Location: Soybean Genomics & Improvement Laboratory
2022 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 thought the effectors may physically interact with bean plant proteins to disrupt the plant immune system. To test this, we fused the DNA of 13 bean rust effector proteins to the green fluorescent protein (GFP) gene and inserted the fusion gene into the soybean mosaic virus expression vector. We then used the virus to deliver the fused protein to bean leaves and extracted the fused protein from the leaves using an antibody to GFP. We reported last year that we were unsuccessful in finding protein-protein interactions between the virus expressed rust effectors and bean plant proteins. There were no specific milestones to report for this year for this hypothesis.
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. We reported the conclusions of this research last year. There were no specific milestones to report for this year. Notwithstanding, we performed additional research that extended from the hypothesis. In prior studies, we demonstrated that benzothiadiazole applied to bean leaves makes the leaves resistant to bean rust infection. Benzothiadiazole functions by activating part of the plant immune system controlled by salicylic acid, a plant hormone. We previously reported that benzothiadiazole treated bean leaves produce many proteins known to be regulated by salicylic acid. Meanwhile in a separate study that we reported last year, we found that beans naturally resistant to the bacterium that causes halo blight disease have increased amounts of a similar set of salicylic acid regulated proteins. This year, we used mass spectrometry to find salicylic acid molecules in bean leaves after inoculation with halo blight bacteria to confirm the salicylic acid hormonal resistance response. We also monitored other hormones including jasmonate, methyljasmonate, indole-3-acetic acid, abscisic acid, cytokinin, gibberellic acid, and 1-aminocyclopropane-1-carboxylic acid. Salicylic acid, but no other examined hormone, consistently increased at sites of infection to greater levels in resistant beans compared to susceptible beans at 4 days after inoculation. We then monitored 10 candidate bean phytoalexins. Phytoalexins are antibiotic molecules produced during disease resistance that are often regulated by salicylic acid. We found that phytoalexins daidzein, genistein, kievitone, phaseollin, phaseollidin, coumestrol, and resveratrol increased alongside salicylic acid in resistant beans but not in susceptible beans. In vitro culture assays revealed that salicylic acid, daidzein, genistein, coumestrol, and resveratrol functioned as antibiotics by inhibiting halo blight bacterial culture growth. These results demonstrate that these phytoalexins may be regulated by salicylic acid and work in conjunction with salicylic acid to restrict bacterial replication in a disease resistance response. To further address this, we used mass spectrometry to monitor the output of disease resistance on the halo blight bacterial proteome after plant infection. We found that resistant beans inhibited the accumulation of bacterial proteins required for virulence, secretion, motility, chemotaxis, quorum sensing, and alginate production. Sets of genes encoding these proteins appeared to function in operons, which implies that resistance altered the coregulated genes in the bacterium. Resistance also reduced amounts of bacterial enzymes needed to detoxify adverse molecules that spontaneously arise during glycolysis. Suspecting that a chemical produced by the bean plant during resistance may adversely affect the bacteria, we treated bacteria with salicylic acid in vitro. The salicylic acid reduced bacterial growth, decreased gene expression of the detoxification enzyme, and increased the amounts of toxic aldehydes that reduced bacterial reproduction. These findings support the hypothesis that plant resistance involves the chemical induction of adverse changes to the bacterial proteome to reduce pathogenicity and replication. These findings also demonstrate that salicylic acid is both a plant hormone and an antibiotic molecule.
The third hypothesis of Objective 1 involves modulating plant proteins identified by mass spectrometry with the goals of deciphering the plant immune system and improving upon disease resistance. This year’s milestones were linked to the protein-protein interaction studies that were part of objective 1. Because we did not identify plant proteins that interacted with the effectors, we followed the back-up plan to perform a proteomics experiment on beans resistant and susceptible to bean rust. For this study, Black Valentine beans were inoculated separately with a bean rust strain that elicits resistance and another strain that causes disease. The leaves were collected at 24 hours after inoculation, a time when the bean rust fungus inserts its infectious haustoria into bean cells and when bean cells initially respond, and at 72 hours after inoculation, a time when the leaf exhibits visible signs of resistance or disease (in the case of susceptibility). We purified protein from the leaves and submitted the samples for quantitative mass spectrometry analysis. More than 4,000 bean proteins were quantified. These data are still being statistically analyzed, but preliminary results reveal that resistant beans produce more glucosylases. We are interested in using our gene silencing system to prove whether any of these proteins are indeed important for resistance to the bean rust fungus.
The goals of Objective 2 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 Objective 3 was to collect seeds, extract proteins, and perform TMT quantitative proteomic analysis of selected nutritional traits in soybean mutants during seed development (2,3,4,5, and 6 WAF). Wild and mutant soybean seeds were grown at the University of Missouri field site. Seeds were collected at three developmental stages and three biological replicates were collected. Proteins were extracted and digested with Lys-C protease and trypsin. We analyzed the resulting peptides using high-throughput TMT mass spectrometry protein profiling. In the early, mid, and late stages of seed development, we quantified approximately 4,500 proteins in wild and mutant soybeans.
The second milestone of Objective 3 was data processing and pathway analysis of data collected from the first milestone. We analyzed the mass spectra using Pinpoint software from Thermo Scientific. R programming was used to visualize the data after a statistical analysis. The identified proteins were mapped to metabolic pathways related to seed development. During the early and mid-stages of seed filling in wild soybeans, we found greater abundances of several kinases. This implies that protein phosphorylation was activated. In early to late stages of seed development, there was greater abundance of proteins associated with the cell wall, oil, and vacuolar-related processes. The seed storage proteins 7S (beta conglycinin) and 11S (glycinin) increased steadily from the early to late stages of seed development. In contrast, 2S albumin storage proteins decreased during the same period of seed development. Likewise, several key enzymes related to amino acid and protein synthesis differentially accumulated during seed development. The soybean mutants with duplicated genes exhibited greater accumulation of iron-and sulfur-containing compounds along with 7S and 11S storage proteins during seed development compared to wild soybean.
Accomplishments
1. Resistant beans alter a bacterial proteome. Halo blight disease, caused by a bacterium, reduces harvests of the dry, edible common bean. A natural resistance gene in the bean stops the spread of the bacteria and kills them, but how this occurs is unknown. ARS scientists in Beltsville, Maryland, used mass spectrometry to measure bacterial proteins in bean leaves inoculated with a halo blight strain that triggers resistance and compared it to another halo blight strain that causes disease. We previously found that resistant plants produce the plant hormone salicylic acid at the site of infection. Following up on this finding, we monitored seven other hormones, but no other examined hormone, consistently increased at sites of infection to greater levels in resistant beans compared to susceptible beans. Bacteria from resistant beans exposed to salicylic acid exhibited reductions in bacterial proteins required for virulence, secretion, motility, chemotaxis, quorum sensing, alginate production, and methylglyoxal detoxification. These proteins control pathogenicity, motility, and metabolic homeostasis. Hence, the results show that the resistant plant reduces bacterial proteins needed for infection and spread and that the plant alters the metabolism of the bacterium by increasing toxic molecules inside the bacterium. These results will be of interest to scientists in the government, at universities, and at private institutions who want to understand the mechanisms of disease resistance in beans and other plants.
Review Publications
Islam, N., Krishnan, H.B., Natarajan, S.S. 2021. Quantitative proteomic analyses reveal the dynamics of protein and amino acid accumulation during soybean seed development. Proteomics. Article e2100143. https://doi.org/10.1002/pmic.202100143.
Cooper, B., Beard, H.S., Yang, R., Garrett, W.M., Campbell, K. 2021. Bacterial self-toxicity induced by a plant immune system. Journal of Proteome Research. 20:3664-3677. https://doi.org/10.1021/acs.jproteome.1c00232.
Islam, N., Krishnan, H.B., Natarajan, S.S. 2022. Protein profiling of fast neutron soybean mutant seeds reveal differential accumulation of seed and iron storage proteins. Phytochemistry. 200. Article 113214. https://doi.org/10.1016/j.phytochem.2022.113214.
Cooper, B. 2022. The detriment of salicylic acid to the Pseudomonas savastanoi pv. phaseolicola proteome. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-05-22-0104-R.
Cooper, B., Campbell, K., Garrett, W.M. 2022. Salicylic acid and phytoalexin induction by a bacterium that causes halo blight in beans. Phytopathology. https://doi.org/10.1094/PHYTO-12-21-0496-R.
