Browsing by Author "Linda K. Hanley-Bowdoin, Committee Member"

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    Analysis of Begomoviruses-Mediated Gene Silencing
    (2003-03-02) Muangsan, Nooduan; Linda K. Hanley-Bowdoin, Committee Member; William F. Thompson, Committee Member; Judith F. Thomas, Committee Member; Dominique Robertson, Committee Chair
    Viral vectors carrying a host-derived sequence insert induce silencing of the corresponding gene in infected plants. This virus-induced gene silencing (VIGS) is a defense mechanism that is related to post-transcriptional gene silencing (PTGS) in transgenic plants. Here I describe modified vectors derived from two members of the Begomoviruses: Tomato Golden Mosaic Virus (TGMV) and Cabbage Leaf Curl Virus (CbLCV). In order to gain insight into the geminivirus-induced gene silencing (G-VIGS) mechanism, I tested six known silencing-deficient Arabidopsis mutants: sgs1, sgs2, sgs3, ago1, som and mom. Sgs1, sgs2, sgs3 and ago1 mutants are impaired for PTGS of transgenes and endogenous genes, whereas som and mom mutants release transcriptional gene silencing (TGS). SGS2 and AGO1 encode an RNA-dependent RNA polymerase (RdRP) and an eIF2C-like protein belonging to PAZ/PIWI family, respectively. SGS1 and SGS3 map to genes with unknown function. SOM and MOM are chromatin-associated proteins. All mutants were bombarded with CbLCV carrying a gene fragment homologous to an endogenous gene required for chlorophyll biosynthesis, magnesium chelatase (CH42). G-VIGS of CH42 endogenous gene occurred efficiently in the sgs1, ago1, som and mom mutants, but it was inhibited (not abolished) in sgs2 and sgs3 mutants. These results indicate that SGS2 and SGS3 are required for G-VIGS, whereas SGS1, AGO1, SOM and MOM have minimal or no effect. The findings that SOM and MOM are not required for G-VIGS support the ideas that G-VIGS is PTGS-specific or that geminiviruses counteract TGS. Considering these results, G-VIGS has unique requirements distinct from previously described silencing pathways in plants. I also showed that TGMV- and CbLCV-derived vectors triggered VIGS of endogenous genes in Nicotiana benthamiana and Arabidopsis thaliana, respectively. G-VIGS silencing of RdRP affected silencing while G-VIGS of a gene upregulated by AL1 negatively affected geminivirus replication or movement, suggesting that G-VIGS could be used as a preliminary screen for resistance genes. This research has provided an insight into genetic silencing mechanisms mediated by the Begomoviruses, and their potential tools as episomal vectors in reverse genetic studies.
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    Insect response to Alphavirus infection
    (2007-11-03) Mudiganti, Usharani; Dennis T. Brown, Committee Chair; Carla Mattos, Committee Member; Linda K. Hanley-Bowdoin, Committee Member; Gregory C. Gibson, Committee Member
    Invertebrate cells survive Alphavirus infections to establish viral persistence, in contrast to cell death seen soon after infection in mammalian cells. Invertebrate response to prototype alphavirus, Sindbis, has been studied to a certain extent, using mosquitoes and cell lines derived from mosquitoes. Some of the observations made in studies using mosquito systems include formation of intracellular vesicles soon after infection with Sindbis, identification of antiviral activity in the media used to grow the mosquito cell lines and in Sindbis-infected mosquito cell lysates, controlled levels of virus production as persistence is established and superinfection exclusion by Sindbis-infected cells. The study presented here is designed to utilize array of genomic and genetic information available in Drosophila model to identify the candidate genes ⁄ gene products playing a role in establishment of alphavirus persistence. Observations described in Chapter I establish Drosophila S2 cells as a suitable invertebrate system to study alphavirus-insect interactions. Gene expression analysis identified increased expression of 18 transcripts coding for membrane trafficking and cytoskeletal components and 10 transcripts coding for Notch pathway components, at 5 days post-infection. Identification of upregulation of Notch pathway suggests similarities between mechanism of establishment of persistence of Alphaviruses and Herpesviruses. Transcript coding for TEP II, a wide-spectrum protease inhibitor is increased in expression at 5 days post-infection and upon superinfection at 5 days post-infection. We probed for inhibition of viral protease activity during early persistence and upon superinfection of Sindbis-infected cells with Sindbis. Inhibition of Sindbis viral protease nsP2 is identified to be involved in establishment of viral persistence and superinfection exclusion in cells derived from Mosquito and Drosophila