Studies on specific and broad spectrum virus resistance in transgenic plants

http://www.ester.ee/record=b1053278~S1*es

Sobemovirus RNA linked to VPg over a threonine residue

AbstractPositive sense ssRNA virus genomes from several genera have a viral protein genome-linked (VPg) attached over a phosphodiester bond to the 5′ end of the genome. The VPgs of Southern bean mosaic virus (SBMV) and Ryegrass mottle virus (RGMoV) were purified from virions and analyzed by mass spectrometry. SBMV VPg was determined to be linked to RNA through a threonine residue at position one, whereas RGMoV VPg was linked to RNA through a serine also at the first position. In addition, we identified the termini of the corresponding VPgs and discovered three and seven phosphorylation sites in SBMV and RGMoV VPgs, respectively. This is the first report on the use of threonine for linking RNA to VPg

Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins.

Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5' end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP) moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.Funding is provided by Wellcome Trust (Ref: 097997/Z/11/Z) and the institutional research funding IUT19-3 from the Estonian Ministry of Education and Research.This is the final version of the article. It first appeared from PeerJ via https://doi.org/10.7717/peerj.213

Class XI Myosins Contribute to Auxin Response and Senescence-Induced Cell Death in Arabidopsis

The integrity and dynamics of actin cytoskeleton is necessary not only for plant cell architecture but also for membrane trafficking-mediated processes such as polar auxin transport, senescence, and cell death. In Arabidopsis, the inactivation of actin-based molecular motors, class XI myosins, affects the membrane trafficking and integrity of actin cytoskeleton, and thus causes defective plant growth and morphology, altered lifespan and reduced fertility. To evaluate the potential contribution of class XI myosins to the auxin response, senescence and cell death, we followed the flower and leaf development in the triple gene knockout mutant xi1 xi2 xik (3KO) and in rescued line stably expressing myosin XI-K:YFP (3KOR). Assessing the development of primary inflorescence shoots we found that the 3KO plants produced more axillary branches. Exploiting the auxin-dependent reporters DR5::GUS and IAA2::GUS, a significant reduction in auxin responsiveness was found throughout the development of the 3KO plants. Examination of the flower development of the plants stably expressing the auxin transporter PIN1::PIN1-GFP revealed partial loss of PIN1 polarization in developing 3KO pistils. Surprisingly, the stable expression of PIN1::PIN1-GFP significantly enhanced the semi-sterile phenotype of the 3KO plants. Further we investigated the localization of myosin XI-K:YFP in the 3KOR floral organs and revealed its expression pattern in floral primordia, developing pistils, and anther filaments. Interestingly, the XI-K:YFP and PIN1::PIN1-GFP shared partially overlapping but distinct expression patterns throughout floral development. Assessing the foliar development of the 3KO plants revealed increased rosette leaf production with signs of premature yellowing. Symptoms of the premature senescence correlated with massive loss of chlorophyll, increased cell death, early plasmolysis of epidermal cells, and strong up-regulation of the stress-inducible senescence-associated gene SAG13 in 3KO plants. Simultaneously, the reduced auxin responsiveness and premature leaf senescence were accompanied by significant anthocyanin accumulation in 3KO tissues. Collectively, our results provide genetic evidences that Arabidopsis class XI myosins arrange the flower morphogenesis and leaf longevity via contributing to auxin responses, leaf senescence, and cell death

An attempt to identify recombinants between two sobemoviruses in doubly infected oat plants

Recombination in RNA viruses is considered to play a major role as a driving force in virus variability to counterbalance loss in fitness that can be due to the accumulation of detrimental mutations. Studies on mixed infections are pertinent for understanding the role of recombination in virus evolution. They also provide important baseline information for studying the biosafety of plants expressing viral sequences. To investigate the possibility of RNA recombination occurrence between two sobemoviruses under little or no selection pressure, we co-infected test plants with Cocksfoot mottle virus (CfMV) and Ryegrass mottle virus (RGMoV). CfMV and RGMoV were selected because of their overlapping host range and geographical distribution. First, symptom development of both viruses in barley (Hordeum vulgare) and oat (Avena sativa) was examined. Both viruses generated quite strong infection symptoms in oat, but synergism was not detected. RGMoV was lethal for barley, whereas CfMV infection in barley was nearly symptomless. RT-PCR analysis revealed 100% infection with both viruses in oat but not in barley. Therefore, an RNA recombination study of CfMV and RGMoV was performed in oat. 105 plants were co-inoculated with both viruses and putative recombinational hot spot regions were screened for recombination events by RT-PCR analysis at a sensitivity level down to 0.1–100 pg of viral genomic RNA. No recombination events between the two sobemoviruses were detected

Incorporation of mammalian actin into microfilaments in plant cell nucleus

<p>Abstract</p> <p>Background</p> <p>Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now.</p> <p>Results</p> <p>Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin.</p> <p>Conclusions</p> <p>Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.</p

ABCE1 is essential for S phase progression in human cells

<p>ABCE1 is a highly conserved protein universally present in eukaryotes and archaea, which is crucial for the viability of different organisms. First identified as RNase L inhibitor, ABCE1 is currently recognized as an essential translation factor involved in several stages of eukaryotic translation and ribosome biogenesis. The nature of vital functions of ABCE1, however, remains unexplained. Here, we study the role of ABCE1 in human cell proliferation and its possible connection to translation. We show that ABCE1 depletion by siRNA results in a decreased rate of cell growth due to accumulation of cells in S phase, which is accompanied by inefficient DNA synthesis and reduced histone mRNA and protein levels. We infer that in addition to the role in general translation, ABCE1 is involved in histone biosynthesis and DNA replication and therefore is essential for normal S phase progression. In addition, we analyze whether ABCE1 is implicated in transcript-specific translation via its association with the eIF3 complex subunits known to control the synthesis of cell proliferation-related proteins. The expression levels of a few such targets regulated by eIF3A, however, were not consistently affected by ABCE1 depletion.</p