Progress 01/01/05 to 12/31/05
Outputs
The University of Kentucky Advanced Genetic Technologies Center (UK-AGTC) increased its capacity by 50%. Currently the facility can accommodate over 1000 DNA-sequencing samples per day for each of two different chemistries. The upgraded facility was key to two successful genomics grants: one from the National Science Foundation, the other from the U.S.D.A. National Research Initiative. The laboratory information management system (LIMS) was further refined, providing web interface for all sample submission forms, and automated uploading of all data to password-protected client pages. The internship program provided research experiences for five undergraduate students, one postbaccalaureate student, and three high school students, who participated in DNA sequencing, library cloning, or computer programming.
Impacts
The deep-coverage libraries of genomic DNA and cDNA clones are essential reagents for genetic research on the plants, fungi, bacteria and bacteriophage from which they were derived. As such, they will leverage competitive grants and advance the accomplishments and reputation of Kentucky researchers in genomics research. The unique organisms investigated with these reagents are important in the health and profitability of agriculture in Kentucky. Knowlege gained about their genetic constitution and gene expression will facilitate improved management of agricultural plants and livestock, their beneficial symbionts, and their potential pests.
Publications
- Blankenship, J.D., Houseknecht, J.B., Pal, S., Bush, L.P., Grossman, R.B. and Schardl, C.L. 2005. Biosynthetic precursors of fungal pyrrolizidines, the loline alkaloids. Chembiochem 6:1016-1022.
- Du, M., Schardl, C.L., Nuckles, E.M. and Vaillancourt, L.J. 2005. Using mating-type gene sequences for improved phylogenetic resolution of Collectotrichum species complexes. Mycologia 97:641-658.
- Funnell, D.L., Lawrence, C.B., Pedersen, J.F. and Schardl, C.L. 2005. Expression of the tobacco beta-1,3-glucanase gene PR-2d, following induction of SAR with Peronospora tabacina. Physiol. Mol. Plant Pathol. 65:285-296.
- Gentile, A., Rossi, M.S., Cabral, D., Craven, K.D. and Schardl, C.L. 2005. Origin, divergence, and phylogeny of epichloe endophytes of native Argentine grasses. Mol. Phylogen. Evol. 35:196-208.
- Haarmann, T., Machado, C., Luebbe, Y., Correia, T., Schardl, C.L., Panaccione, D.G. and Tudzynski P. 2005. The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution. Phytochemistry 66:1312-1320.
- Prapassorn, D., Sedlock, A.B., Young, C.A., Johnson, R.D., Goetz1, K.E., Scott, B., Schardl, C.L. and Panaccione, D.G. 2005. Structural analysis of a peptide synthetase gene required for ergopeptine production in the endophytic fungus Neotyphodium lolii. DNA Seq. 16:379-385.
- Schardl, C.L. and Leuchtmann, A. 2005. The epichloe endophytes of grasses and the symbiotic continuum. pp. 475-503 In Dighton, J., White, J.F., Oudemans, P., eds., The Fungal Community: Its Organization and Role in the Ecosystem, Boca Raton, Florida: CRC Press.
- Spiering, M.J., Moon, C.D., Wilkinson, H.H. and Schardl, C.L. 2005. Gene clusters for insecticidal loline alkaloids in the grass-endophytic fungus Neotyphodium uncinatum. Genetics 169:1403-1414.
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Progress 01/01/04 to 12/31/04
Outputs
To facilitate high throughput genetic analyses of plants, plant symbionts, and animal pathogens, several clone libraries have been constructed representing genomic DNA and copy DNA (cDNA) derived from mRNA. A genomic library of 6 million small insert (4kb) and 200,000 large insert (40 kb) clones was constructed for the bioprotective plant symbiont, Epichloe festucae (a fungal endophyte). Seven cDNA libraries of approx. 2 million clones each were constructed from mRNA of grasses at various developmental stages, five of these representing symbiosis with E. festucae. The genome of the horse pathogen, Streptococcus zooepidemicus W60, and of Streptococcus equii bacteriophage P9, were cloned to high redundancy. These libraries are reagents sufficient for complete analysis of the genes and the mRNAs of the respective organisms. Thus, they are crucial for leveraging grants in genomics research. With upgrades to the sequencing facilities and refinement of techniques, the
success rate of sequencing from typical genomic clones exceeds 90%, and read lengths exceed 800 bases. Costs were reduced to $0.00125 per base, and bioinformatic support was expanded for the users of the facility. Three undergraduate research interns were employed and trained in use of robots and high-throughput sequencing in genomic research.
Impacts
The deep-coverage libraries of genomic DNA and cDNA clones are essential reagents for genetic research on the plants, fungi, bacteria and bacteriophage from which they were derived. As such, they will leverage competitive grants and advance the accomplishments and reputation of Kentucky researchers in genomics research. The unique organisms investigated with these reagents are important in the health and profitability of agriculture in Kentucky. Knowlege gained about their genetic constitution and gene expression will facilitate improved management of agricultural plants and livestock, their beneficial symbionts, and their potential pests.
Publications
- Afitlhile, M.M., Fukushige, H., and Hildebrand, D. 2004. Labeling of major plant lipids and jasmonic acid using [1-14C] lauric acid. Phytochemistry 65: 2679-2684.
- Bashir, N.S., Sanger, M., Jarlfors, U., and Ghabrial, S.A. 2004. Expression of Peanut stunt virus coat protein gene is essential and sufficient for production of host-dependent ribbon-like inclusions in infected plants. Phytopathology 94: 722-729.
- Boonrod, K., Galetzka, D., Nagy, P.D., Conrad, U. and Krczal, G. 2004. Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus resistance. Nature Biotechnolology 22: 856-862.
- Chandra-Shekara A. C., Navarre, D., Kachroo, A., Kang, H-G., Klessig, D.F., Kachroo, P. 2004. Signaling requirements and role of salicylic acid in HRT - and rrt-mediated resistance to turnip crinkle virus in Arabidopsis. Plant Journal 40: 647-659.
- Downie, B., Dirk, L.M.A., Xu, Q., Drake, J., Zhang, D., Dutt, M., Levy, A., Butterfield, DA., Geneve, R.L., Corum, III J.W., Lindstrom, K.G., Snyder, J.C. 2004. A physical, enzymatic, and genetic characterization of perturbations in the brownseed mutants of tomato. Journal of Experimental Botany 55: 961-973.
- Hatanaka, T., Shimizu, R., and Hildebrand, D. 2004. Expression of a Stokesia laevis epoxygenase gene. Phytochemistry 65: 2189-2196.
- Hildebrand, D., Yu, K., McCracken, C., and Rao, S. 2004. Chapter 3. Fatty Acid Changes. In: Plant Lipids: Biology, Utilization and Manipulation, Ed., Denis J Murphy, Blackwell Publishing, Oxford, UK.
- Jiang, D., and Ghabrial, S.A. 2004. Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus. Journal of General Virology 85: 2111-2121.
- Tang, W. 2004. Regulatory networks of the plant MADS domain protein AGL15: Characterization of DNA binding and isolation of direct downstream targets. Ph.D. Dissertation.
- Wang, H., Caruso, L.V., Downie, A.B. and Perry, S.E. 2004. The embryo MADS-domain protein AGAMOUS-like 15 directly regulates expression of a gene encoding an enzyme involved in gibberellin metabolism. Plant Cell 16: 1206-1219.
- Wang, H., Hill, K. and Perry, S.E. 2004. An Arabidopsis RNA lariat debranching enzyme is essential for embryogenesis. Journal of Biological Chemistry 279: 1468-1473.
- Wang, J., Machado, C., Panaccione, D.G., Tsai, H.-F., Schardl, C.L. 2004. The determinant step in ergot alkaloid biosynthesis by an endophyte of perennial ryegrass. Fungal Genetics and Biology 41: 189-198.
- White, K.A., and Nagy, P.D. 2004. Advances in the Molecular Biology of Tombusviruses: Gene Expression, Genome Replication and Recombination. Progress in Nucleic Acid Research and Molecular Biology 78: 187-226.
- Xu, Q., Belcastro, M., Dolan, S., Dinkins, R., Clarke, S., Downie, B. 2004. Isolation and characterization of a novel nuclear localized protein repair methyltransferase gene from Arabidopsis thaliana. Plant Physiology 136: 2652-2664.
- Zhao, T.-Y., Thacker, R.R., Corum, III J.W., Snyder, J.C., Meeley, R.B., Obendorf, R.L., Downie, B. 2004. Expression of the maize GALACTINOL SYNTHASE gene family: I) Expression of two different genes during seed development and germination. Physiologia Plantarum 121: 634-646.
- Kachroo, A., Srivathsa, C. V., Lapchyk, L., Falcone, D., Hildebrand, D., Kachroo, P. (2004) Oleic acid levels regulated by glycerolipid metabolism modulate defense gene expression in Arabidopsis. Proc. Natl. Acad. Sci. USA. 101: 5152-5257.
- Moon, C.D., Craven, K.D., Leuchtmann, A., Clement, S.L., Schardl, C.L. 2004. Prevalence of interspecific hybrids amongst asexual fungal endophytes of grasses. Molecular Ecology 13: 1455-1467
- Panaviene, Z., Panavas, T., Serva, S. and Nagy, P.D. 2004. Purification of the Cucumber necrosis virus replicase from yeast: The role of co-expressed viral RNA in stimulation of replicase activity. Journal of Virology 78: 8254-8263.
- Rajendran, K. S. and Nagy, P.D. 2004. Interaction between the replicase proteins of Tomato Bushy Stunt Virus in vitro and in vivo. Virology 326: 250-261.
- Shapka, N. and Nagy, P.D. 2004. The AU-rich RNA recombination hot spot sequence of Brome mosaic virus is functional in tombusviruses: Implications for the mechanism of RNA recombination. Journal of Virology 78: 2288-2300.
- Sri Putta, J. Smith, J., Walker, J., Rondet, M., Weisrock, D., Monaghan, J., Kump, K., King, D.C., Maness, N. J., Habermann, B., Tanaka, E., Bryant, S.V., Gardiner, D.M., Parichy, D.M., and Voss, S.R.. 2004. From Biomedicine to Natural History Research: Expressed Sequence Tag Resources for Ambystomatid Salamanders. BMC Genomics 5: 54, 17 pp.
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