Elucidation of the functional role of oligopeptide transporters in bacterial virulence

Abstract only availableThe oligopeptide transporter (OPT) family is a relatively poorly characterized family of peptide/modified peptide transporters found in archebacteria, bacteria, fungi and plants. Plant and yeast OPTs were shown to transport tetra- and pentapeptides as well as the modified peptide glutathione. Our database analysis of sequenced bacterial genomes indicated that OPT proteins are encoded in the genomes of important human pathogens such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, Neisseria meningitidis, and Haemophilus influenzae. However, functional analysis of this family of peptide transporters, especially their possible function in bacterial pathogenesis, is lacking. We obtained three P. aeruginosa strains harboring transposon insertions in the PA3934 locus, the gene predicted to encode the putative orthologous OPT protein (OptA) in P. aeruginosa PA01. Two of the optA mutant strains have in-frame fusion between PaOptA and the PhoA protein encoded within the transposon. Expression of OptA-PhoA is induced by the addition of 20 mM arginine, whereas the expression of OptA-PhoA is not affected by iron availability. The lack of iron-regulated expression of optA would indicate that it is unlikely involved in iron nutrition in P. aeruginosa. We also found that 20 mM arginine and 0.4% peptone enhanced biofilm formation by wild type PA01 strain. However, enhanced biofilm formation by arginine was not observed in the optA mutant strains. Addition of 20 mM lysine had no effect on biofilm formation. We also determined the possible function of OptA in the ability of P. aeruginosa to produce pyocyanin. We found that the optA mutant strains produced higher amounts of pyocyanin than the wild type strain. The presence or absence of arginine in the growth medium had no effect on pyocyanin production. Taken together, these results indicate that OptA is important for biofilm formation by P. aeruginosa in response to arginine and peptides, but is unlikely involved in pyocyanin production.NSF grant to G. Stace

Analysis of Arabidopsis thaliana mutants defective in the oligopeptide transporter OPT3

Abstract only availableThe transport of peptides across membranes is a phenomenon found in both prokaryotes and eukaryotes as a method of obtaining amino acids, nitrogen, and carbon. Peptides can be transported by ATP-dependent transporters, as well as proton-coupled transporters. Among the latter are members of the oligopeptide transport (OPT) family, which transport tetra- and pentapeptides. Sequence comparisons led to the identification of nine OPT genes in Arabidopsis and our laboratory is investigating the role of these transporters in plant growth and development. Previous studies showed that mutations in the OPT3 gene resulted in embryo lethality. More recently, OPT3 expression was shown to increase under conditions of iron limitation, suggesting a possible role for OPT3 in transporting iron-chelates. The lethal nature of OPT3 T-DNA insertion mutation makes them difficult to study in a homozygous condition. Therefore, we sought non-lethal mutations within the OPT3 gene sequence, which can be maintained as homozygous plants. To create such mutations, we used the process of Targeted Induced Local Lesions IN Genomes (TILLING) to identify non-lethal, point mutations in the OPT3 gene. Eight mutant alleles, opt3-1 to opt3-8, were identified by TILLING. These mutants were sequenced and aligned with the other members of the OPT family to determine whether the mutations occurred within conserved regions of the protein. The mutations opt3-5 (P628S) and opt3-8 (P547L) were the first homozygous mutants identified which occurred within a highly conserved region and, therefore, were the likely candidates to disturb OPT3 function. These mutations were followed in segregating populations by CAPS (Cleaved Amplified Polymorphic Sequence) markers. Homozygous mutant lines and wild-type controls were grown on medium containing limited, moderate, or excess iron. The iron effects on the plant were determined by assaying the chlorophyll content in whole plants. These assays revealed no measurable effect of the OPT3 mutations on chlorophyll content under the conditions tested. We are now examining other opt3 alleles for a role in iron transport and other possible phenotypes displayed during plant growth and development.MU Monsanto Undergraduate Research Fellowshi

The University of Missouri Center for Sustainable Energy

Track II: Transportation and BiofuelsIncludes audio file (17 min.)The University of Missouri Center for Sustainable Energy was formed as a collaboration between the College of Agriculture, Food and Natural Resources and the College of Engineering. Over 70 faculty members, from a wide variety of disciplines, are associated with the center. The Center focuses on five primary areas: energy policy and management, research, education and training, service, and commercialization. The Center utilizes a number of excellent programs already established on the MU campus (e.g., the Food and Agricultural Policy Institute) to support activities in the area of energy policy and management. We are very excited about our education and training programs where we are partnering with Missouri two-year institutions to develop curriculum to support the growth of jobs in energy industries. In the area of transportation fuels, the Center has a variety of on-going projects. For example, work is underway to utilize novel, nanotechnology composites to enhance methane and hydrogen storage. A number of research projects are also focusing on the utilization of biomass for fuel production. Specific projects are focused on enhancing biomass production but also on understanding and reducing any harmful environmental impacts that might arise. The MU Center for Sustainable Energy serves as a single, central portal for access to the full resources of the University of Missouri the goal of addressing the energy challenges that face our state and nation

Distinct, crucial roles of flavonoids during legume nodulation

http://www.soyroothair.org/publications.phpRNA interference-mediated silencing of the key flavonoid and isoflavone biosynthesis enzyme, respectively, by two different research groups has provided direct genetic evidence for the essential roles that these compounds play in nodulation. Anton Wasson et al. have shown that flavonoids are essential for localized auxin transport inhibition during nodulation in the indeterminate legume Medicago truncatula. By contrast, Senthil Subramanian et al. have shown that isoflavones are essential for endogenous nod gene induction in the determinate legume soybean.Research in the Yu laboratory was supported by grants from the National Science Foundation (MCB0519634), USDA (NRI2005-05190), and MSMC (Grant 02-242). The Stacey laboratory was supported by a grant (DBI-0421620) from the National Science Foundation, Plant Genome program

Transcription factors leading the pathway to survival

Abstract only availableAsian Soybean Rust, a foliar disease, is caused by the fungal pathogen Phakospora pachyrhizi, which threatens soybean (Glycine max) production in many countries. In the absence of fungicide treatment, yield losses from ASR can be up to 80%. The use of fungicides significantly drives up production costs for farmers. Four resistant genes, Rpp1-4, have been identified for ASR but none of these provide sustained, field resistance due to adaptation by the pathogen. Soybean cultivar Williams 82 is susceptible to ASR, while cultivar DT2000 exhibits significant levels of tolerance to the pathogen. We utilized these two cultivars to examine the differential response in the expression of various transcription factor genes to ASR inoculation. Our goal is to identify transcription factors that contribute to soybean resistance to ASR and to identify the corresponding genes and pathways responsible for resistance. Due to the -relatively low abundance of TF gene mRNA, we utilized the qRT-PCR technique to accurately assay gene expression. We also examined the progress of ASR infection by staining infected leaves at different time points after inoculation. In this way, we hope to correlate the expression of specific genes with the stage of infection. After some trial and error, we were able to easily visualize ASR infection in soybean leaves by staining with Calcofluor White. This staining method allowed us to track ASR infection and document the various stages of fungal development. Our initial screens for TF gene expression identified a few TF genes that are clearly differentially expressed between the susceptible and resistance soybean cultivars. We hope in further experiments to understand the function of these TF genes in soybean resistance to ASR and, ultimately, contribute to the development of soybean cultivars that will benefit soybean farmers.NSF grant to G. Stace

Genetics and functional genomics of legume nodulation

http://www.soyroothair.org/publications.phpGram-negative soil bacteria (rhizobia) within the Rhizobiaceae phylogenetic family (a-proteobacteria) have the unique ability to infect and establish a nitrogen-fixing symbiosis on the roots of leguminous plants. This symbiosis is of agronomic importance, reducing the need for nitrogen fertilizer for agriculturally important plants (e.g. soybean and alfalfa). The establishment of the symbiosis involves a complex interplay between host and symbiont, resulting in the formation of a novel organ, the nodule, which the bacteria colonize as intracellular symbionts. This review focuses on the most recent discoveries relating to how this symbiosis is established. Two general developments have contributed to the recent explosion of research progress in this area: first, the adoption of two genetic model legumes, Medicago truncatula and Lotus japonicus, and second, the application of modern methods in functional genomics (e.g. transcriptomic, proteomic and metabolomic analyses).Ongoing related research in the Stacey laboratory is funded by grants from the US Department of Energy (DE-FG02-02ER15309), the National Science Foundation (NSF) (DBI-0421620), and the National Research Initiative (NRI) of the US Department of Agriculture (USDA) Cooperative State Research, Education and Extension Service (2005-35319-16192 and 2004-35604-14708)

Expression patterns of oligopeptide transporters in arabidopsis thaliana [abstract]

Abstract only availableFaculty Mentor: Dr. Gary Stacey, Plant Microbiology and Pathology & BiochemistryPeptide transport is a universally observed cellular mechanism employed by both prokaryotes and eukaryotes to transport peptides (2-6 residues) across cellular membranes in an energy-dependent manner. These peptides, upon internalization, are rapidly hydrolyzed into amino acids, which can be used for protein synthesis or as alternative sources of carbon and nitrogen. The OPT (Oligopeptide Transporter) family is involved in transport of tetra- and penta-peptides and was first identified in yeast. A distinct subfamily of nine putative Arabidopsis thaliana OPT orthologs (AtOPT1-AtOPT9) were identified by comparison to fungal OPTs. The function of OPT proteins in plant growth and development is largely unknown. One approach to help elucidate their physiological function is to determine the tissue-specific localization of each AtOPT, as well as factors that affect their expression. To accomplish this, promoter-GUS fusions of seven of the AtOPTs were utilized. The transgenic plants were stained for GUS to characterize gene expression at various stages of development, as well as under different growth conditions. Each of the AtOPT fusions exhibited similar tissue-specific expression. The AtOPTs were preferentially expressed in the vascular tissues of vegetative organs but were not expressed in root hairs and root tips. However, differential expression of the AtOPTs was observed under limiting iron conditions.Plant Genomics Internships @ M

Constructing proteome and metabolome maps for genetic improvement of energy-related traits in soybean [abstract]

Only abstract of poster available.Track V: BiomassAlthough the genetic blueprint of soybean is represented by the genome, its phenotype is a product of that blueprint manifested as the production of proteins and metabolites influencing growth characteristics, stress responses, seed composition, and yield. We are using various tools of genomics and molecular breeding with an aim towards development of value-added soybeans that will help United States farmers to maintain their competitiveness and expand utilization of soybean crops (e.g. functional foods, industrial uses, biodiesel, etc). Profiling soybean gene products will lay the foundation for a systems biology approach to key processes such as seed development, which will lead to the genetic improvement of yield and seed composition. Being one of the major bio-energy crops, building a comprehensive map of proteins and metabolites for soybean will help make connections between regulatory or metabolic pathways not previously characterized. Another major benefit from these studies is the discovery of energy related traits including plant productivity and seed compositional traits for the genetic improvement of soybean. It is well known that environmental cues influence developmental phenotypes in plants. Different biotic stresses such as fungal diseases and abiotic stresses, such as drought and flooding, also elicit phenotypic responses from the genome. Thus, by studying the gene products, a direct correlation between response and specific peptides/metabolites can be made. This will lead to crop improvement either through breeding or transgenic efforts. Major objectives of this study are: a) to identify key soybean seed, leaf, and root proteins involved in development and biotic and abiotic stress responses; b) to establish a comprehensive set of chemical standards for soybean metabolites moving toward construction of a metabolome map with a focus on seed and drought effects on seed development and, c) to compile a database linking proteomic and metabolite information and associate this information to value-added soybean traits and markers for assisted breeding. We are utilizing GC/MS, LC/MS, and NMR approaches to identify key molecules for further characterization

The affects of genistein on the Bradyrhizobium japonicum bacterial transcriptome [abstract]

Abstract only availableFaculty Mentor: Gary Stacey, Plant Microbiology and PathologyBradyrhizobium japonicum forms a symbiotic relationship with the roots of the soybean plant (Glycine max). This bacterium is of great importance because of its ability to provide the soybean with a source of nitrogen by the conversion of atmospheric dinitrogen to ammonia. In order to establish this symbiosis, the bacteria must attach to the root hair surface and initiate development of a root nodule. The bacterium responds to plant flavonoids through production of nod factor, a product of the nod genes, which facilitates bacterial root entry and initiates nodule organogenesis. Therefore, we have investigated the expression of nod genes after treatment with a plant derived flavonoid inducer, genistein. Free-living cultures of Bradyrhizobium japonicum were treated and cells were harvested six hours after treatment and total RNA was extracted. Semi-quantitative and quantitative RT-PCR was performed to confirm induction of the nodY and nodC genes. The RT-PCR results confirmed that nodY and nodC are upregulated in the presence of genistein compared to an ethanol-treated control. To further define genistein regulation on the nod genes, a DNA microarray experiment was performed on the above extracted RNA to define the bacterial transcriptional response to genistein, the results of which will be presented.Bradyrhizobium japonicum forms a symbiotic relationship with the roots of the soybean plant (Glycine max).  This bacterium is of great importance because of its ability to provide the soybean with a source of nitrogen by the conversion of atmospheric dinitrogen to ammonia.   In order to establish this symbiosis, the bacteria must attach to the root hair surface and initiate development of a root nodule.  The bacterium responds to plant flavonoids through production of nod factor, a product of the nod genes, which facilitates bacterial root entry and initiates nodule organogenesis. Therefore, we have investigated the expression of nod genes after treatment with a plant derived flavonoid inducer, genistein. Free-living cultures of Bradyrhizobium japonicum were treated and cells were harvested six hours after treatment and total RNA was extracted.  Semi-quantitative and quantitative RT-PCR was performed to confirm induction of the nodY and nodC genes. The RT-PCR results confirmed that nodY and nodC are upregulated in the presence of genistein compared to an ethanol-treated control.  To further define genistein regulation on the nod genes, a DNA microarray experiment was performed on the above extracted RNA to define the bacterial transcriptional response to genistein, the results of which will be presented

Development and assessment of scoring functions for protein identification using PMF data

PMF is one of the major methods for protein identification using the MS technology. It is faster and cheaper than MS/MS. Although PMF does not differentiate trypsin-digested peptides of identical mass, which makes it less informative than MS/MS, current computational methods for PMF have the potential to improve its detection accuracy by better use of the information content in PMF spectra. We developed a number of new probability-based scoring functions for PMF protein identification based on the MOWSE algorithm. We considered a detailed distribution of matching masses in a protein database and peak intensity, as well as the likelihood of peptide matches to be close to each other in a protein sequence. Our computational methods are assessed and compared with other methods using PMF data of 52 gel spots of known protein standards. The comparison shows that our new scoring schemes have higher or comparable accuracies for protein identification in comparison to the existing methods. Our software is freely available upon request. The scoring functions can be easily incorporated into other proteomics software packages