Location: Soybean Genomics & Improvement Laboratory
2023 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
This is the final report for project 8042-21220-234-000D which ended March 27, 2023. New NP301 OSQR approved project 8042-21220-261-000D, entitled “Proteomic and Metabolomic Characterization of Quality Traits in Soybeans, Beans, and Agricultural Products” has been established. Extensive results were realized over the 5 years of the project.
For Objective 1, to characterize biochemical processes in rust fungi and hosts during infection, we used mass spectrometry to identify candidate effector proteins from the 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. 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. An invention disclosure was submitted to the USDA-ARS Office of Technology Transfer.
Also for Objective 1, we tested benzothiadiazole (BTH), an immune system inducer, on beans. Beans treated with BTH showed no signs of disease by 10 days after rust inoculation while controls died. To understand the effect BTH has on the bean immune system, we measured the changes of accumulation for 3,973 proteins using mass spectrometry. The set of 409 proteins with significantly increased accumulation in BTH-treated leaves included receptor-like kinases SOBIR1, CERK1, and LYK5 that perceive pathogens, and EDS1, a regulator of the salicylic acid defense pathway. Other proteins that likely contributed to resistance included PR-proteins, a full complement of enzymes that catalyze phenylpropanoid biosynthesis, and protein receptors, transporters, and enzymes that modulate other defense responses controlled by jasmonic acid, ethylene, brassinosteroid, abscisic acid, and auxin. Increases in the accumulation of proteins required for vesicle mediated protein secretion and RNA splicing occurred as well. These results revealed a set of proteins needed for rust resistance and reaffirmed the utility of BTH to control disease by amplifying the bean plant’s natural immune system. These experiments also initiated the quest to use mass spectrometry to identify metabolites like phenylpropanoids that might be crucial to resistance as described in the forthcoming research project.
Also for Objective 1, in a collaboration with a visiting scientist from Embrapa (the Brazilian Agricultural Research Corporation), we used mass spectrometry to investigate the bean immune system during infections with halo blight bacteria that elicit hypersensitive resistance. More than 4,000 bean proteins were quantitated and several, including receptor kinases that can sense bacteria and send signals within the plant to stimulate defense, were increased. Virus-induced gene silencing revealed that a G-type lectin-receptor-like kinase is needed to suppress bacterial infection. These experiments also revealed that resistant plants produced increased amounts of enzymes used in salicylic acid mediated immune signaling and for the biosynthesis of phytoalexins.
Also for Objective 1, we used mass spectrometry to detect the production of salicylic acid and resveratrol, a phytoalexin, in bean leaves immune to halo blight bacteria. To see if salicylic acid and resveratrol also affected the bacterium directly, we used mass spectrometry to measure the changes in bacterial proteins after bacteria were exposed separately to salicylic acid and resveratrol. The results showed that each compound imparts different antibiotic activities. Salicylic acid reduced the amounts of type VI secretion system proteins and alginate biosynthesis enzymes needed for infection. By contrast, resveratrol impeded the bacterial tricarboxylic acid cycle and ATP biosynthesis at the electron transport chain and decreased bacterial flagella needed for movement. Together, these compounds produced by immune beans disrupt mechanisms that the bacteria need to infect beans. These findings will be of interest to scientists in the government, at universities, and at private institutions who want to understand how beans and other plants protect themselves from pathogen infection.
For Objective 2, determining the role of root knot nematode secreted MiIDL1 hormone mimic, the scientist who discovered MilDL1 retired from ARS in 2018.
For Objective 3, to assess protein and metabolite profiles in soybean seeds, we used high throughput, large-scale quantitative mass spectrometry to identify different classes of soybean proteins, including storage, allergen, and anti-nutritional proteins. We optimized the protocol to extract the maximum amounts of certain types of proteins and to clean them before separation for the quantitative analyses. For the initial understanding of metabolic control of synthesis and accumulation of proteins and amino acids during seed filling, we investigated protein profiles using tandem mass tag quantitative analysis. We identified 4,172 proteins in three developmental stages, namely early, mid, and late seed filling, and mapped the identified proteins to metabolic pathways associated with seed filling. We found differential accumulation of several essential enzymes/proteins related to amino acid and protein synthesis during seed development. Also, the storage proteins, 7S (beta-subunit)/11S (Gy3, Gy4, Gy5) steadily increased in abundance, and 2S decreased during the early to late stages of seed filling. In addition, the results revealed some protein and sulfur-containing amino acid accumulation pathways that had not been previously recognized. These findings will help guide scientists and breeders wanting to alter the metabolic pathways and to develop new value-added soybeans with improved protein quality.
Also for Objective 3, we used large-scale protein analyses to study soybeans with a silenced fatty acid desaturase gene (GmFAD3). We quantified 6,074 proteins, 1,036 of which exhibited higher abundance and which were associated with photosynthetic pathways and glycolysis. Among the 1,079 proteins with lower abundance were proteins and enzymes associated with fatty acid and secondary metabolite production. We confirmed desaturase gene silencing and its collateral effects on the differential expression of key enzymes of fatty acid metabolic pathways. Understanding these metabolic pathways will aid researchers and biotechnologists in efforts to alter the oil quality needed for the food industries.
Also, for Objective 3, we secured ten (L1-L10) soybean mutants in which the genome compositions were altered by fast neutron (FN) mutagenesis. The mutants' initial seed composition analyses were performed by near-infra-red scanning. These mutants also were characterized by next generation sequencing and comparative genomic hybridization to locate gene duplication and deletions. One mutant (L06) had the highest number of genes located within heterozygous deletions (246 genes) in chromosome 6, followed by L09 (103 genes) in chromosome 15. Large duplications encompassing 1,743 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) and then investigated which deleted or added genes were responsible for the altered phenotype of each line. Large-scale proteomics analyses were performed for two selected mutants that exhibited 15-18% higher protein content. Using mass spectrometry, we observed 155 compounds with increased abundance and 298 with decreased abundance in one FN mutant compared to the wild type. S-methylmethionine derivatives were among metabolites that increased in the mutant. The increased sulfur content will be of interest to the animal feed industry.
This research project produced more than 23 scientific manuscripts published in scholarly journals and included collaborations with more than 42 national and 8 international scientists comprising students, post-doctoral scientists, senior scientists, and visiting scientists. Formal collaborative agreements were executed with the University of Maryland, College Park, Maryland, the Brazilian Agricultural Research Corporation (Embrapa), Brazil, and the Oak Ridge Institute for Science and Education (ORISE).
Accomplishments
1. Resveratrol adversely alters a bacterial proteome. Halo blight disease, caused by a bacterium, reduces harvests of the dry, edible common bean. Natural resistance genes in the bean plant can provide protection against some strains by producing phytoalexins like resveratrol, an antibiotic. To learn more about the antibiotic properties of resveratrol, USDA-ARS scientists in Beltsville, Maryland, used mass spectrometry, an analytical technique, to measure the changes in proteins after bacteria were exposed to resveratrol. The results show that the bacterium is actively engaged in trying to eliminate resveratrol from cells and that resveratrol decreases enzymes the bacterium needs to produce energy and to move. Hence, the antibiotic properties of resveratrol include reducing bacterial energy needed for reproduction and slowing bacterial motility. This explains, in part, why beans that induce production of resveratrol exhibit resistance to halo blight. These findings will be of interest to scientists in the government, at universities, and at private institutions who want to understand how beans and other plants resist pathogen infection.
2. Soybeans with more protien are a knock-out. Fast-neutron mutagenesis was employed to alter genes in soybean seeds. Changes were investigated by USDA-ARS scientists in Beltsville, Maryland, using a mass spectrometry-based analytical technique. The results showed that a fast-neutron mutant with 21 deleted genes had increased protein, isoflavones, and sulfur-containing metabolites in the soybean seed. The results contributed to a better understanding of the perturbation of the metabolic pathways due to gene deletion and how gene deletion contributed to the synthesis and accumulation of these metabolites. These results will aid breeders, geneticists, and plant physiologists in further developing soybeans with enhanced nutritional profiles for value-added food products.
Review Publications
Cooper, B. 2022. Disruptive effects of resveratrol on a bacterial pathogen of beans. Journal of Proteome Research. 22(1):204-214. https://doi.org/10.1021/acs.jproteome.2c00633.
Islam, N., Krishnan, H.B., Slovin, J.P., Natarajan, S.S. 2023. Metabolic profiling of a fast neutron soybean mutant reveals increased abundance of isoflavones. Journal of Agricultural and Food Chemistry. 71(26):9994-10003. https://doi.org/10.1021/acs.jafc.3c01493.
