Differentially expressed genes of Sophrolaeliacattleya Ginny Champion "Riverbend" in response to the odontoglossum ringspot virus

Due to the rapid destruction of native orchid habitats it has become necessary to house many endangered orchid species in greenhouse environments where enhanced spread of viral disease occurs due to the close contact between plants. This research was concerned with the construction of a library of genes whose expression is induced in response to viral challenge. In uncovering the genes that are activated during plant-pathogen interactions, it may be possible to manipulate these pathways to develop virus resistant orchids. Furthermore, this research will contribute additional information for the existing framework of plant-pathogen interactions of all plant species.In order to construct a library of genes expressed in response to viral infection, suppression subtractive hybridization was performed using the PCR-Select cDNA Subtraction Kit (CLONTECH, Palo Alto, CA) on Sophrolaeliacattleya Ginny Champion 'Riverbend' clones. RNA was isolated from plants that had been inoculated with the Odontoglossum ringspot virus (ORSV) and from control plants that had not been inoculated with ORSV. Following reverse transcription-PCR (RT-PCR) to obtain cDNA, cDNAs of the tester population (those cDNAs containing differentially expressed messages in response to ORSV) and the driver population (reference cDNAs from uninfected plants) were obtained. The two different cDNA populations are mixed together and hybridized. The sequences common to both populations were subtracted, leaving only the differentially expressed sequences available for PCR amplification.A library containing these genes was constructed, and one clone, chosen at random, was sequenced. Based on homology comparisons to known genes, we have cloned a gene that may contain a nucleotide binding site similar to that of the tobacco N gene, important for plant resistance to pathogens. In the near future, this clone will be used to construct probes for use in northern analysis to determine the timing and localization of the products of this gene. This information will aid in characterizing the function of the orchid N-gene and identifying other members of this signal cascade. In addition, many other clones await sequencing and similar characterization.Department of BiologyThesis (M.S.