Eugene V. Skurat

Role of the leader sequence in tobacco pectin methylesterase secretion

We report that unprocessed tobacco pectin methylesterase (PME) contains N‐terminal pro‐sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro‐sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro‐sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.

A novel function for a ubiquitous plant enzyme pectin methylesterase: The enhancer of RNA silencing

Co‐agroinjection of Nicotiana benthamiana leaves with the pectin methylesterase (proPME) gene and the TMV:GFP vector resulted in a stimulation of virus‐induced RNA silencing (inhibition of GFP production, virus RNA degradation, stimulation of siRNAs production). Conversely, co‐expression of TMV:GFP with either antisense PME construct or with enzymatically inactive proPME restored synthesis of viral RNA. Furthermore, expression of proPME enhanced the GFP transgene‐induced gene silencing accompanied by relocation of the DCL1 protein from nucleus to the cytoplasm and activation of siRNAs and miRNAs production. It was hypothesized that DCL1 relocated to the cytoplasm may use as substrates both miRNA precursor and viral RNA. The capacity for enhancing the RNA silencing is a novel function for the polyfunctional PME.

Immunogenicity and Protective Efficacy of Candidate Universal Influenza A Nanovaccines Produced in Plants by Tobacco Mosaic Virus-based Vectors

A new approach for super-expression of the influenza virus epitope M2e in plants has been developed on the basis of a recombinant Tobacco mosaic virus (TMV, strain U1) genome designed for Agrobacterium-mediated delivery into the plant cell nucleus. The TMV coat protein (CP) served as a carrier and three versions of the M2e sequence were inserted into the surface loop between amino acid residues 155 and 156. Cysteine residues in the heterologous peptide were thought likely to impede efficient assembly of chimeric particles. Therefore, viral vectors TMV-M2e-ala and TMV-M2e-ser were constructed in which cysteine codons 17 and 19 of the M2e epitope were substituted by codons for serine or alanine. Agroinfiltration experiments proved that the chimeric viruses were capable of systemically infecting Nicotiana benthamiana plants. Antisera raised against TMV-M2e-ala virions appear to contain far more antibodies specific to influenza virus M2e than those specific to TMV carrier particle (ratio 5:1). Immunogold electron microscopy showed that the 2-epitopes were uniformly distributed and tightly packed on the surface of the chimeric TMV virions. Apparently, the majority of the TMV CP-specific epitopes in the chimeric TMV-M2e particles are hidden from the immune system by the M2e epitopes exposed on the particle surface. The profile of IgG subclasses after immunization of mice with TMV-M2e-ser and TMV-M2e-ala was evaluated. Immunization with TMV-M2e-ala induced a significant difference between the levels of IgG1 and IgG2a (IgG1/IgG2a=3.2). Mice immunized with the chimeric viruses were resistant to five lethal doses (LD50) of the homologous influenza virus strain, A/PR/8/34 (H1N1) and TMV-M2e-ala also gave partial protection (5LD50, 70% of survival rate) against a heterologous strain influenza A/California/04/2009 (H1N1) (4 amino acid changes in M2e). These results indicate that a new generation candidate universal nanovaccine against influenza based on a recombinant TMV construct has been obtained.

Novel Inter-Subunit Contacts in Barley Stripe Mosaic Virus Revealed by Cryo-Electron Microscopy

Summary Barley stripe mosaic virus (BSMV, genus Hordeivirus) is a rod-shaped single-stranded RNA virus similar to viruses of the structurally characterized and well-studied genus Tobamovirus. Here we report the first high-resolution structure of BSMV at 4.1 Å obtained by cryo-electron microscopy. We discovered that BSMV forms two types of virion that differ in the number of coat protein (CP) subunits per turn and interactions between the CP subunits. While BSMV and tobacco mosaic virus CP subunits have a similar fold and interact with RNA using conserved residues, the axial contacts between the CP of these two viral groups are considerably different. BSMV CP subunits lack substantial axial contacts and are held together by a previously unobserved lateral contact formed at the virion surface via an interacting loop, which protrudes from the CP hydrophobic core to the adjacent CP subunit. These data provide an insight into diversity in structural organization of helical viruses.

Pectin methylesterase as a factor of plant transcriptome stability

Pectin methylesterase (PME) is a cell-wall enzyme that acts as a growth and morphogenesis factor in higher plants and is involved in gene silencing, plant virus reproduction, and transgenesis. A study was made of the role of PME as a stress protein in host plant-virus interactions. PME enzymatic activity was induced, not only by an additional PME gene copy, but also by an empty vector. PME suppressed tobacco mosaic virus (TMV) reproduction, including short-and long-distance virus movement in plants. Surprisingly, elevated PME activity was observed in intact stably transformed transgenic plants. For example, PME activity was increased in transgenic Nicotiana tabacum and N. benthamiana plants expressing the genes for the TMV movement protein and GFP and in tomato plants with cosuppression of the polygalacturonase gene. Activation of light-inducible psbO induced transcription of the PME gene. It was suggested that PME is involved in maintaining the stability of the plant transcriptome and restores its status quo upon viral infection, transformation with a foreign gene, or excess transcription of the cell genome.

Reciprocal dependence between pectinmethylesterase gene expression and tobamovirus reproduction effectiveness in Nicotiana benthamiana.

The transport protein (TP) of tobacco mosaic virus (TMV) ensures intercellular transport of viral RNA through plasmodesms, apparently by interacting with the cell proteins of endoplasmic reticulum, cytoskeleton, and cell wall [1]. During the past years, several cell proteins that specifically interact with VTM TP have been identified. These are cytoskeletal tubulin, myosin, and actin [2, 3]; protein kinanses [4‐6]; transcriptional coactivator KELP [7]; and cytoskeletal protein MPB2C [8]. The functional role of these proteins in the transport of viral infection remains unknown.

Molecular Diagnostics of Potato Infections with PVY and PLRV by Immunochromatography

An immunochromatography test system has been developed for molecular diagnostics of the potato virus Y and PLRV infection. To increase a low yield of PLRV and raise antibodies against the PLRV antigen, chimerical virus was constructed comprising the PLRV coat protein and recombinant RNA of a tobamovirus, in which capsid protein gene was replaced by the PLRV coat protein gene. Binary vector containing the DNA copy of the recombinant RNA was infectious, and yield of the chimerical virus increased up to 800 times in comparison with the wild type PLRV. On the basis of experience in the development of the diagnostics of viral and viroid infections, rational tactics are proposed for the mass laboratory and field diagnosis of viral infections on the molecular level.

Development of Male Reproductive Organs in an Insertion Mutant TPD1 of Tobacco Characterized by Extended Flowering

A tobacco clone TPD1 (Tobacco Pollen Development), characterized by an extended flowering period, was obtained using a serial agrobacterial transformation of tobacco (Nicotiana tabacum L., cv. Samsun) by constructing a DNA carrying the kanamycin resistance gene inserted into the binary pBin19 vector. However, the characteristics of the vegetative growth of these plants were similar to those of other tobacco clones and the wild type. In the insertion mutant, pollen was 1.5 times smaller than in the wild type and germinated on the stigmata of neither its own clone nor the wild-type plants. Cytochemical investigation of pollen of the TPD1-mutant did not reveal any activity of respiratory enzymes, succinate dehydrogenase and peroxidase, indicating that the pollen was nonviable. Unlike the wild type, theTPD1 mutant exhibited disturbed trophic interactions within the anther tissues, particularly suppressed starch hydrolysis in the anther wall tissues at the stage of rapidly growing microspores. We conclude that the insertion of T-DNA into the TPD1 gene produced structural and metabolic changes during the development of anther tissues in the mutant clone, resulting in pollen sterility.

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.