This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. My main research project is focused on the interaction of Turnip Crinkle Virus (TCV) and its host plants. We are analyzing the genetic interactions of TCV and the model plant Arabidopsis. Currently, we are using two strains of TCV, a wild type strain and a strain developed by the Morris lab at UNL with one amino acid altered in the viral coat protein called R6a. Wild type TCV coat protein shows a binding with a transcriptional activator in Arabidopsis belonging to the NAC family of transcription factors. This interaction causes RNA silencing in the infected plants. The mutated R6a TCV does not show this interaction and causes a more severe and symptomatically different disease. We are analyzing this interaction by inoculating wild type Arabidopsis with normal and mutant TCV individually, as well as a mock inoculation with no virus. We then harvested leaves after 24 and 48 hrs form all three plant populations. We have extracted total RNA from all six tissues in triplicate. These RNAs will soon be used in microarray analysis of Arabidopsis whole genome cDNA arrays. Transcripts identified as being the most consistently up or down regulated from these arrays will be further studied in the near future using real-time PCR to verify the microarray results. I also have several other projects underway mostly in collaboration with Dr. Gautam Sarath, but also with Dr. Christian Tobias and Dr. John Vogel all of the USDA. With Drs. Sarath and Tobias, I have been working on a genomics inititative characterizing the genetics of the biofuel plant Switchgrass (Panicum virgatum). My students and I have to date constructed cDNA libraries from Switchgrass callus tissue, stems, leaves, crowns, and seedlings. Together with Dr. Tobias, we have sequenced and analyzed the 5'end of 15,272 cDNA clones from the leaf, callus, stem ,and crown libraries. All of these sequences are soon to be deposited in GenBank bearing my and my students names as well as the appropriate INBRe acknowledgement. From these sequences, we have gleaned many genes important in a variety of metabolic processes for further characterization. Chief amongst these are the genes involved in lignin biosynthesis which will soon be the basis of our first publications on this material. I am currently beginning a similar genomics initiative with Dr. John Vogel studying another biofuel grass called Bradypodium distachyon. This project is in its infancy, but we are currently constructing four cDNA libraries from various tissues from this plant. I am also working with Dr. Sarath on a genomics study of a prairie legume called Partridge Pea (Chamaecrista fasciculata). This plant is an important prairie species that participates in a symbiotic nitrogen-fixing symbiosis with Rhizobium forming root nodules. We have constructed a cDNA library from the nodule tissue of this plant, and have begun to characterize the 5'end of various sequences. We have isolated a clone for a symbiotic protein called leghemoglobin which we have selected for further characterization. We are currently concentrating on subcloning the open-reading frame into an appropriate expression vector for the production of recombinant protein. We intend to analyze the oxygen-binding behavior and structure of the leghemoglobin protein. This work will shortly produce its first manuscripts. My last project involves a plant called corn lily (Veratrum californicum). This plant is of interest due to its production of the teratogenic and carcinogenic steroidal alkaloid called cyclopamine. This compound is largely concentrated in the roots of the corn lily, but consumption of its leaves by pregnant ewes can produce somewhat gruesome offspring with only one eye or both eyes merged into one socket. To study this interesting plant, we have constructed cDNA libraries from the roots and leaves. We have also analyzed the differences in the transcriptomes of these tissues using cDNA-AFLP analysis. We have isolated and sequenced in excess of 100 individual AFLP fragments which are unique to one tissue or the other. We are currently analyzing these sequences with the hope of identifying some of the genetic factors contributing to cyclopamine synthesis. We hope to publish this work shortly.
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