Browsing by Subject "VPG"

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  • Saha, Shreya; Hafren, Anders; Makinen, Kristiina (2019)
    One large open reading frame (ORF) encodes 10 potyviral proteins. We compared the accumulation of cylindrical inclusion (CI) protein from the middle, coat protein (CP) from the 3'end, and Renilla luciferase (RLUC) from two distinct locations in potato virus A (PVA) RNA. 5' RLUC was expressed from an rluc gene inserted between the P1 and helper component proteinase (HCPro) cistrons, and 3' RLUC was expressed from the gene inserted between the RNA polymerase and CP cistrons. Viral protein and RNA accumulation were quantitated (i) when expressed from PVA RNA in the presence of ectopically expressed genome-linked viral protein (VPg) and auxiliary proteins and (ii) at different time points during natural infection. The rate and timing of 3' RLUC and CP accumulation were found to be different from those of 5' RLUC and Cl. Ectopic expression of VPg boosted PVA RNA, 3' RLUC, and, together with HCPro, CP accumulation, whereas 5' RLUC and CI accumulation remained unaffected regardless of the increased viral RNA amount. In natural infection, the rate of the noteworthy minute early accumulation of 3' RLUC accelerated toward the end of infection. 5' RLUC accumulation, which was already pronounced at 2 days postinfection, increased moderately and stabilized to a constant level by day 5, whereas PVA RNA and CP levels continued to increase throughout the infection. We propose that these observations connect with the mechanisms by which potyvirus infection limits CP accumulation during early infection and specifically supports its accumulation late in infection, but follow-up studies are required to understand the mechanism of how this occurs. IMPORTANCE The results of this study suggest that the dynamics of potyviral protein accumulation are regulated differentially from the 3' end of viral RNA than from the rest of the genome, the significance of which would be to satisfy the needs of replication early and particle assembly late in infection.
  • Rajamaki, Minna-Liisa; Sikorskaite-Gudziuniene, Sidona; Sarmah, Nandita; Varjosalo, Markku; Valkonen, Jari P. T. (2020)
    BackgroundInfection of plants by viruses interferes with expression and subcellular localization of plant proteins. Potyviruses comprise the largest and most economically damaging group of plant-infecting RNA viruses. In virus-infected cells, at least two potyviral proteins localize to nucleus but reasons remain partly unknown.ResultsIn this study, we examined changes in the nuclear proteome of leaf cells from a diploid potato line (Solanum tuberosum L.) after infection with potato virus A (PVA; genus Potyvirus; Potyviridae) and compared the data with that acquired for healthy leaves. Gel-free liquid chromatography-coupled to tandem mass spectrometry was used to identify 807 nuclear proteins in the potato line v2-108; of these proteins, 370 were detected in at least two samples of healthy leaves. A total of 313 proteins were common in at least two samples of healthy and PVA-infected leaves; of these proteins, 8 showed differential accumulation. Sixteen proteins were detected exclusively in the samples from PVA-infected leaves, whereas other 16 proteins were unique to healthy leaves. The protein Dnajc14 was only detected in healthy leaves, whereas different ribosomal proteins, ribosome-biogenesis proteins, and RNA splicing-related proteins were over-represented in the nuclei of PVA-infected leaves. Two virus-encoded proteins were identified in the samples of PVA-infected leaves.ConclusionsOur results show that PVA infection alters especially ribosomes and splicing-related proteins in the nucleus of potato leaves. The data increase our understanding of potyvirus infection and the role of nucleus in infection. To our knowledge, this is the first study of the nuclear proteome of potato leaves and one of the few studies of changes occurring in nuclear proteomes in response to plant virus infection.
  • Eskelin, Katri; Varjosalo, Markku; Ravantti, Janne; Makinen, Kristiina (2019)
    Nicotiana benthamiana is an important model plant for plant-microbe interaction studies. Here, we compared ribosome profiles and riboproteomes of healthy and infected N. benthamiana plants. We affinity purified ribosomes from transgenic leaves expressing a FLAG-tagged ribosomal large subunit protein RPL18B of Arabidopsis thaliana. Purifications were prepared from healthy plants and plants that had been infiltrated with Agrobacterium tumefaciens carrying infectious cDNA of Potato virus A (PVA) or firefly luciferase gene, referred to here as PVA- or Agrobacterium-infected plants, respectively. Plants encode a number of paralogous ribosomal proteins (r-proteins). The N. benthamiana riboproteome revealed approximately 6600 r-protein hits representing 424 distinct r-proteins that were members of 71 of the expected 81 r-protein families. Data are available via ProteomeXchange with identifier PXD011602. The data indicated that N. benthamiana ribosomes are heterogeneous in their r-protein composition. In PVA-infected plants, the number of identified r-protein paralogues was lower than in Agrobacterium-infected or healthy plants. A. tumefaciens proteins did not associate with ribosomes, whereas ribosomes from PVA-infected plants co-purified with viral cylindrical inclusion protein and helper component proteinase, reinforcing their possible role in protein synthesis during virus infection. In addition, viral NIa protease-VPg, RNA polymerase NIb and coat protein were occasionally detected. Infection did not affect the proportions of ribosomal subunits or the monosome to polysome ratio, suggesting that no overall alteration in translational activity took place on infection with these pathogens. The riboproteomic data of healthy and pathogen-infected N. benthamiana will be useful for studies on the specific use of r-protein paralogues to control translation in infected plants.