INTERACTION OF TURNIP CRINKLE VIRUS (TCV) AND ITS HOST PLANTS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我的主要研究方向是芜菁皱病毒（TCV）与寄主植物的相互作用。我们正在分析TCV与模式植物拟南芥的遗传相互作用。目前，我们正在使用两种TCV菌株，一种是野生型菌株，另一种是由UNL的莫里斯实验室开发的菌株，病毒外壳蛋白R6a中的一个氨基酸发生了变化。野生型TCV外壳蛋白在拟南芥中与一个转录激活因子结合，属于NAC转录因子家族。这种相互作用导致受感染植物的RNA沉默。突变的R6a TCV没有表现出这种相互作用，并引起更严重和症状不同的疾病。我们分别用正常和突变TCV接种野生型拟南芥，以及不接种病毒的模拟接种来分析这种相互作用。然后在24和48小时后采集所有三个植物种群的叶片。我们从所有六个组织中提取了总RNA，一式三份。这些rna将很快用于拟南芥全基因组cDNA阵列的微阵列分析。在不久的将来，将使用实时PCR技术进一步研究这些芯片中最一致的上调或下调转录本，以验证微阵列结果。我还有其他几个项目正在进行主要是和高塔姆·萨拉特博士合作，也和美国农业部的克里斯蒂安·托比亚斯博士和约翰·沃格尔博士合作。Drs。萨拉特和托拜厄斯，我一直在研究一项基因组学计划，研究生物燃料植物柳条稷（Panicum virgatum）的遗传学特征。我和我的学生已经从柳枝稷愈伤组织、茎、叶、冠和幼苗中构建了cDNA文库。与Tobias博士一起，我们对来自叶片，愈伤组织，茎和冠文库的15,272个cDNA克隆的5'端进行了测序和分析。所有这些序列很快就会存入基因银行，上面有我和我的学生的名字以及相应的INBRe确认。从这些序列中，我们收集了许多在各种代谢过程中重要的基因，以进一步表征。其中最主要的是参与木质素生物合成的基因，这将很快成为我们在该材料上首次发表的基础。我现在和约翰·沃格尔博士一起开始了一个类似的基因组计划，研究另一种生物燃料草，叫做长尾草。这个项目还处于起步阶段，但我们目前正在从这种植物的不同组织中构建4个cDNA文库。我还与Sarath博士合作，对一种叫做鹧鸪豆（Chamaecrista fasciculata）的草原豆科植物进行基因组学研究。本植物是与根瘤菌进行共生固氮共生，形成根瘤的重要草原物种。我们已经从该植物的根瘤组织中构建了cDNA文库，并开始对各种序列的5'端进行表征。我们已经分离出一种叫做豆血红蛋白的共生蛋白的克隆体，我们选择它进行进一步的表征。我们目前正专注于将开放阅读框亚克隆成一个合适的表达载体，用于生产重组蛋白。我们打算分析血红蛋白的氧结合行为和结构。这部作品不久就会出第一批手稿。我的最后一个项目涉及一种叫做玉米百合（Veratrum california）的植物。这种植物是感兴趣的，因为它的生产致畸和致癌的类固醇生物碱称为环巴胺。这种化合物主要集中在玉米百合的根部，但是怀孕的母羊食用玉米百合的叶子会产生一些可怕的后代，这些后代只有一只眼睛或两只眼睛合并在一个眼窝里。为了研究这种有趣的植物，我们从根和叶中构建了cDNA文库。我们还使用dna - aflp分析分析了这些组织的转录组差异。我们已经分离和测序了超过100个单独的AFLP片段，这些片段是一个组织或另一个组织所独有的。我们目前正在分析这些序列，希望确定一些有助于环巴胺合成的遗传因素。我们希望不久就能发表这项工作。

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.