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Biomedical subjects

Thomas L German

Publications and source records attributed to Thomas L German.

6 recordsLinked to original sources
PubMed2006

Methods for effective real-time RT-PCR analysis of virus-induced gene silencing.

We applied real-time RT-PCR to the analysis of Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) of the phytoene desaturase (PDS) gene in Nicotiana benthamiana and tomato. Using a combination of direct measurement and mathematical assessment, we evaluated three plant genes, ubiquitin (ubi3), elongation factor-1 alpha (EF-1), and actin, for use as internal reference transcripts and found that EF-1 and ubi3 were least variable under our experimental conditions. Primer sets designed to amplify the 5' or 3' regions of endogenous PDS transcripts in tomato yielded similar reductions in transcript levels indicating a uniform VIGS-mediated degradation of target RNA. By measuring the ratio of the abundance of the PDS insert transcript to the TRV coat protein RNA, we established that the PDS insert within TRV was stable in both hosts. VIGS in N. benthamiana resulted in complete photo-bleaching of all foliar tissue compared to chimeric bleaching in tomato. PDS transcript levels were decreased eleven- and seven-fold in photobleached leaves of N. benthamiana and tomato, respectively, while sampling tomato leaflets on the basis of age rather than visible bleaching resulted in only a 17% reduction in PDS coupled with a large leaf-to-leaf variation. There was a significant inverse relationship (r2=76%, P=0.01) between the relative abundance of CP RNA and the amount of PDS transcript in rTRV::tPDS-infected tomato suggesting that virus spread and accumulation are required precursors for successful VIGS in this host.

Gene Silencing↗
PubMed2005

Tomato spotted wilt virus glycoprotein G(C) is cleaved at acidic pH.

Tomato spotted wilt virus (TSWV) is a plant-infecting member of the family Bunyaviridae. TSWV encodes two envelope glycoproteins, G(N) and G(C), which are required for virus infection of the arthropod vector. Other members of the Bunyaviridae enter host cells by pH-dependent endocytosis. During this process, the glycoproteins are exposed to conditions of acidic pH within endocytic vesicles causing the G(C) protein to change conformation. This conformational change renders G(C) more sensitive to protease cleavage. We subjected TSWV virions to varying pH conditions and determined that TSWV G(C), but not G(N), was cleaved under acidic pH conditions, and that this phenomenon did not occur at neutral or alkaline pH. This data provides evidence that G(C) changes conformation at low pH which results in altered protease sensitivity. Furthermore, sequence analysis of G(C) predicts the presence of internal hydrophobic domains, regions that are characteristic of fusion proteins. Like studies with other members of the Bunyaviridae, this study is the first step towards characterizing the nature of cell entry by TSWV.

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

Tospovirus-thrips interactions.

The complex and specific interplay between thrips, tospoviruses, and their shared plant hosts leads to outbreaks of crop disease epidemics of economic and social importance. The precise details of the processes underpinning the vector-virus-host interaction and their coordinated evolution increase our understanding of the general principles underlying pathogen transmission by insects, which in turn can be exploited to develop sustainable strategies for controlling the spread of the virus through plant populations. In this review, we focus primarily on recent progress toward understanding the biological processes and molecular interactions involved in the acquisition and transmission of Tospoviruses by their thrips vectors.

Animals↗
PubMed2004

Expression and characterization of a soluble form of tomato spotted wilt virus glycoprotein GN.

Tomato spotted wilt virus (TSWV), a member of the Tospovirus genus within the Bunyaviridae, is an economically important plant pathogen with a worldwide distribution. TSWV is transmitted to plants via thrips (Thysanoptera: Thripidae), which transmit the virus in a persistent propagative manner. The envelope glycoproteins, G(N) and G(C), are critical for the infection of thrips, but they are not required for the initial infection of plants. Thus, it is assumed that the envelope glycoproteins play important roles in the entry of TSWV into the insect midgut, the first site of infection. To directly test the hypothesis that G(N) plays a role in TSWV acquisition by thrips, we expressed and purified a soluble, recombinant form of the G(N) protein (G(N)-S). The expression of G(N)-S allowed us to examine the function of G(N) in the absence of other viral proteins. We detected specific binding to thrips midguts when purified G(N)-S was fed to thrips in an in vivo binding assay. The TSWV nucleocapsid protein and human cytomegalovirus glycoprotein B did not bind to thrips midguts, indicating that the G(N)-S-thrips midgut interaction is specific. TSWV acquisition inhibition assays revealed that thrips that were concomitantly fed purified TSWV and G(N)-S had reduced amounts of virus in their midguts compared to thrips that were fed TSWV only. Our findings that G(N)-S binds to larval thrips guts and decreases TSWV acquisition provide evidence that G(N) may serve as a viral ligand that mediates the attachment of TSWV to receptors displayed on the epithelial cells of the thrips midgut.

Amino Acid Sequence↗
PubMed2003

Association of L protein and in vitro tomato spotted wilt virus RNA-dependent RNA polymerase activity.

We have previously described an in vitro assay for RNA-dependent RNA polymerase activity in virions of tomato spotted wilt virus (TSWV). Here we report antibody inhibition of virion-associated RNA synthesis in vitro with an L-protein-specific polyclonal antibody raised against the carboxy-terminus of the L protein. In contrast, RNA synthesis was not inhibited by a heterologous antiserum and was unaffected by antiserum raised against an internal portion of the L protein. Our results directly associate the TSWV L protein, the putative viral polymerase, with RNA synthesis functions in vitro.

Antibodies, Viral↗