Location: Soybean Genomics & Improvement Laboratory
2019 Annual Report
Objectives
Objective 1: Characterize biochemical processes in rust fungi and hosts during infection, determine relationships with currently used resistance genes, and work with breeders or pathologists to insert multiple resistance genes. [NP301, C1, PS1A; C3, PS3A]
Objective 2: Determine the role of root knot nematode secreted proteins in soybean growth alterations, such as the recently discovered MiIDL1 hormone mimic, to develop genetic resistance to the nematode. [NP301, C3, PS3A]
Objective 3: Assess proteins and metabolite profiles in soybean seeds, determine associations of metabolic pathways with nutritional traits, and identify germplasm or genes that breeders can use for genetic improvement of quality traits. [NP301, C2, PS2A]
Approach
For Objective 1, candidate rust fungus effector proteins identified in infected beans and soybeans will be characterized. A plant virus gene silencing system will be used to deliver fungal effector gene silencing RNAs from the plant to the fungus to block rust fungus infection. The fungal effector genes will be inserted into a plant virus for protein expression in plant leaves, and mass spectrometry will be used to identify plant proteins that interact with the fungal protein. Plants will be treated with plant hormones to induce disease resistance, and mass spectrometry will be used to identify plant proteins that contribute to disease resistance. Transgenic plants expressing proteins that may confer resistance to rust fungi will be screened by mass spectrometry and tested for resistance. For Objective 2, immunocytochemistry on thin root-gall sections will be performed to determine if an effector protein from a nematode pathogenic to soybean is secreted into the plant. The nematode effector gene will be expressed in plant roots, and mass spectrometry will be used to identify plant proteins that interact with the nematode protein. RNA sequencing and mass spectrometry will be used to identify differential transcript and protein accumulation in the galls formed on nematode infected roots. For Objective 3, a systems approach will be used to identify the protein and metabolic pathways that produce protein, oil, and carbohydrate seed traits in soybeans and to ensure that allergens and anti-nutritional proteins do not exceed normal levels. Comparative genomic hybridization will be used to map gene deletions associated with traits. Seeds with high protein content will be investigated by mass spectrometry for changes in the protein profiles with special attention being paid to assure the presence of low amounts of allergens or high methionine content. Seeds selected for oil, carbohydrates, and other (isoflavones, amino acids) traits will be investigated for changes in the metabolite profiles and to identify mutants with low anti-nutritional compounds/high isoflavone content.
Progress Report
Several hypotheses were evaluated as part of Objective 1. For the first hypothesis, we postulated that modulation of fungal effectors will lead to less pathogen accumulation and improved disease resistance. This hypothesis was based on prior reported research where we used mass spectrometry to identify effector proteins from Uromyces appendiculatus, the common bean rust fungus. Suspecting that these effector proteins are required by the fungus to infect plants, we utilized a gene-silencing mechanism to reduce the amount of effector protein RNA expressed in the fungus. Specifically, we inserted a 258 base pair DNA fragment from each of five candidate effector genes in bean pod mottle virus, infected beans with the virus, and then challenged beans with bean rust. Virus-infected plants expressing gene fragments for four of five candidate effectors accumulated lower amounts of rust and had dramatically less rust disease. By contrast, controls that included a fungal gene fragment for a non-effector protein died from the fungus. The results implied that RNA generated in the plant moved across the cell into the fungus to silence the fungal effector genes important to fungal pathogenicity. The experiments revealed that four bean rust genes encode pathogenicity determinants and that the expression of fungal RNA in the plant can be an effective method for protecting bean plants from rust.
For the second hypothesis of Objective 1, we postulated that expression of fungal growth inhibitors will lead to less pathogen accumulation and improved disease resistance. One fungal growth inhibitor, KP4, is made by a virus that infects the corn smut fungus. Scientists have previously expressed KP4 in transgenic wheat and maize to confer resistance to smut. Therefore, we hypothesized that KP4 could confer protection to soybean rust in transgenic plants. We fused the KP4 gene to open reading frames to cytokinin oxidase and separately to a defensin N-terminal signal peptide to facilitate secretion of KP4 into the leaf apoplast where fungi spread. We made transgenic soybean plants harboring the KP4 gene constructs. To test whether the transgenic plants produced KP4, we purified protein from the leaves, digested the protein with trypsin, spiked in heavy-labeled commercially synthesized KP4 standard peptides, and set a mass spectrometer to search for KP4 peptides and the heavy-labeled standards as the total peptide population was separated and eluted into the instrument. This “targeted proteomics” method is known as parallel reaction monitoring. We then used Skyline software to analyze the ratios between the transgenic peptides and the spiked standards. Our data showed that we could monitor a linear range of 6 orders of magnitude of targeted peptide and could detect as little as 0.6 attomoles of peptide in 1 microgram total peptide analyzed. Using this method, we screened more than 100 plants from 10 different transgenic lines and discovered that some lines accumulated more transgenic KP4 protein than others. We will allow the plants with the greatest amounts of KP4 to self-fertilize, and then we will select homozygote progeny with the greatest amounts of KP4. We intend to deliver these plants to the ARS containment greenhouse at Ft. Detrick and challenge the plants with soybean rust to determine if KP4 accumulation confers resistance to soybean rust.
The third hypothesis was not planned to be addressed this year. For the fourth hypothesis of Objective 1, we postulated that modulation of plant growth regulators will lead to less pathogen accumulation and improved disease resistance. Brassinosteroid (BR) is one such plant growth regulator and was the first steroid hormone found in plants. A newly EPA-approved plant product related to BR, homobrassinolide (hBR), accelerates plant growth and improves yield. It is not yet known if hBR affects disease resistance. Current research by other scientists shows that BR application on tobacco decreases susceptibility to fungi. To test the activity of hBR, we applied 0.1% hBR to bean seedlings. Specifically, a soaking spray of hBR was applied to fully opened primary leaves of 8 to 10 day-old bean plants. Prior to hBR treatment, primary leaves on each plant were measured length by width. These measurements were taken again 7 days after hBR treatment. In addition, the distances from the soil surface to the cotyledon, the cotyledon to the primary leaf node, and the primary leaf node to the secondary leaf node were measured 7 days after treatment. In three separate experiments, we observed no change in primary leaf area after hBR treatment compared to the control. Also, we observed no changes in distance from soil to cotyledon, cotyledon to first node, or first node to second node after hBR treatment. To determine whether hBR treatment enhances resistance to rust, we spray-inoculated beans with bean rust spores 8 hours after hBR treatment. The inoculated plants were placed in a dew chamber overnight to induce spore germination and infection, and the plants were placed on a light cart the next morning. These experiments were repeated 3 times. Rust fungus pustules were counted on each primary leaf 7 days after inoculation. We observed no reduction in numbers of rust pustules on hBR treated leaves 7 days after inoculation. We are currently investigating other formulations of BR and other plant hormones that could increase rust resistance.
The goals of the second objective were to determine if the root-knot nematode MilDL1 peptide is secreted from the nematode into its plant host, evaluate where in the host it is secreted, identify if it interacts with plant host proteins, and determine if it acts as a hormone to affect plant cell development. The scientist responsible for this objective retired, and the work was not completed due to a critical vacancy.
The first milestone of the third objective was to collect seeds, grow mutant plants, extract DNA and perform CGH analysis on ten fast neutron (FN) mutant soybean lines. The FN mutants were provided by a collaborator at the University of Minnesota. The ten lines (L01-L10) were known to have altered seed compositions such as high total protein and methionine, high oleic oil, and low raffinose and stachyose. These traits are important to the soybean industry and the plants warrant additional investigation. Therefore, we acquired these seeds and grew all ten mutants alongside reference Williams 82 soybeans in the greenhouse. Young leaf tissues were collected and DNA was extracted for CGH-microarray analysis. The CGH-microarray consists of unique oligonucleotide probes designed from the reference soybean sequence (Williams 82) and spaced at approximately 1.1-kilo base pair intervals. The ten FN samples were labeled with a fluorescent cyanine dye, Cy3, and reference samples were labeled with Cy5. The reactions were performed using genomic DNA from all ten mutants and Williams 82. The labeled DNA was hybridized on the CGH microarrays and then the relative fluorescence intensities were quantified.
The second milestone of the third objective was to perform the bioinformatics analysis of the CGH data. We used NimbleScan software to extract signal intensities from scanned images of the arrays. The intensities of the hybridized probes were converted to log2 ratios and spatial correction and qspline normalization were applied. The significant changes in the gene copy number were determined and statistical analyses were performed on corrected log2 ratios with three standard deviations. We found significant variations of gene duplication and gene deletions in the ten mutants across twenty soybean chromosomes. In some lines, several hundred genes were deleted or duplicated (homozygous or heterozygous) due to the FN radiation. Larger heterozygous deletions were observed in other lines. Interestingly, L06 showed the highest number of genes located within heterozygous deletions (246 genes) in the chromosome 6 followed by L09 (103 genes) in the chromosome 15. Large duplications encompassing a total of 1743 genes in chromosomes 5 and 14 were observed in L10. Using these data, we mapped the position of the gene deletions/duplications in each mutant (L01-L10). We are investigating which deleted or added genes are responsible for the altered phenotype of each line. A detailed quantitative proteomic analysis of these mutants using high throughput mass spectrometry is in progress.
Accomplishments
1. Lengthening the shelf life of soybean oil. Commercial soybean oil is composed of up to 10 percent linolenic acid. This percentage is undesirable because linolenic acid lowers shelf life and stability of the oil at higher temperatures. To overcome this problem, researchers have improved soybeans using genes needed to produce linolenic acid. ARS scientists in Beltsville, Maryland, along with other ARS and university collaborators, used different analytical technique, to observe changes in the amounts of enzymes in low linolenic acid soybean seeds. They found reductions in the enzymes altered by gene silencing decreased linolenic acid production. This new knowledge will be useful to scientists at universities, companies, and public institutions who work to develop new and improved soybean products of higher quality.
Review Publications
Islam, N., Bates, P.D., John, M.K., Krishnan, H.B., Zhang, Z., Luthria, D.L., Natarajan, S.S. 2019. Quantitative proteomic analysis of low linolenic acid transgenic soybean reveals perturbations of fatty acid metabolic pathways. Proteomics. 19:1-11. https://doi.org/10.1002/pmic.201800379.
Maria John, K.M., Luthria, D.L., Natarajan, S.S. 2018. An overview of soybean seed protein analysis. Journal of the Science of Food and Agriculture. 98:5572-5580.
Krishnan, H.B., Oehrle, N.W., Alaswad, A.A., Stevens, W., John, M.K., Luthria, D.L., Natarajan, S.S. 2019. Biochemical and anatomical investigation of Sesbania herbacea (Mill.) McVaugh nodules grown under flooded and non-flooded conditions. International Journal of Molecular Sciences. 20(8):1824. https://doi.org/10.3390/ijms20081824.
Meir, S., Philosoph-Hadas, S., Riov, J., Tucker, M., Patterson, S.E., Roberts, J.A. 2019. Re-evaluation of the ethylene-dependent and -independent pathways in the regulation of floral and organ abscission. Journal of Experimental Botany. 70(5):1461-1467. https://doi.org/10.1093/jxb/erz038.
