoters of genes expressed in soybean roots during soybean cyst nematode infection. We prepared minimal promoter constructs with forward and reverse 3x tandem repeats of the SNCbox1 motif linked to GUS and florescent reporter genes as well as minimal promoter constructs without the SCNbox1 sequence which served as a control. These constructs were transformed into soybean to produce transgenic hairy roots. We are in the process of propagating transgenic roots for subsequent analysis of expression of the reporter genes in cyst nematode infected roots. We are interested in learning whether the nematode secretes proteins that can modulate soybean genes by recognition of this DNA sequence motif. In a related project, we previously 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 Inflorescence Deficient in Abscission (IDA) signaling peptide. We demonstrated that expression of the nematode IDA-like (IDL) gene in Arabidopsis rescued the delayed abscission and reduced lateral rooting phenotypes in the ida mutant that lacks the Arabidopsis IDA peptide. Moreover, when the full-length MiIDL1 gene was transformed into wild-type Arabidopsis there were a greater number of lateral roots. Enhanced lateral root initiation supports our hypothesis that the nematode MiIDL1 peptide alters root cell differentiation when the peptide is secreted from the nematode into the host. An MiIDL-RNAi construct designed for suppression of the nematode MiIDL1 gene was transformed into Arabidopsis and tomato and into hairy roots of soybean and tomato. Hairy roots of soybean with or without the MiIDL1 RNAi did not infect well with the root-knot nematode and this experimental approach was discontinued. This year we completed studies with transgenic Arabidopsis, but are still working to obtain stably transformed tomato and hairy roots of tomato. The results for transgenic Arabidopsis demonstrated that the MiIDL-RNAi molecules are, as expected, processed into smaller double-stranded RNAs that enter the nematode during feeding and suppress expression of the nematode MiIDL1 gene. Suppression of the MiIDL1 gene reduced the number of galls that formed on the roots by approximately 50%, and at 35 days post inoculation the galls that did form were reduced in diameter. 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. In another related project, we studied soybean proteins involved in disease resistance to soybean rust. We 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 resistance to soybean rust. We also previously reported that the genetic silencing of five of these transcription factors using an RNAi mechanism led to soybean rust susceptibility in previously resistant plants, hence demonstrating that these transcription factors help control resistance. To determine if over expression of these soybean transcription factors might lead to increased resistance in otherwise susceptible cultivars, two approaches were taken: 1) soybeans were genetically transformed to over-express the soybean transcription factors, and 2) the soybean transcription factors were transiently over-expressed using Bean pod mottle virus (BPMV) and Soybean mosaic virus (SMV) vectors. Transgenic plants selected for herbicide resistance (expressing the co-transformed herbicide resistance selectable marker) were challenged with soybean rust at the containment greenhouse at Ft. Detrick. None of the transgenic plants showed improved resistance over susceptible controls. Because no antibodies exist for the transcription factors, we did not know if the transgenic plants had indeed accumulated additional amounts of active soybean transcription factors. Hence, for this year we tabled the transgenic experiments and turned our attention to transient transcription factor expression with BPMV and SMV. We discovered that the BPMV icosahedron limits the size of foreign genes that can be inserted into the BPMV genome and that the virus deleted the inserted soybean transcription factor genes during plant infection. Hence, no over-expression occurred with BPMV. The SMV system can tolerate longer genes, so the soybean transcription factors were tested in SMV. But before we moved forward, we also wanted to tag the transcription factors with a common epitope to which commercial antibodies exist. This would allow us to differentiate between the transient soybean transcription factors and the endogenous analogs and verify that over-expression occurred. Several tags including FLAG, 3xFLAG, MYC and 6xHIS at amino and carboxyl- terminal positions on the soybean transcription factors were tested. We have determined that carboxyl-terminal positioning of a 3xFLAG epitope and a specific source of commercial antibody to 3xFLAG are sufficient for resolving transiently expressed soybean transcription factors from SMV. It remains to be tested in future experiments if the tagged soybean transcription factors retain their native biological activity when expressed from SMV and if the extra amounts of transcription factors will improve resistance to soybean rust. Resource challenges and renovations at the Ft. Detrick containment greenhouse are currently keeping us from testing plants for resistance. In the meantime, we will determine if the soybean transcription factors interact with other soybean proteins. The tagged versions, SMV transient expression, the antibodies, and mass spectrometry may together reveal protein interaction partners. It is possible that the identity of interacting protein partners may reveal how the nuclear regulatory proteins are controlled or how they mediate gene transcription to coordinate disease resistance to rust. The second objective is to identify genes and proteins expressed during interactions of soybeans 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. Plant cellulases (endo-beta-1,4-glucanases; EGases) include cell wall-modifying enzymes that are involved in nematode-induced growth of syncytia (feeding structures) in nematode-infected roots. EGases in the alpha- and beta-subfamilies contain signal peptides and are secreted, whereas those in the gamma-subfamily have a membrane-anchoring domain and are not secreted. The Arabidopsis alpha-EGase At1g48930, designated as AtCel6, is known to be down-regulated by beet cyst nematode (Heterodera schachtii) in Arabidopsis roots, whereas another alpha-EGase, AtCel2, is up-regulated. We showed that the ectopic expression of AtCel6 in soybean roots reduced susceptibility to both soybean cyst nematode (SCN; Heterodera glycines) and root knot nematode (Meloidogyne incognita). Suppression of GmCel7, the soybean homologue of AtCel2, in soybean roots also reduced the susceptibility to SCN. In contrast, in studies on two gamma-EGases, both ectopic expression of AtKOR2 in soybean roots and suppression of the soybean homologue of AtKOR3 had no significant effect on SCN parasitism. Our results suggest that secreted alpha-EGases are likely to be more useful than membrane-bound gamma-EGases in the development of an SCN-resistant soybean through gene manipulation. This research also provided evidence that Arabidopsis shares molecular events of cyst nematode parasitism with soybean. 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 extracted and separated proteins from medium Maturity Group soybean genotypes grown in greenhouse and field conditions. The proteins were separated on two dimensional gels, and 300 protein spots were analyzed and compared for differential expression. We are beginning to identify the 300 protein spots by mass spectrometry. In addition, we have analyzed primary metabolites using gas-chromatography mass spectrometry and secondary metabolites using liquid chromatography mass spectrometry from soybean genotypes grown in greenhouse and field conditions. We identified and characterized ten primary metabolites (amino acids, organic acids, and sugars) and ten secondary metabolites (isoflavones, fatty acid methyl esters). Multivariate analysis (PCA and PLS-DA) showed distinct separation based on varieties and growing conditions. Sugar molecules such as glucose, sucrose, and pinitol were increased under greenhouse cultivation whereas amino acids and organic acids varied between the varieties. Among the identified isoflavones, the level of glucosides decreased in most of the greenhouse cultivated plants but the levels of aglycons were marginally increased. The results show that clustering patterns of soybean metabolites were significantly influenced by genetic variation and growing conditions.


Accomplishments
1. Why rust never sleeps. Little is known about the proteins made by two related species of fungi that cause rust diseases on beans and soybeans, but experts suspect that these proteins are secreted from the fungi into plants to dismantle resistance and to take control of plant biochemistry. ARS scientists at Beltsville, Maryland, used mass spectrometry (an analytic technique) to identify fungal proteins in infected beans and soybeans. They 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 and 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. Knowledge of these proteins may help scientists at universities, government agencies and companies discover new chemicals that could prevent the infection of beans and soybeans.


An ARS scientist at Beltsville, Maryland mentored a graduate student from the department of Biology, Morgan State University, Baltimore, Maryland, 2015. An ARS scientist at Beltsville, Maryland served on the Northeast Area Diversity Taskforce, Special Emphasis Committee.


Review Publications
Tucker, M.L., Yang, R., Sundaresan, S., Meir, S., Kim, J., Phlosoph-Hadas, S. 2015. Examination of the abscission-associated transcriptomes for soybean, tomato, and Arabidopsis highlights the conserved biosynthesis of an extensible extracellular matrix and boundary layer. Frontiers in Plant Science. 6(1109):1-15.
Kim, J., Barnaby, J.Y., Yang, R., Sicher Jr, R.C., Chang, C., Tucker, M.L. 2016. Transcriptome analysis of soybean leaf abscission identifies transcriptional regulators of organ polarity and cell fate. Frontiers in Plant Science. 7:125.
Cooper, B., Campbell, K., Garrett, W.M. 2015. Expression of a synthetic rust fungal virus cDNA in yeast. Archives of Virology. 161:111-123.
Natarajan, S.S., Khan, F., Song, Q., Lakshman, S., Cregan, P., Scott, R.A., Shipe, E., Garrett, W.M. 2016. Characterization of soybean storage and allergen protein affected by environmental and genetic factors. Journal of Agricultural and Food Chemistry. 64(6):1433-1445.
Cooper, B., Campbell, K., Beard, H.S., Garrett, W.M., Islam, N. 2016. Putative rust fungal effector proteins in infected bean and soybean leaves.Phytopathology. doi: 10.1094/PHYTO-11-15-0310-R.
Sundaresan, S., Philosoph-Hadas, S., Riov, J., Mugasimangalam, R., Kuravadi, N.A., Kochanek, B., Salim, S., Tucker, M.L., Meir, S. 2016. De novo transcriptome sequencing and customized abscission zone-specific microarray as a new molecular tool for analysis of tomato organ abscission. Frontiers in Plant Science. 6(1258):1-29.
Tucker, M.L., Kim, J. 2015. Abscission, organ separation, is more complex than you might think. Stewart Postharvest Review. 11:1-7.
Wen, C., Tucker, M.L. 2015. Research tools: ethylene preparation. In: Chi-Kuang Wen editor. Ethylene in plants. Springer Netherlands. Springer Link. Ethylene in Plants. p. 245-261.
Matthews, B.F., Youssef, R.M. 2016. Agrobacterium rhizogenes - based transformation of soybean roots to form composite plants. Bio-protocol. 6:e1708.
Matthews, B.F., Youssef, R.M. 2016. Soybean cyst nematode, Heterodera glycines, infection assay using soybean roots. Bio-protocol. 6:e1707.
Alzohairy, A.M., Macdonald, M.H., Amer, R., Matthews, B.F. 2015. Root transformation of Glycine max with responsive promoters to nematode infection. Egyptian Journal of Genetics and Cytology. 44:149-164.
Lakshman, D.K., Roberts, D.P., Natarajan, S.S., Garrett, W.M., Alkharouf, N., Darwish, O., Khan, F.H., Paine, A., Mitra, A. 2016. Proteomic investigation of Rhizoctonia solani AG 4 identifies secretome and mycelial proteins with roles in plant cell wall degradation and virulence. Journal of Agricultural and Food Chemistry. 64:3101-3110.