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]
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.
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 into bean pod mottle virus a 258 base pair DNA fragment from each of five candidate effector genes, 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. We previously reported these results. Our plan for this year included assessing five more candidates, but because of flooding in 2018 and drought in 2019, we were not able to produce enough Black Valentine bean seed from the field plots to perform this part of the project. To overcome this limitation, we developed raised planting beds at the end of 2019 to put seed production under tighter control. In the meantime, we moved ahead with cloning 13 bean rust effector genes, linking them to the green fluorescent protein gene, and inserting them into the soybean mosaic virus expression vector. We are proceeding to increase our seed production for 2020, and have begun testing these bean rust effector genes in soybeans to see where they localize in cells and to identify which soybean immune system proteins they physically interact with. 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 made transgenic soybean plants harboring the KP4 gene constructs and the Basta resistance gene (for selection). To test whether the transgenic plants produced KP4, we used a “targeted” mass spectrometry method known as parallel reaction monitoring. Last year we reported that we could monitor a linear range of 6 orders of magnitude of KP4. We then screened more than 100 plants from 10 different transgenic lines and discovered that some lines accumulated more transgenic KP4 protein than others. This year, we allowed the plants with the greatest amounts of KP4 to self-fertilize. These included three T1 plants from line St268-40, two from line St268-45, one from line St268-52, and three from St268-62. If T1 plants have a single transgene, then T2 plants should segregate 3:1 for Basta resistance and KP4. We germinated 10 each of the T2 seeds on Basta, and tested the survivors by targeted proteomics. We found select plants from 1 St268-40 line, 2 St268-45 lines, and 1 St268-62 line with KP4 levels as great or greater than their parents. We then allowed each plant to self-fertilize to produce T3 seed. If the T2 parent was homozygous for Basta/KP4, then all T3 seeds will be Basta resistant. If the T2 parent was heterozygous, then we expect to observe a 3:1 Basta resistance ratio. We will test Basta resistant T3 progeny for 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. Last year, we reported that we found that that beans treated with BR were not resistant to rust. So, we began investigating other hormones like salicylic acid (SA). We found that beans pretreated with SA had a 39% reduction in rust pustules. Because SA is phytotoxic, we tested benzothiadiazole (BTH), a less-toxic inducer of the SA hormone defense pathway. Protection occurred as soon as 72 hours after treatment with BTH and resulted in no signs of disease 10 days after inoculation with rust spores. By contrast, the susceptible control plants sustained heavy infections and died. To understand the effect BTH has on the bean proteome, 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 SA 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. By contrast, more than half of the 168 decreases belonged to chloroplast proteins and proteins involved in cell expansion. 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. 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 extract proteins from fast neutron (FN) soybean mutants and quantify proteins using high throughput tandem mass tag (TMT) mass spectrometry. The FN mutants were grown in the field. These FN mutants have altered genome compositions that yield soybean seeds with high total protein content (58%) when compared to the parental (42%) and other U.S. soybean cultivars (40%). The high total protein trait is important to soybean-product industries. Additional investigation is needed to understand the relationship between protein content and its metabolic control. Therefore, we extracted proteins from FN mutant seeds and their parents and analyzed them by TMT based mass spectrometry. We quantified a total of 3,502 proteins. Several proteins were differentially expressed. A total of 206 proteins exhibited increased abundance and 214 proteins showed decreased abundance in the mutants. Among the abundant proteins, one of the major storage proteins, basic 7S globulin, increased four-fold, followed by a vacuolar-sorting receptor, and protein transporters. The second milestone of the third objective was to conduct pathway analysis using differentially expressed proteins observed in the high protein FN mutant line. The list of differentially expressed protein IDs was submitted to the singular enrichment analysis of Gene Ontology (GO) against the background of Glycine max using a web-based tool, AgriGO v2.0. We found GO enrichment of the abundant proteins mostly in ribosome and endoplasmic reticulum (ER). We found a higher enrichment of protein syntheses pathways starting from translation initiation factors to the storage vacuole. Based on our results, we anticipate that the deletion of a sequence-specific DNA binding transcription factor, three kinase genes, and another 21 genes in the FN mutant might have created a cascaded effect on protein synthesis, resulting in an overall increase in seed protein content. The third milestone of the third objective was to conduct bioinformatic analysis of these protein data. A Thermo Scientific Pinpoint Software tool developed by Thermo Center at Harvard Medical School was used for quantification of the MS data. A t-test was performed to identify differentially expressed proteins identified with more than 1 spectrum and the Benjamini and Hochberg correction was applied to limit false discovery to = 0.05. These data were subjected to several bioinformatic tools/programming to understand the trends and to interpret the biological relevance of the data in relation to protein synthesis. This included KEGG, AgriGO, AmiGO, Biomart and R programming. These results helped produce results from the second milestone.
1. Induced resistance to common bean rust. Common bean rust, caused by a fungus, reduces harvests of the dry, edible common bean and reduces food security for the people who rely on beans for a primary source of nutrition; however, natural resistance genes in the plant can provide protection until a strain of the fungus breaks resistance. In this study, ARS scientists in Beltsville, Maryland, demonstrated that a chemical called benzothiadiazole (BTH) can be sprayed on susceptible beans to induce resistance to common bean rust. Plants sprayed with BTH showed no signs of disease 10 days after inoculation with rust spores while plants without BTH sustained heavy infections and died. To better understand the effect BTH has on the bean leaf, the scientists measured proteins using mass spectrometry, an analytical technique. Proteins that increased in accumulation included receptor-like kinases that perceive pathogens, and enzymes that produce lignin to make leaf cell walls stronger and phytoalexins that are toxic to fungi. These results are important to farmers who are interested in using BTH to protect beans from rust and to breeders interested in the genes and proteins amplified by BTH that result in protection.
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