Anna Sheveleva

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.

Occurrence and Genetic Diversity of Winona-Like Plum pox virus Isolates in Russia

In studying the distribution and genetic diversity of Plum pox virus (PPV) in Russia, over a dozen new PPV isolates belonging to the strain Winona (PPV-W) were identified by immunocapture reverse-transcription polymerase chain reaction with the PPV-W-specific primers 3174-SP-F3/3174-SP-R1. Isolates were detected in two geographically distant regions of European Russia (Northern Caucasus and Moscow regions) in naturally infected plum (Prunus domestica), blackthorn (P. spinosa), Canadian plum (P. nigra), and downy cherry (P. tomentosa). The new PPV-W isolates were shown to be serologically related but not identical by triple-antibody sandwich enzyme-linked immunosorbent assay and Western blotting analysis using the monoclonal antibody (MAb) 5B-IVIA and MAbs specific to the N-terminal epitopes of PPV-W isolate 3174. Analysis of nucleotide and deduced amino acid sequences of the (C-ter)NIb-(N-ter)CP genome region indicate great genetic diversity among isolates, with phylogenetic analysis revealing seven clades. Isolates P1 and P3 found in plum in the south of Russia clustered closely with the putative ancestral PPV-W isolate LV-145bt from Latvia, while isolate 1410-7 found in P. nigra in Moscow appears to be closely related to the Canadian isolate W3174. The data obtained indicate wide dissemination of PPV-W isolate in stone fruit in the European part of the former USSR.

Genome Sequence Analysis of New Isolates of the Winona Strain of Plum pox virus and the First Definitive Evidence of Intrastrain Recombination Events.

Plum pox virus (PPV) is genetically diverse with nine different strains identified. Mutations, indel events, and interstrain recombination events are known to contribute to the genetic diversity of PPV. This is the first report of intrastrain recombination events that contribute to PPVs genetic diversity. Fourteen isolates of the PPV strain Winona (W) were analyzed including nine new strain W isolates sequenced completely in this study. Isolates of other strains of PPV with more than one isolate with the complete genome sequence available in GenBank were included also in this study for comparison and analysis. Five intrastrain recombination events were detected among the PPV W isolates, one among PPV C strain isolates, and one among PPV M strain isolates. Four (29%) of the PPV W isolates analyzed are recombinants; one of which (P2-1) is a mosaic, with three recombination events identified. A new interstrain recombinant event was identified between a strain M isolate and a strain Rec isolate, a known recombinant. In silico recombination studies and pairwise distance analyses of PPV strain D isolates indicate that a threshold of genetic diversity exists for the detectability of recombination events, in the range of approximately 0.78×10(-2) to 1.33×10(-2) mean pairwise distance. RDP4 analyses indicate that in the case of PPV Rec isolates there may be a recombinant breakpoint distinct from the obvious transition point of strain sequences. Evidence was obtained that indicates that the frequency of PPV recombination is underestimated, which may be true for other RNA viruses where low genetic diversity exists.

Binding of monoclonal antibodies to the movement protein (MP) of Tobacco mosaic virus: influence of subcellular MP localization and phosphorylation.

Monoclonal antibodies (mAbs) to recombinant movement protein (MP(REC)) of Tobacco mosaic virus (TMV) were used to reveal the dependence of MP epitope accessibility to mAbs on subcellular MP localization and post-translational MP phosphorylation. Leaves of Nicotiana benthamiana or N. tabacum were inoculated mechanically with TMV or agroinjected with an MP expression vector. At different time post-inoculation, ER membrane- and cell wall-enriched fractions (ER-MP and CW-MP, respectively) were isolated and analysed. The N-terminal region (residues 1-30) as well as regions 186-222 and 223-257 of MP from the CW and ER fractions were accessible for interaction with mAbs. By contrast, the MP regions including residues 76-89 and 98-129 were not accessible. The C-terminal TMV MP region (residues 258-268) was inaccessible to mAbs not only in CW-MP, but also in ER-MP fractions. Evidence is presented that phosphorylation of the majority of TMV MP C-terminal sites occurred on ER membranes at an early stage of virus infection, i.e. not after, but before reaching the cell wall. C-terminal phosphorylation of purified MP(REC) abolished recognition of C-proximal residues 258-268 by specific mAbs, which could be restored by MP dephosphorylation. Likewise, accessibility to mAbs of the C-terminal MP epitope in ER-MP and CW-MP leaf fractions was restored by dephosphorylation. Substitution of three or four C-terminal Ser/Thr residues with non-phosphorylatable Ala also resulted in abolition of interaction of mAbs with MP.

Occurrence and characterization of plum pox virus strain D isolates from European Russia and Crimea

Numerous plum pox virus (PPV) strain D isolates have been found in geographically distant regions of European Russia and the Crimean peninsula on different stone fruit hosts. Phylogenetic analysis of their partial and complete genomes suggests multiple introductions of PPV-D into Russia. Distinct natural isolates from Prunus tomentosa were found to bear unique amino acid substitutions in the N-terminus of the coat protein (CP) that may contribute to the adaptation of PPV-D to this host. Serological analysis using the PPV-D-specific monoclonal antibody 4DG5 provided further evidence that mutations at positions 58 and 59 of the CP are crucial for antibody binding.

Analysis of genetic diversity of Russian sour cherry Plum pox virus isolates provides evidence of a new strain

Plum pox virus (PPV) exists as a complex of nine strains adapted to different Prunus hosts. Unusual PPV isolates that do not belong to the known cherry-adapted strains were discovered on sour cherry in Russia. Here, two complete genomes of isolates Tat-2 and Tat-4 were determined by sequencing on the Illumina HiSeq 2500 platform. Both were composed of 9,792 nucleotides, excluding the poly(A) tail, with the organization typical of PPV and had 99.4 and 99.7% identity between each other at the nucleotide and amino acid levels. The sequence identities between Tat-2/Tat-4 and known PPV strains ranged from 77.6 to 83.3% for genomic RNA and from 80.0 to 93.8% for polyprotein. Phylogenetic analysis placed Tat-2 and Tat-4 in a separate clade, distinct from the C and CR strains. Three more Tat-2/Tat-4-like isolates were detected in local cherry plantings using the newly developed, specific RT-PCR assay. Based on the phylogenetic analysis, sequence identities, and environmental distribution, Tat-2, Tat-4, and related isolates represent a new cherry-adapted PPV strain for which the name PPV-CV (Cherry Volga) is proposed.

Molecular biological properties of new isolates of Plum pox virus strain Winona

Plum pox virus (PPV, genus Potyvirus, family Potyviridae) is the most important virus pathogen of stone fruit cultures of the genus Prunus from an economical standpoint. Winona strain (PPV-W) is the most variable of nine known virus strains and one of the most widespread in the European part of Russia. Six new PPV-W isolates were first discovered in green plantations of Moscow (Kp2U, Avang, Pulk, Pulk-1), in Taldomsky district of Moscow region (Karm), and in Kovrovsky district of Vladimir region (Vlad-4) on wild trees of plum Prunus domestica. 3’-Terminal segment of genome of new isolates was notable for high variability level. The study on the relationship with other isolates of this strain by means of phylogenetic analysis of gene sequence of the coat protein showed the lack of clusterization of Russian PPV-W isolates according to geographical principle. Inoculation of Nicotiana benthamiana plants by hop plant louse Phorodon humili from plum trees infected with Avang and Pulk isolates and by thistle aphid Brachycaudus cardui from the tree infected with Kp2U isolate led to the systemic viral infection in indicator plants, suggesting the possibility of PPV-W spread by both aphid species in nature. Transmission of PPV-W through seeds was not observed.

Mapping of Epitopes of the Recombinant Movement Protein of the Tobacco Mosaic Virus with the Use of Monoclonal Antibodies

The movement protein (MP) of the tobacco mosaic virus (TMV) provides the intercellular transport of the viral RNA through plasmodesmata. MP fulfils its function while interacting with host cell factors on the whole way of its intracellular movement from the subcellular site of its synthesis to the plasmodesmata of cellular walls. The MP conformation during its intracellular movement and fulfilling the transport function still remains unknown. In this study, we describe the preparation of murine monoclonal antibodies (MAs) to TMV MP and mapping of the MP epitopes. Stable hybridoma lines that produce MAs to the partially denatured recombinant MP (MPr) were obtained. MAs were tested by the immunoblotting and ELISA with the use of deletion variations of MPr. The epitopes of TMV MPr that recognize specific MAs were determined.

Sequence Analysis of Plum pox virus Strain C Isolates from Russia Revealed Prevalence of the D96E Mutation in the Universal Epitope and Interstrain Recombination Events

The understanding of genetic diversity, geographic distribution, and antigenic properties of Plum pox virus (PPV) is a prerequisite to improve control of sharka, the most detrimental viral disease of stone fruit crops worldwide. Forty new PPV strain C isolates were detected in sour cherry (Prunus cerasus) from three geographically distant (700–1100 km) regions of European Russia. Analysis of their 3’-terminal genomic sequences showed that nineteen isolates (47.5%) bear the D96E mutation in the universal epitope of the coat protein. Almost all of them cannot be detected by the monoclonal antibody 5B in triple antibody sandwich enzyme-linked immunosorbent assay and Western blot analysis that may potentially compromise serological PPV detection in cherries. Full-length genomes of seven PPV-C isolates were determined employing next-generation sequencing. Using the Recombination Detection Program (RDP4), the recombination event covering the region from (Cter)P1 to the middle of the HcPro gene was predicted in all the available PPV-C complete genomes. The isolates Tat-4, belonging to the strain CV, and RU-17sc (PPV-CR) were inferred as major and minor parents, respectively, suggesting possible pathways of evolution of the cherry-adapted strains. Downy cherry (P. tomentosa) was identified as the natural PPV-C host for the first time.

Molecular characterization of Plum pox virus Rec isolates from Russia suggests a new insight into evolution of the strain

Field isolates of Plum pox virus (PPV), belonging to the strain Rec, have been found for the first time in Russia. Full-size genomes of the isolates K28 and Kisl-1pl from myrobalan and plum, respectively, were sequenced on the 454 platform. Analysis of all known PPV-Rec complete genomes using the Recombination Detection Program (RDP4) revealed yet another recombination event in the 5′-terminal region. This event was detected by seven algorithms, implemented in the RDP4, with statistically significant P values and supported by a phylogenetic analysis with the bootstrap value of 87%. A putative PPV-M-derived segment, encompassing the C-terminus of the P1 gene and approximately two-thirds of the HcPro gene, is bordered by breakpoints at positions 760–940 and 1838–1964, depending on the recombinant isolate. The predicted 5′-distal breakpoint for the isolate Valjevka is located at position 2804. The Dideron (strain D) and SK68 (strain M) isolates were inferred as major and minor parents, respectively. Finding of another recombination event suggests more complex evolutionary history of PPV-Rec than previously assumed. Perhaps the first recombination event led to the formation of a PPV-D variant harboring the PPV-M-derived fragment within the 5′-proximal part of the genome. Subsequent recombination of its descendant with PPV-M in the 3′-proximal genomic region resulted in the emergence of the evolutionary successful strain Rec.