Progress 09/01/04 to 08/31/07
Outputs
OUTPUTS: 1) T. Bradley: Novel osmotic stress genes were isolated from salmon transferred from freshwater to seawater or during the course of parr-smolt transformation. OSP1.8c is a novel gene that has structural characteristics similar to protein kinase c and might be involved in signal transduction during osmotic stress. Efforts were begun using multiple approaches to clone the transcript coding for a betaine transporter. 2) P. Cohen: We investigated the role of the conversion of succinate to fumarate in the virulence of Salmonella enterica serovar Typhimurium in BALB/c mice. We have shown that a serovar Typhimurium ΔfrdABCD ΔsdhCDA double mutant is totally avirulent and immunogenic in BALB/c mice, i.e. infecting BALB/c mice with the Typhimurium ΔfrdABCD ΔsdhCDA double mutant, protects against subsequent infection with wildtype Typhimurium. 3) M. Gomez-Chiarri: Matrix metalloproteinases are involved in immunity and inflammation in vertebrates. We have isolated genes for two MMPs in oysters and developed assays to detect MMPs in infected and non-infected oysters. We have also developed a method for quantification of the protozoan parasite Perkinsus marinus parasites in different tissues of the oyster using stereological techniques. Our research shows that MMPs are produced by oyster hemocytes and epithelial cells in mucosal surfaces. 4) A. Kausch: We have demonstrated the utility of the FLP/FRT site specific recombination system for generating male sterility in transgenic rice plants and then recovery of fertility by crossing to constitutively expressing FLP expressing plants. This system establishes a basis for hybrid rice production and establishes a proof of concept for the utility of this system for gene confinement strategies in GM plants. We have also begun to develop this system in maize and switchgrass. 5) T. Mather: We investigated the role of vesicle and plasma membrane-bound protein receptors (SNAREs) in regulating protein and pathogen secretion in tick salivary gland cells. Using inhibitory RNA (RNAi) we reduced transcript levels of target genes and demonstrated the effect of specific SNARE proteins on tick blood-feeding success. We have begun experiments to target genes in the secretion pathway of ticks for silencing, in order to identify potential targets for tick-borne pathogen blocking vaccines and therapeutics. 6) D. Nelson: Flavobacterium columnare causes columnaris disease, a highly destructive bacterial infection of fish. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth. Growth, biofilm formation, extracellular protease production, and changes in protein expression of F. columnare cultured in media supplemented with fish skin mucus were compared to the same media without mucus. F. columnare was able to grow using mucus as sole nutrient source. Also, mucus induced biofilm formation and the production of extracellular proteins. PARTICIPANTS: Postdocs: Dr.Subrata Chowdhury, Dr.Shahid Karim, Dr.Sudha Pichu, Dr. Merlin Tchawa-Yimga, Dr. Eric J. Gauger, Dr. Andrew M. Staroscik, Dr.Jacques Zarate. Principal investigators: Dr. T. Bradley, Dr. P. Cohen, Dr. A. Kausch, Dr. M. Gomez Chiarri, Dr. T. Mather, Dr. D. Nelson. TARGET AUDIENCES: The target audience includes a broad array of scientists, researchers, extension personnel, numerous agricultural interests, students,public health workers and consumers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) The findings of this research have contributed to the understanding of smoltification and osmotic adaptation of salmon to seawater. Ultimately, the genes identified might be used as markers of the optimum time for transfer of salmon to seawater. 2) We now know that serovar Typhimutium requires the conversion of succinate to fumarate for virulence in BALB/c mice. This knowledge has allowed the construction of a live oral vaccine strain that protects mice against virulent infection by wildtype virulent serovar Typhimurium. It would appear that identifying key metabolic reactions that pathogens require for pathogenesis has merit for preventing infectious diseases. 3) Control of protozoan and bacterial diseases threatening the eastern oyster Crassostrea virginica, requires a good understanding of the defense mechanisms of the oyster and sensitive methods to study host-pathogen interactions. Characterization of the role of MMPs in immune responses and pathology in oysters will provide tools for management of infectious diseases in oyster aquaculture. 4) The functional utility of FLP/FRT in rice establishes this system for the commercial production of hybrid rice using this system and lays the groundwork for practical applications on gene confinement. 5) Ticks are notorious as vectors of human and animal pathogens and cause annual economic losses in the hundreds of millions of dollars to cattle throughout the world. Ticks also transmit several human pathogens, including the agents that cause Lyme disease, babesiosis and anaplasmosis. Identifying viable biological targets and dissecting signaling pathways will enhance development of vaccination or pharmaceutical therapies for tick control and protection of humans and animals. 6) These results demonstrate that salmon surface mucus promotes growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen.
Publications
- Bjornsson, B.T. and Bradley, T.M. 2007. Past successes, present misconceptions and future milestones in salmon smoltification research. Aquaculture 273: 384-391.
- Hu, Q., Kononowicz-Hodges,H., Nelson-Vasilchik, K., Viola, D., Zeng, P., Liu, H., Kausch, A.P., Chandlee, J.M., Hodges, T.K. and Luo, H. 2007. FLP-mediated site-specific recombination in rice. Plant Biotechnology Journal 6:2:176-188
- Karim, S., Kenny, B., Troiano, E. and Mather, T.N. 2008. RNAi-mediated gene silencing in tick synganglia: a proof of concept study. BMC Biotech. 8:30.
- Remacha-Trivino A, Borsay-Horowitz D., Dungan, C., Gual-Arnau, X., Gomez-Leon, J., Villamil, L. and Gomez-Chiarri, M. 2008. Quantification of Perkinsus marinus in the american oyster Crassostrea virginica (Gmelin, 1791) by modern stereology. J. Parasitol. 94(1):125-136.
- Mercado-Lubo, R., Gauger, E.J., Leatham, M.P., Conway, T., Cohen, P.S. A Salmonella enterica serovar Typhimurium succinate dehydrogenase/fumarate reductase double mutant is avirulent and immunogenic in BALB/c mice. General Meeting of the American Society for Microbiology, Boston, MA, 2008.
- Mercado-Lubo, R., Gauger, E.J., Leatham, M.P., Conway, T., and Cohen, P.S. A Salmonella enterica serovar Typhimurium succinate dehydrogenase/fumarate reductase double mutant is avirulent and immunogenic in BALB/c mice. Infect. Immun. 76:1128-1134, 2008.
- Remacha-Trivino A, Borsay-Horowitz D, Dungan C, Gual-Arnau X, Gomez-Leon J, Gomez-Chiarri M. Quantification of Perkinsus marinus in the eastern oyster Crassostrea virginica using modern stereological techniques. National Shellfisheries Association meeting, Monterey, CA March 2006. J. Shellfish Res 25:765
- Proestou D.A., Munson A. and Gomez-Chiarri. M. 2007. Isolation and characterization of matrix metalloproteinases in oyster genomes. Aquaculture 2007, San Antonio, Texas.
- Vaughn, C.F., Remacha-Trivino, A., Gomez-Leon, J., Schmeidel, C., Guy, A., Gomez-Chiarri M. Expression and activation of matrix metalloproteinases in the eastern oyster, Crassostrea virginica, in response to experimental infections with Perkinsus marinus and Roseovarius crassostreae. Aquaculture 2007, San Antonio, Texas
- Staroscik, A.M. and Nelson, D.R. 2007. The influence of salmon surface mucus on the growth of Flavobacterium columnare. J. Fish Disease 31:59-69.
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Progress 01/01/06 to 12/31/06
Outputs
OUTPUTS: This reports covers progress made during the period of 1/1/06 - 12/31/06. 1) T. Bradley: Characterization of OSP 1.8c continued during this period. In addition to osmotic stress, the gene was found to be upregulated by heat stress and H2O2, similar to heat shock proteins. Transcription of the gene was upregulated in the kidney and in smolts in freshwater prior to exposure to seawater. Efforts were initiated to clone the transporter for betaine, an osmolyte that could play a role in dealing with osmotic stress. 2) Previously, we showed that a Salmonella enterica serovar Typhimurium succinate dehydrogenase mutant (ΔsdhCDA) which can't convert succinate to fumarate was slightly attenuated in BALB/c mice, but that fumarate reductase can substitute for succinate dehydrogenase, i.e. a serovar Typhimurium ΔfrdABCD ΔsdhCDA double mutant was totally avirulent in BALB/c mice. We observed that not only is the serovar Typhimurium ΔfrdABCD ΔsdhCDA double mutant avirulent, it is immunogenic. 3) M. Gomez-Chiarri: Matrix metalloproteinases are involved in immunity and inflammation in vertebrates. To determine the role of oyster MMPs in host-pathogen interactions in oysters, we have developed methods for the immunohistochemical detection of MMPs in oyster tissues. We have also developed a stereological method for quantification of the protozoan parasite Perkinsus marinus in different tissues of the oyster. 4) A. Kausch: We have verified the use of site specific recombination in conjunction with male sterility in transgenic grasses as a viable strategy for gene confinement. We now aim to field test these results. We have introduced molecular constructs that induce late stage pollen abortion connected to herbicide tolerance conferred by express ion of the bar gene. This system may also offer a convenient and advanced breeding strategy for recovery of rare hybrids in wide crosses. The transgene could then be removed by crossing it out and selecting for male fertile non-herbicide resistant progeny. 5) T. Mather: Nymphal blacklegged ticks have become our favored model system for assessing anti-tick vaccine and drug therapy candidates and for experimental disease transmission, but their small size has made introducing dsRNA to silence individual gene expression challenging. Previously, it has been necessary to inject dsRNA directly into the tick hemocoele, limiting the use of this tool for studying tick gene function. We have developed a high throughput electroporation technique for introducing dsRNA into nymphal ticks for gene silencing. In vivo silencing of an I. scapularis PLA2 gene was accomplished using the electroporation method. 6) D. Nelson: Virulence factors in the fish pathogen Flavobacterium columnare were examined. Using a fluorescently labeled protein substrate (BODIPY-casein), extracellular protease activity was found to be present during all phases of growth. Exposure of F. columnare cells to salmon skin mucus caused extracellular protease activity to increase and induced cells to grow as a biofilm. Manipulation of carbohydrate and divalent cation concentrations induced biofilm formation. PARTICIPANTS: Postdocs: Dr.Subrata Chowdhury, Dr.Shahid Karim, Dr.Sudha Pichu, Dr. Merlin Tchawa-Yimga, Dr. Eric J. Gauger, Dr. Andrew M. Staroscik, Dr.Jacques Zarate. Principal investigators: Dr. T. Bradley, Dr. P. Cohen, Dr. A. Kausch, Dr. M. Gomez Chiarri, Dr. T. Mather, Dr. D. Nelson. TARGET AUDIENCES: The target audience includes: scientists, researchers, extension personnel, an array of agricultural interests, students,public health workers and consumers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) The current work continues to provide basic information on smoltification of salmon and on the genes involved in successful adaptation to osmotic stress. 2) A Salmonella enterica serovar Typhimurium ΔfrdABCD ΔsdhCDA double mutant is an effective live oral vaccine strain in BALB/c mice. 3) Control of protozoan and bacterial diseases threatening the eastern oyster Crassostrea virginica, requires a good understanding of the defense mechanisms of the oyster and sensitive methods to study host-pathogen interactions. Characterization of the role of MMPs in immune responses and pathology in oysters will provide tools for management of infectious diseases in oyster aquaculture. 4) Male (pollen) sterility may be a solution to the prevention of transgene escape issues for deregulation of GM perennial plants. 5) This study provides a positive proof of principle that a non-invasive technique for applying RNAi can be a useful tool for identifying critical tick blood feeding and TBD transmission pathways. Identifying viable biological targets and dissecting signaling pathways will enhance development of vaccination or pharmaceutical therapies for tick control and protection of humans and animals. 6) The exposure of F. columnare to fish skin mucus increases growth, biofilm formation, and extracellular protease production. These data suggest that colonization of the fish by F. columnare is initiated on the skin mucus and that this may induce the expression of virulence factors in that bacterium.
Publications
- Hu, Q., Nelson, K. and Luo, H. 2006. FLP-mediated site-specific recombination for genome modification in turfgrass. Biotechnology Letters 28:1793-1804.
- Remacha-Trivino, A., Borsay-Horowitz, D., Dungan, C., Gual-Arnau, X., Gomez-Leon, J. and Gomez-Chiarri, M. 2006. Quantification of Perkinsus marinus in the eastern oyster Crassostrea virginica using modern stereological techniques. National Shellfisheries Association meeting, Monterey, CA. J. Shellfish Res 25:765
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Progress 01/01/05 to 12/31/05
Outputs
OUTPUTS: This report covers the period from 1/1/05-12/31/05. The accomplishments of the projects for the six investigators are presented below. 1) T. Bradley: A novel cDNA was isolated from the gills of Atlantic salmon subjected to osmotic stress using a differential display of mRNA assay. The cDNA coded for a protein of 243 amino acids with a deduced molecular weight of 27.5 kDa and theoretical pI of 5.92. The gene was designated osmotic stress protein 1.8c and was markedly upregulated in lamellae in response to supplemental NaCl concentrations of 50 and 100 mM as well as in fish transferred from freshwater to seawater. 2) P. Cohen: Previously, we found that a complete TCA cycle is required for full Salmonella enterica serovar Typhimurium virulence in BALB/c mice; however a -sucCD mutant which cannot convert succinyl-CoA to succinate was found to be far more attenuated than a -sdhCDA mutant which can't convert succinate to fumarate. During this period, we found that in a serovar Typhimurium succinate dehydrogenase mutant (-sdhCDA), fumarate reductase, can substitute for succinate dehydrogenase, i.e. a -frdABCD -sdhCDA double mutant was found to be totally avirulent in BALB/c mice. 3) M. Gomez-Chiarri: Matrix metalloproteinases are involved in immunity and inflammation in vertebrates. We have sequenced the cDNAs coding for 2 matrix metalloproteinases (MMPs) in oysters. The predicted sizes of these oyster MMPs suggest that these genes code for the proteins responsible for proteolytic activity detected in oyster serum by zymography. 4) A. Kausch: Research has focused on the development of novel mechanisms for gene confinement of genetically engineered corn, rice and grasses. These capabilities have allowed us to develop research on gene confinement in monocots. To accomplish this goal we developed male sterile grasses and rice plants to test the efficacy of controlling gene escape by eliminating pollen flow. 5) T. Mather: Previously, we demonstrated the role of vesicle and plasma membrane-bound protein receptors (SNAREs) in regulating protein secretion in tick salivary gland cells. To begin assessing the potential role of SNARES in secretion of tick-borne pathogens, we used nested PCR to demonstrate the presence of detectable levels of Anaplasma phagocytophilum in questing males, nymphs, unfed and partially fed female adult midguts and salivary glands. Additionally, dissected salivary glands were stimulated with PGE2, pilocarpine, or dissecting buffer, then centrifuged at 1000xg for 1 min, and the supernatant used for preparing A. phagocytophilum DNA. PGE2 successfully induced secretion of A. phagocytophilum secretion from infected tick salivary glands in vitro. 6) D. Nelson: The growth of the fish pathogen Flavobacterium columnare on Atlantic salmon skin mucus was characterized as it is hypothesized that these bacteria invade the fish host through the skin. Additionally, it has been shown that fish skin mucus contains antibacterial peptides. We found that F. columnare grows extremely well on skin mucus exhibiting more rapid growth with more abundant biofilm formation than on other media. PARTICIPANTS: As well as the PIs working on the specific projects the funding allowed for the hire of one postdoctoral fellow for each of the projects. Additionally, graduate students were recruited to work on specific projects. Both postdocs and graduate students received significant training opportunities as part of the projects. TARGET AUDIENCES: The results of this research have been presented at an array of regional, national and international meetings to scientists, researchers, extension personnel, agricultural interests and students. Additionally, publications in popular and peer-reviewed journals have allowed dissemination to a variety of interested parties. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) Information on genes involved in osmoregulation have the potential to enhance the success of transfer of salmon to seawater in commercial aquaculture increasing economic gain to the producer. 2) The nutrients and metabolic pathways that serovar Typhimutium requires for pathogenesis are unknown. Such knowledge should allow innovative approaches to preventing pathogenesis. 3) Control of protozoan and bacterial diseases threatening the eastern oyster Crassostrea virginica, requires a good understanding of the defense mechanisms of the oyster. Characterization of the role of MMPs in immune responses and pathology in oysters will provide tools for management of infectious diseases in oyster aquaculture. 4) Gene confinement is a major regulatory conundrum facing the genetic improvement of perennial plants, including those currently considered as dedicated bioenergy crops, such as corn stover and switchgrass. A solution to the gene confinement issue would broadly affect agricultural introduction of traits into perennial crop species. 5) Since PGE2 induced A. phagocytophilum secretion from infected tick salivary glands in vitro, it seems likely that A. phagocytophilum secretion may involve the tick salivary gland exocytosis machinery. 6) We have now demonstrated that this pathogenic bacterium is prefers to grow on fish skin mucus suggesting that it is adapted to colonizing the fish host skin surface as a prelude to infection.
Publications
- Luo,H., Hu, Q., Nelson, K., Longo, C., Chandlee, J.M. and Kausch, A.P. 2005. Agrobacterium tumefaciens-mediated turfgrass transformation. In: Methods in Molecular Biology-Agrobacterium Protocols (2nd edition), Wang K (ed.) The Humana Press Inc., Totowa, NJ.
- Hu, Q., Nelson, K. and Luo, H. 2005 FLP-mediated site-specific DNA recombination in turfgrass. Biotechnology Letters 28:22:1793-1804.
- Hu, Q., Nelson, K., Viola, D., Lickwar, C., Chandlee, J.M., Hodges, T. K. and Luo, H. 2005 FLP-mediated site-specific DNA recombination for use in hybrid rice production. The Plant Journal 23:3:423-430
- Hu, Q, Nelson, K., Zheng, P., Lickwar, C., Kausch, A.P., Chandlee, J.M. and Luo, H. 2005. Stable expression of yeast FLP site-specific recombinase in rice. Plant Biology 2004, Annual Meetings of the American Society of Plant Biologists, Florida, USA.
- Luo,H., Hu, Q., Nelson, K., Longo, C. and Kausch, A.P. 2005 Strategies for prevention of transgene escape in genetically modified perennials. The 55th annual meeting of the American Institute of Biological Sciences, Washington D.C., USA.
- Luo, H., Kausch, A.P., Hu, Q., Nelson, K., Wipff, J.K., Fricker, C.C.R., Owen, T.P., Moreno, M.A., Lee, J-Y. and Hodges, T.K. 2005 Controlling transgene escape in GM creeping bentgrass. Molecular Breeding 16:185-188.
- Luo, H., Lee, J.Y., Hu, Q., Nelson, K., Kausch, A.P., Chandlee, J.M., Eitas, T.K., Lickwar, C. and Hodges, T.K. 2005. RTS, an anther-specific gene isolated from rice is required for male fertility and its promoter sequence directs tissue-specific transcription in dicotyledonous species. Plant Molecular Biology 62:397-408.
- Lee, J-Y., Hodges, T. K., Eitas, T.K., Lickwar, C., Hu, Q., Nelson, K., Kausch, A.P., Chandlee, J.M. and Luo, H. 2005. RTS, an anther-specific gene isolated from rice is required for male fertility and its promoter sequence directs tissue-specific transcription in dicotyledonous species. Plant Biology, Annual Meetings of the American Society of Plant Biologists, Florida.
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Progress 01/01/04 to 12/31/04
Outputs
Funds were awarded in September allowing retention of experienced postdoctoral fellows to continue research on six research projects. Funds from the award have been used for the purchase of a Vibratome Cryostat which is currently in use for two of the projects. Coupled with funds from the previous award, a Qiagen BioRobot 8000 was purchased for automated isolation of RNA and DNA using a 96 well plate format. The robot is being utilized by several groups at this time. Given the short time since receipt of the award, specific research findings will be provided in the next progress report.
Impacts
The retention of experienced postdoctoral fellows will generate a critical mass of researchers with expertise in molecular biology addressing problems germane to the mission of USDA. Purchase of critical instrumentation will further facilitate research in these areas at the University.
Publications
- No publications reported this period
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