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.
A goal of the first objective was to demonstrate that the IDA-like gene that we discovered in the genomic sequence of the root knot nematode Meloidogyne incognita is transcribed and translated in the nematode and secreted into the soybean cell where it initiates a signaling pathway similar to that described in Arabidopsis. The IDA-like sequence in M. incognita encodes a peptide (small protein) similar to the plant Inflorescence Deficient in Abscission (IDA) signaling peptide. We fully confirmed this year that exogenous peptide treatments and transgenic expression of the nematode IDA-like (IDL) gene rescued the delayed abscission and reduced lateral rooting phenotypes of the ida Arabidopsis mutant that lacks the IDA peptide. An MiIDL-RNAi construct designed to suppress the nematode MiIDL1 gene was transformed into Arabidopsis and tomato, two experimental systems used to quickly model nematode infectivity of soybean. Suppression of the MiIDL1 gene in the nematode through ingestion of the RNAi expressed in the plant cell cytoplasm reduced the number of galls that formed on infected Arabidopsis roots by approximately 50%. In tomato, we screened multiple transgenic events that contain the MiIDL1-RNAi construct and currently have identified two independent transgenic events that are homozygous, single-copy transformants. The seeds of these homozygous tomato plants are being grown and infected with nematodes to determine if the suppression of MiIDL1 reduces the number galls that form on the tomato roots as it did for Arabidopsis and to identify any changes in gene expression associated with suppression of the MiIDL1 gene. In situ hybridization to localize MiIDL1 expression within a secretory organelle in the nematode did not produce a reproducible signal. Therefore, an alternative approach was initiated to immuno-localize the MiIDL1 protein within the nematode and to identify its putative secretion from the nematode and accumulation in the host plant cells. Antiserum was obtained and successfully tested on an immunoblot. In addition, we have partially completed an in situ binding assay for MiIDL1 that demonstrates that the MiIDL1 peptide can bind to the same receptors (HAESA and HAESA-like-2) that the Arabidopsis IDA peptide binds to. This suggests that the MiIDL peptide functions though the same biochemical signal receptor system as plant IDA. Overall, research on nematode and plant receptor and 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. Another goal was to study soybean proteins involved in disease resistance to the soybean rust fungus. 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 (TF) using an RNAi mechanism led to soybean rust susceptibility in previously resistant plants, hence demonstrating that these TFs help control resistance. This year, we wanted to validate whether the soybean TFs migrated to the soybean cell nucleus where they could activate genes. The plan was to fuse the TFs to a peptide tag (3xFLAG) to help visualize or immuno-localize them in soybean cell nuclei. As reported last year, we needed to develop a system by which we could express fusion proteins in soybean cells. We selected the Soybean mosaic virus (SMV) transient fusion protein expression system, but the system required further optimization because we also needed to confirm that we could isolate (purify) fused proteins to be able to address the next year’s research goals which include determining if other soybean proteins physically attach to or interact with the over-expressed TFs. We successfully fused test genes to 3xFLAG and expressed the fusion proteins from SMV in soybean, but we were unsuccessful in recovering significant amounts of fused test proteins from leaves. We were concerned that the length and size of the test genes could have been a factor such that longer, larger fusion proteins were unstable. We tested different length fusions, but found no better recovery with small fusions compared to larger fusions. We then turned to mass spectrometry (MS) to try to detect if fusion protein peptide fragments existed as evidence for protein degradation. We were fortunate to have acquired a new Lumos mass spectrometer, the fastest and most sensitive one that is commercially available. We first had to set up this new instrument. We learned how to control the software and how to clean, service, and calibrate the instrument. We also built a computer to run MS data analysis software and learned how to use the software to interpret results. Research and development included 1) implementation of a custom chromatography method to separate peptides and deliver them to the mass spectrometer; 2) implementation of a chemical tagging method to allow relative quantitation of peptides; 3) development of an HPLC off-line fractionation method to improve separations and the detection of chemically tagged peptides; 4) optimization of methods, parameters, procedures, and software to maximize the total number of high-quality peptide and protein identifications; and 5) development of a targeted method with attomole sensitivity. Initial tests led to the discovery of ~6,000 proteins from common bean leaves (Phaseolus vulgaris). Implementation of the chemical tagging method led to the quantitation of ~4,000 proteins from soybean roots, ~3,000 proteins from nitrogen-fixing bacteria (Bradyrhizobium elkanii), and ~1,000 proteins from Staphylococcus aurea (a bacterial pathogen of humans). Improvements made with HPLC off-line fractionation led to the quantitation of ~8,000 proteins in flowers (Arabidopsis thaliana). We are hoping to use this new mass spectrometer to detect the presence of fusion proteins expressed from SMV in soybeans, to troubleshoot the expression system, and to detect other soybean proteins that physically interact with the fusion proteins to better understand the function and activity of soybean proteins that control and regulate disease resistance. On a related note, we successfully used our prior mass spectrometer before it was decommissioned to determine the in situ localization (in plant leaves) of proteins that the soybean rust and bean rust fungi use to infect soybeans and beans. These findings were described as an accomplishment in the FY16 annual report and are published in the scientific journal Phytopathology. A goal of the second objective was to develop genetically transformed plants with over-expressed and silenced genes to improve resistance to nematodes. As reported last year, we showed that the ectopic expression of Arabidopsis alpha-EGase At1g48930 (designated 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. These studies were performed in model root systems and not in wholly genetically transformed plants. The plan for this year was to test in wholly genetically transformed plants, but the lead scientist for this objective retired and the work was not completed due to a critical vacancy. 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 in medium Maturity Group soybean genotypes grown under greenhouse and field conditions. The proteins were separated on two dimensional gels, and 400 proteins were analyzed and compared for differential expression and identified by mass spectrometry. We found significant variation in the accumulation of different isoforms of storage proteins, allergens, and anti-nutritional proteins in seeds of soybeans of the same genotypes grown in the greenhouse and the field. These comparative studies provided information about the influence of environmental conditions on the nutritional quality of different soybean varieties. In addition, we compared proteins and metabolite abundance in transgenic soybean seeds compared with seeds of non-transgenic controls (Jack). The transgenic soybean was developed through siRNA-mediated gene silencing of the omega-3 fatty acid desaturase (FAD3), which is responsible for the synthesis of a -linolenic acids (18:3) in the polyunsaturated fatty acid pathway. The significant reduction of this fatty acid increased the stability of the seed oil, enhancing the seed agronomical value. Our metabolic analysis revealed that in addition to having a reduced a-linolenic acid content, the transgenic soybean seed had a decreased amount of isoflavones. We also applied a new quantitative high-throughput mass spectrometry method to evaluate changes in protein abundance between the same seeds. We identified and quantified 6,079 proteins of which 1,319 exhibited statistically significant accumulation differences (703 up and 616 down). Our analysis suggests that proteins involved in photosynthetic electron transport, carbon assimilation, carbon cycling and arginine biosynthesis were altered in the transgenic soybean. A better understanding of the network of regulated proteins and the changes in secondary metabolites will allow breeders to more confidently develop new value-added soybeans and other agricultural crops.
1. Root knot nematodes don’t like these plants. Nematodes, minute soil worms, are the most economically damaging pathogens of soybean. Pathogenic nematodes infect roots and induce the formation of a feeding structure, a specialized set of cells from which the nematodes acquire nutrients from the plant. The nematodes change the plant root cell biochemistry and physiology by co-opting developmental programs that are natural to the plant root. ARS scientists at Beltsville, Maryland, discovered and characterized a protein from a root-knot nematode that mimics the natural plant IDA cellular development protein but alters cellular development in favor of the nematode. The scientists engineered plants that inhibit the protein from the nematode using a gene silencing mechanism and consequently reduced the nematode’s ability to infect plants. These results are expected to be useful to scientists at universities, government agencies and companies who are trying to fight nematodes and the disease they cause on soybeans and other agriculturally important plants.
2. Location affects allergens in soybean seeds. Soybean seeds are a good source of dietary protein, so it is important to investigate the factors that influence protein accumulation in seeds. ARS scientists at Beltsville, Maryland, used mass spectrometry (an analytic technique) to monitor seed protein accumulation in seeds of 27 different soybean varieties grown in Maryland, South Carolina, and South Dakota during early and late sowing seasons. The data showed that there was significant compositional variation in proteins including known allergens and anti-nutritional proteins that upset digestion and that these differences correlated with the varieties but also correlated with geography. In addition, the data showed a correlation between the accumulation of anti-nutritional proteins and an early sowing season. These results suggest that the nutritional quality of soybean seeds is partly dependent upon soybean field locations and planting times. This information could be useful to growers wanting to maximize the nutritional quality of their yields.
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