Pathogen resistance genes and proteins in orchids

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Marchione, Wesley A.
Vann, Carolyn N.
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Thesis (M.S.)
Department of Biology
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To study resistance (R) genes that are expressed when Sophrolaeliacattleya Ginny Champion 'Riverbend' orchid tissue was infected with the tobacco mosaic virus (TMV0), a subtraction library of cDNA clones was previously constructed using mRNA isolated before and after infection (Shuck, unpublished). From 200 clones collected, 5 clones were randomly selected, DNA was isolated, and the cDNA insert was sequenced. These sequences were imported into BLAST to search for homology to other R genes. This search revealed clone 4A to have an 84% homology to a 54 nucleotide region from the Arabidopsis thaliana oligouridylate binding protein which is highly expressed and known to bind RNA Polymerase III transcripts and adenovirus associated RNAs. Further bioinformatics analysis was performed utilizing databases and analysis packages available on the Internet, software such as Vector NTI (Informax, Bethesda, MD), and manual searches. However, no additional domains or motifs indicative of pathogen resistance genes were located in any of the 5 clones. Subsequently, total proteins expressed at various time points following infection were examined on denaturing 5-20% gradient polyacrylamide gels stained with the ProteoSilver Plus TM silver stain kit (Sigma, St. Louis, MO) in order to examine the timing and duration of expression of proteins involved in TMV-O resistance. One protein of-18 kDa was highly expressed at 4 hr after infection that was not seen in the negative control. By 8 hr the band was no longer expressed, it was expressed again from 30 - 48 hr, but was not seen again in later time points. Finally, total mRNA isolated from pooled time points and subjected to in vitro translation indicated a reduction in translation products after infection, providing evidence of posttranscriptional gene silencing (PTGS) following TMV-O infection.