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Browsing by Subject "Reverse genetics"

Now showing 1 - 2 of 2
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    Characterization of the multidomain Nsp2 Replicase protein of porcine reproductive and respiratory syndrome virus.
    (2008-12) Han, Jun
    This dissertation focused on understanding the biology of the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV), the etiological agent of porcine reproductive and respiratory syndrome (PRRS). PRRSV nsp2 is a multidomain protein, containing a putative N-terminal cysteine protease PL2 domain, a 500-700aa middle region of unknown function, a transmembrane domain and a C-terminal tail with uncertainty. In this dissertation, we report the following. (i) PRRSV nsp2 is undergoing rapid evolution in field strains exemplified by viral isolates MN184A and B. (ii) We showed that PRRSV nsp2 hypervariable regions aa12-35 and aa324-813 were not essential for viral replication in MARC-145 cells by using a reverse genetics system based on strain VR-2332. In contrast, deletion of the cysteine protease PL2 core domain, the PL2 downstream flanking sequence (aa181-323), the predicted transmembrane domain and the C-terminal domain were lethal to the virus. (iii) We provided evidence that the nsp2 protein encodes an active PL2 protease and mediates nsp2/3 processing in CHO cells with a substrate preference for the dipeptide G1196|G1197. The PL2 protease possessed both trans- and cis-cleavage activities, which could be distinguished by point mutations. Site-directed mutagenesis studies revealed that mutations that caused a specific loss of trans function of the PL2 protease, but not cis activity, were detrimental to the virus. In addition, we showed that the conserved aspartic acid residues (e.g., Asp89) played an important role in the PL2 trans-cleavage activity. (iv) We investigated the proteolytic processing of nsp2 in MARC-145 cells using recombinant PRRSV expressing foreign epitope-tagged nsp2 protein. We showed the presence of the nsp2 protein as different isoforms in PRRSV-infected cells, which appeared to share the same N terminus but differed in their respective C-termini. The nsp2 species emerged almost simultaneously in the early stage of PRRSV infection, were stable and had low turnover rates. Deletion mutagenesis suggested that the smaller nsp2 species (e.g. nsp2d, e and f) were not essential for viral replication in cell culture. Lastly, a cellular protein, heat shock 70kDa protein 5 (HSPA5), was identified as a coimmunoprecipitate of nsp2.
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    A study of Fusarium graminearum virulence factors.
    (2011-05) Menke, Jon R.
    The plant pathogen F. graminearum (Gibberella zeae) presents a two-fold threat to farmers and consumers. Not only does this filamentous fungus cause the disease Fusarium head blight (FHB) that results in significant yield loss in infected grains, it also taints these grains with potent mycotoxins harmful to humans, animals, and plants alike. Equally alarming is the evidence that grain can appear to be physically sound while still being significantly contaminated with trichothecene mycotoxins. Tri12 encodes a predicted major facilitator superfamily transporter protein suggested to play a role in the export of trichothecene mycotoxins produced by the Fusarium species. However, the role of Tri12p in toxin sensitivity and plant pathogenicity of Fusarium graminearum was previously unknown. In this study, the correct intron positions for Tri12 in F. graminearum (FgTri12) were established using cDNA sequencing, EST data, and comparative genomics. Reverse genetics was used to establish that FgTri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta. To identify the subcellular location of FgTri12p during toxin production in culture, FgTri12p was tagged with eGFP. FgTri12p::eGFP was localized in small motile vesicles, the plasma membrane, and the lumen of vacuoles within fungal cells. Treatment of cells with latrunculin A resulted in the absence of motile vesicles labeled with FgTri12p::eGFP, suggesting their formation relies upon actin polymerization. To determine if FgTri12p co-localizes with enzymes involved in trichothecene metabolism, its cellular fate was compared with FgTri1p::eGFP, a fluorescently tagged oxygenase catalyzing a key intermediate step in trichothecene biosynthesis. While FgTri1p::eGFP initially localizes to small motile vesicles and later accumulates in the vacuole, during the period of initial trichothecene biosynthesis it is targeted to the periphery of intermediate sized vesicles, presumed to be the site of toxin synthesis. These results indicate FgTri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta. The interactions between F. graminearum and its hosts – wheat, rice, or barley – differ in disease severity and the levels of trichothecenes that accumulate in response to infection. The transcriptome of the fungus in rice and wheat was examined in order to identify genes expressed in planta. The hypothesis that fungal genes expressed in planta, but not during growth in culture, could include those that determine the plant infection phenotype was tested by reverse genetics: Four genes expressed exclusively in planta were deleted and two of these were determined to significantly alter disease phenotype. FGSG_03539, also called Tri9, is a previously uncharacterized gene in the major trichothecene biosynthetic gene cluster. A mutant with a tri9 deletion has attenuated virulence and lower trichothecene levels in wheat compared to wild type or a mutant strain complemented with the intact Tri9 gene. FGSG_11164 encodes a predicted trypsin protease and deletion of this gene also results in a small but significant reduction in pathogenicity toward wheat. The results demonstrate that a reverse genetic approach using in planta gene expression data may supplement forward genetic screens for identifying genes encoding virulence factors.