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Potyviruses move to neighboring cells in the form of virus particles or a coat protein (CP)-containing ribonucleoprotein complex. However, the precise roles of RNA-binding residues in potyviral CP in viral cell-to-cell movement remain to be elucidated. In this study, we predicted the three-dimensional model of tobacco vein banding mosaic virus (TVBMV)-encoded CP and found nine residues presumably located in the CP RNA-binding pocket. Substitutions of the two basic residues at positions 192 and 225 (R192 and K225) with either alanine, cysteine, or glutamic acid abolished TVBMV cell-to-cell and systemic movement in Nicotiana benthamiana plants. These substitutions also reduced the replication of the mutant viruses. Results from the electrophoretic mobility shift assay showed that the RNA-binding activity of mutant CPs derived from R192 or K225 substitutions was significantly lower than that of wild-type CP. Analysis of purified virus particles showed that mutant viruses with R192 or K225 substitutions formed RNA-free virus-like particles. Mutations of R192 and K225 did not change the CP plasmodesmata localization. The wild-type TVBMV CP could rescue the deficient cell-to-cell movement of mutant viruses. Moreover, deletion of any of the other seven residues also abolished TVBMV cell-to-cell movement and reduced the CP RNA-binding activity. The corresponding nine residues in watermelon mosaic virus CP were also found to play essential roles in virus cell-to-cell movement. In conclusion, residues R192 and K225 in the CP RNA-binding pocket are critical for viral RNA binding and affect both virus replication and cell-to-cell movement. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Key message PSV infection changed the abundance of host plant’s transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and cytosol, affecting photosynthesis, translation, transcription, and splicing. Abstract Virus infection is a process resulting in numerous molecular, cellular, and physiological changes, a wide range of which can be analyzed due to development of many high-throughput techniques. Plant RNA viruses are known to replicate in the cytoplasm; however, the roles of chloroplasts and other cellular structures in the viral replication cycle and in plant antiviral defense have been recently emphasized. Therefore, the aim of this study was to analyze the small RNAs, transcripts, proteins, and phosphoproteins affected during peanut stunt virus strain P (PSV-P)–Nicotiana benthamiana interactions with or without satellite RNA (satRNA) in the context of their cellular localization or functional connections with particular cellular compartments to elucidate the compartments most affected during pathogenesis at the early stages of infection. Moreover, the processes associated with particular cell compartments were determined. The ‘omic’ results were subjected to comparative data analyses. Transcriptomic and small RNA (sRNA)–seq data were obtained to provide new insights into PSV-P–satRNA–plant interactions, whereas previously obtained proteomic and phosphoproteomic data were used to broaden the analysis to terms associated with cellular compartments affected by virus infection. Based on the collected results, infection with PSV-P contributed to changes in the abundance of transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and the cytosol, and the most affected processes were photosynthesis, translation, transcription, and mRNA splicing. Furthermore, sRNA-seq and phosphoproteomic analyses indicated that kinase regulation resulted in decreases in phosphorylation levels. The kinases were associated with the membrane, cytoplasm, and nucleus components.

Tomato spotted wilt virus (TSWV) is economically prominent disease for its impact on tobacco (Nicotiana tabacum L.) production worldwide. An increase of the incidence of symptoms typical of TSWV has been observed in tobacco production areas in Denizli province of Turkey where tobacco is significantly grown. Surveys were conducted to determine the prevalence status of TSWV in tobacco cultivars and its possible sources of infections in four tobacco growing districts of Denizli province. A total of 501 plant samples from field-grown tobaccos, weeds, potential intermediate hosts, seedlings and seeds were collected during 2019 and tested by DAS-ELISA. Of these plants, 243 belong to 55 different weed species from 26 different families with intermediate host potential. Throughout the study, 40 crop plant samples which could be intermediate hosts and 39 tobacco seed samples were also taken for testing. Adult thrips specimens were picked up from the fields and brought to the laboratory for preparations. Four vector virus species were detected when adult thrips individuals were diagnosed: Thrips tabaci Lindeman (Thysanoptera: Thripidae), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), Aeolothrips intermedius Bagnall (Thysanoptera: Aeolothripidae) and Thrips major Uzel (Thysanoptera: Thripidae). Of the 179 tobaccos sampled, 31.2% was positive; besides, of 243 weeds tested 10 were found to be infected. Echinochloa crus-,alli and Tordylium apulum were determined to be new host recordings for TSWV infection. Only one tomato plant from the crop plants as intermediate also confirmed in tobacco fields., Aegean Exporters'Associations, This work was supported by Aegean Exporters'Associations.

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., 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 CI. 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.

In this study, we demonstrate a novel pro-viral role for the Nicotiana benthamiana ARGONAUTE 1 (AGO1) in potyvirus infection. AGO1 strongly enhanced potato virus A (PVA) particle production and benefited the infection when supplied in excess. We subsequently identified the potyviral silencing suppressor, helper-component protease (HCPro), as the recruiter of host AGO1. After the identification of a conserved AGO1-binding GW/WG motif in potyviral HCPros, we used site-directed mutagenesis to introduce a tryptophan-to-alanine change into the HCPro (HCProAG) of PVA (PVAAG) and turnip mosaic virus (TuMVAG). AGO1 co-localization and co-immunoprecipitation with PVA HCPro was significantly reduced by the mutation suggesting the interaction was compromised. Although the mutation did not interfere with HCPro’s complementation or silencing suppression capacity, it nevertheless impaired virus particle accumulation and the systemic spread of both PVA and TuMV. Furthermore, we found that the HCPro-AGO1 interaction was important for AGO1’s association with the PVA coat protein. The coat protein was also more stable in wild type PVA infection than in PVAAG infection. Based on these findings we suggest that potyviral HCPro recruits host AGO1 through its WG motif and engages AGO1 in the production of stable virus particles, which are required for an efficient systemic infection., Author summary In the course of an infection viral proteins come regularly into contact with host factors. Knowledge of these virus-host interactions in plants is essential for understanding the progress of infections and for addressing challenges in the production of healthy plants, which is important for global food security. In this study we report that the potyviral Helper component proteinase (HCPro) recruits host ARGONAUTE1 protein and engages it in pro-viral functions. We found that the interaction of potato virus A HCPro with ARGONAUTE1 benefited the infection by promoting the stability and accumulation of virus particles. When HCPro was engineered to disrupt the interaction, we detected very few virus particles and observed that the mutated virus did not spread efficiently. Thus, we propose that the interaction between HCPro and ARGONAUTE1 has biological relevance as it is important for formation of stable particles and achieving optimal systemic infection.

The interaction between the viral protein genome-linked (VPg) and eukaryotic initiation factor 4E (eIF4E) or eIF(iso)4E of the host plays a crucial role in potyvirus infection. The VPg of potato virus A (PVA) contains the Tyr-X-X-X-X-Leu-phi (YXXXL&Phi, ) binding motif for eIF(iso)4E. In order to investigate its role in PVA infection, we substituted the conserved tyrosine and leucine residues of the motif with alanine residues in the infectious cDNA of PVA (PVAVPgmut). PVAVPgmut RNA replicated in infiltrated leaves, but RNA accumulation remained low. Systemic infection occurred only if a reversion to wild type PVA occurred. VPg was able to stabilize PVA RNA and enhance the expression of Renilla luciferase (3&rsquo, RLUC) from the 3&rsquo, end of the PVA genome. VPgmut could not support either PVA RNA stabilization or enhanced 3&rsquo, RLUC expression. The RNA silencing suppressor helper-component proteinase (HCPro) is responsible for the formation of PVA-induced RNA granules (PGs) during infection. While VPgmut increased the number of PG-like foci, the percentage of PVA RNA co-localization with PGs was reduced from 86% to 20%. A testable hypothesis for future studies based on these results is that the binding of eIF(iso)4E to PVA VPg via the YXXXL&Phi, motif is required for PVA RNA stabilization, as well as the transfer to the RNA silencing suppression pathway and, further, to polysomes for viral protein synthesis.

Infection caused by the synergistic interaction of two plant viruses is typically manifested by severe symptoms and increased accumulation of either virus. In potex-potyviral synergism, the potyviral RNA silencing suppressor helper component proteinase (HCPro) is known to enhance the pathogenicity of the potexvirus counterpart. In line with this, Potato virus X (PVX; genus Potexvirus) genomic RNA (gRNA) accumulation and gene expression from subgenomic RNA (sgRNA) are increased in Nicotiana benthamiana by Potato virus A (PVA; genus Potyvirus) HCPro expression. Recently, we have demonstrated that PVA HCPro interferes with the host cell methionine cycle by interacting with its key enzymes S-adenosyl-l-methionine synthetase (SAMS) and S-adenosyl-l-homocysteine hydrolase (SAHH). To study the involvement of methionine cycle enzymes in PVX infection, we knocked down SAMS and SAHH. Increased PVX sgRNA expression between 3 and 9 days post-infiltration (dpi) and upregulation of (-)-strand gRNA accumulation at 9 dpi were observed in the SAHH-silenced background. We found that SAMS and SAHH silencing also caused a significant reduction in glutathione (GSH) concentration, specifically in PVX-infected plants between 2 and 9 dpi. Interestingly, HCPro expression in PVX-infected plants caused an even stronger reduction in GSH levels than did SAMS+SAHH silencing and a similar level of reduction was also achieved by knocking down GSH synthetase. PVX sgRNA expression was increased in the GSH synthetase-silenced background. GSH is a major antioxidant of plant cells and therefore GSH shortage may explain the strong oxidative stress and severe symptoms observed during potex-potyvirus mixed infection.

[EN] Tomato spotted wilt virus (TSWV) is worldwide spread and causes production losses in many important horticultural crops such as tomato and pepper. Breeding resistant cultivars has been the most successful method for disease control. So far, only two resistance genes have been found to confer resistance against a wide spectrum of TSWV isolates: Sw-5 in tomato and Tsw in pepper. However, TSWV resistance-breaking isolates have emerged in different countries after a few years of implementation of resistant cultivars. In this paper we report the first complete nucleotide sequence of three TSWV isolates from Spain with different biotypes according to the ability of overcoming resistance: LL-N.05 (wild type, WT), Pujol1TL3 (Sw-5 resistance breaking, SBR) and PVR (Tsw resistance-breaking, TBR). The genome of these TSWV isolates consisted of three genomic segments: L (8913-8914 nt), M (4752-4825 nt) and S (2924-2961 nt). Variations in nucleotide sequences and genomic lengths among the different virus biotypes are reported here. Phylogenetic analysis of the five TSWV open reading frames showed evidences of reassortment between genomic segments of LL-N.05 and Pujol1TL3, which was supported by analysis with different recombination-detecting algorithms. Genetic distances were uncorrelated to biotype, host or geographic location., D.E.D. was the recipient of a predoctoral FPU fellowship from the Spanish Ministry of Education, Culture and Sports. This work was funded in part by grants RTA2008-00010-C03 and RTA2013-00047-C02 from Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA) and grants ACOMP/2009/103, ACOMP/2010/085 and ACOMP/2011/078 from Generalitat Valenciana. Work in the J.A.D. laboratory was supported by grant BIO2011-26741 from the Spanish Ministerio de Economia y Competitividad. We thank Veronica Aragones (IBMCP, CSIC-UPV) for technical assistance.

In the melon exotic accession PI 161375, the gene cmv1, confers recessive resistance to Cucumber mosaic virus (CMV) strains of subgroup II. cmv1 prevents the systemic infection by restricting the virus to the bundle sheath cells and impeding viral loading to the phloem. Here we report the fine mapping and cloning of cmv1. Screening of an F2 population reduced the cmv1 region to a 132 Kb interval that includes a Vacuolar Protein Sorting 41 gene. CmVPS41 is conserved among plants, animals and yeast and is required for post-Golgi vesicle trafficking towards the vacuole. We have validated CmVPS41 as the gene responsible for the resistance, both by generating CMV susceptible transgenic melon plants, expressing the susceptible allele in the resistant cultivar and by characterizing CmVPS41 TILLING mutants with reduced susceptibility to CMV. Finally, a core collection of 52 melon accessions allowed us to identify a single amino acid substitution (L348R) as the only polymorphism associated with the resistant phenotype. CmVPS41 is the first natural recessive resistance gene found to be involved in viral transport and its cellular function suggests that CMV might use CmVPS41 for its own transport towards the phloem., The TILLING platform is supported by the Program Saclay Plant Sciences (SPS, ANR-10-LABX-40) and the European Research Council (ERC-SEXYPARTH). This work was supported by grants AGL2009-12698-C02-01 and AGL2012-40130-C02-01 from the Spanish Ministry of Science and Innovation, the Spanish Ministry of Econom and Competitiveness, through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV‐2015‐0533)” and the CERCA Programme/Generalitat de Catalunya.

BACKGROUND Polygodial, a sesquiterpene dialdehyde of the drimane family, has been shown to have deterrent and antifeedant effects on various insect species, including Myzus persicae (Sulzer), Spodoptera spp. and Leptinotarsa decemlineata (Say). This compound may have potential as a broad-spectrum biocontrol agent, similar to pyrethrins, given that it was previously reported to improve yield when sprayed on barley fields. RESULTS This study compares the deterrent effect of polygodial and pyrethrins against the silverleaf whitefly Bemisia tabaci (Gennadius) and the green peach aphid M. persicae in dual-choice assays using compound-coated tomato leaf discs. B. tabaci adults were deterred by polygodial at an ED50 (effective dose at which 50% of the insects are deterred) of about 25 µg g-1 fresh weight (FW), and green peach aphids at about 54 µg g-1 FW. Bioassays were benchmarked with pyrethrins that had a 20-fold lower ED50 of approximately 1.4 µg g-1 FW against whiteflies, but only a twofold lower ED50 (about 28 µg g-1 FW) against peach aphids. Polygodial showed moderate phytotoxic effects (score of 2 on a scale of 1–5) on tomato leaves at concentrations above the ED50 concentrations (=90 µg g-1 FW). CONCLUSION The sesquiterpene dialdehyde polygodial is 2–20 times less deterrent than pyrethrins, depending on the insect species, but it could provide a useful complement to pyrethrin sprays as it has a different mode of action, is food grade and has low volatility. However, a formulation that reduces the risks of phytotoxic effects should be developed. © 2013 Society of Chemical Industry

Barley stripe mosaic virus (BSMV) induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB) proteins were coexpressed in cells with the actin marker DsRed: Talin. TGB ectopic expression experiments revealed that TGB3 is a major elicitor of filament thickening, that TGB2 resulted in formation of intermediate DsRed:Talin filaments, and that TGB1 alone had no obvious effects on actin filament structure. Latrunculin B (LatB) treatments retarded BSMV cell-to-cell movement, disrupted actin filament organization, and dramatically decreased the proportion of paired TGB3 foci appearing at the cell wall (CW). BSMV infection of transgenic plants tagged with GFP-KDEL exhibited membrane proliferation and vesicle formation that were especially evident around the nucleus. Similar membrane proliferation occurred in plants expressing TGB2 and/or TGB3, and DsRed: Talin fluorescence in these plants colocalized with the ER vesicles. TGB3 also associated with the Golgi apparatus and overlapped with cortical vesicles appearing at the cell periphery. Brefeldin A treatments disrupted Golgi and also altered vesicles at the CW, but failed to interfere with TGB CW localization. Our results indicate that actin cytoskeleton interactions are important in BSMV cell-to-cell movement and for CW localization of TGB3.

Viral diseases are an important limiting factor in many crop production systems. Because antiviral products are not available, control strategies rely on genetic resistance or hygienic measures to prevent viral diseases, or on eradication of diseased crops to control such diseases. Increasing international travel and trade of plant materials enhances the risk of introducing new viruses and their vectors into production systems. In addition, changing climate conditions can contribute to a successful spread of newly introduced viruses or their vectors and establishment of these organisms in areas that were previously unfavorable. Tomato is economically the most important vegetable crop worldwide and many viruses infecting tomato have been described, while new viral diseases keep emerging. Pepino mosaic virus is a rapidly emerging virus which has established itself as one of the most important viral diseases in tomato production worldwide over recent years. Begomovirus species and other whitefly-transmitted viruses are invading into new areas, and several recently described new viruses such as Tomato torrado virus and new Tospovirus species are rapidly spreading over large geographic areas. In this article, emerging viruses of tomato crops are discussed.

The complete nucleotide sequence of Peanut stunt virus strain Mi (PSV-Mi) from China was determined and compared to other viruses of the genus Cucumovirus. The tripartite genome of PSV-Mi encoded five open reading frames (ORFs) typical of cucumoviruses. Distance analyses of four ORFs indicated that PSV-Mi differed sufficiently in nucleotide sequence from other PSV strains of subgroups I and II to warrant establishment of a third subgroup of PSV.

The tuber necrotic strain of Potato virus Y (PVY NTN ) causes potato tuber necrotic ringspot disease in sensitive potato cultivars. Gene expression in the disease response of the susceptible potato ( Solanum tuberosum L.) cultivar Igor was investigated at different times after infection, using subtractive hybridization, cDNA microarrays and real-time PCR. The most pronounced change in the expression pattern of functionally diverse groups of genes was detected in systemically infected leaves 14 days after inoculation, and in leaves of plants grown from infected tubers. The expression of several stress-related genes during the infection process, including those for heat shock proteins, catalase 1, β-1,3-glucanase, wound inducing gene, and genes involved in photosynthesis, suggests their role in the susceptible potato–PVY NTN interaction.

The aphid transmissibility of seven Plum pox virus (PPV) isolates and the amino acid sequences of their coat proteins were analysed Two aphid transmissible isolates PPV-A and PPV-P contained the DAG amino triplet, while DAL or NAG replaced this triplet in the coat proteins of non-aphid transmissible isolates. In addition, a sequence of 15 or 17 amino acids was deleted in the coat proteins of PPV-P48, PPV-P45 and PPV-II/7. Helper components with Mr. of 52 kDa were successfully purified from N. benthamiana plants inoculated with aphid transmissible and non-aphid transmissible isolates. The helper components of two coat-proteins-defective isolates mediated aphid transmission of non-coat-protein defective isolates, indicating that the helper components were biologically active.

BACKGROUND Polygodial, a sesquiterpene dialdehyde of the drimane family, has been shown to have deterrent and antifeedant effects on various insect species, including Myzus persicae (Sulzer), Spodoptera spp. and Leptinotarsa decemlineata (Say). This compound may have potential as a broad-spectrum biocontrol agent, similar to pyrethrins, given that it was previously reported to improve yield when sprayed on barley fields. RESULTS This study compares the deterrent effect of polygodial and pyrethrins against the silverleaf whitefly Bemisia tabaci (Gennadius) and the green peach aphid M. persicae in dual-choice assays using compound-coated tomato leaf discs. B. tabaci adults were deterred by polygodial at an ED50 (effective dose at which 50% of the insects are deterred) of about 25 µg g-1 fresh weight (FW), and green peach aphids at about 54 µg g-1 FW. Bioassays were benchmarked with pyrethrins that had a 20-fold lower ED50 of approximately 1.4 µg g-1 FW against whiteflies, but only a twofold lower ED50 (about 28 µg g-1 FW) against peach aphids. Polygodial showed moderate phytotoxic effects (score of 2 on a scale of 1–5) on tomato leaves at concentrations above the ED50 concentrations (=90 µg g-1 FW). CONCLUSION The sesquiterpene dialdehyde polygodial is 2–20 times less deterrent than pyrethrins, depending on the insect species, but it could provide a useful complement to pyrethrin sprays as it has a different mode of action, is food grade and has low volatility. However, a formulation that reduces the risks of phytotoxic effects should be developed. © 2013 Society of Chemical Industry

International audience; Obtaining pure virus suspensions is an essential step in many applications, such as vaccine production, antibody production, sample preparation for procedures requiring enrichment in viruses and other in vitro characterizations. Purification procedures usually consist of complex, long lasting and tedious protocols involving several ultracentrifugation steps. Such complexity is particularly evident in the case of plant viruses, where the virus needs to be isolated from the complex plant tissue matrix. Convective Interaction Media (CIM) monoliths are chromatographic supports that have been successfully utilized for the purification of large bio-molecules such as viruses, virus like particles and plasmids from various matrixes. In this study a CIM monolith based procedure was developed for the fast purification from plant tissue of the filamentous Potato virus Y(PVY) (virion size, 740 nm x 11 nm), which is one of the most important plant viruses causing great economical losses in potato production. Different mobile phases, chemistries and sample preparation strategies were tested. The presence of the virus in the chromatographic fraction was monitored with viral RNA quantification (RT-qPCR), viral protein purity estimation (SDS-PAGE) and viral particle integrity observation (transmission electron microscopy). The optimized procedure involves initial clarification steps, followed by chromatography using CIM quaternary amine (QA) monolithic disk column. In comparison to classical purification procedure involving ultracentrifugation through sucrose and caesium chloride, the developed CIM-QA purification achieved comparable yield, concentration and purity. Plant nucleic acids were successfully removed. Purification showed good reproducibility and moreover it reduced the purification time from four working days required for classic purification to a day and a half. This is the first study where a filamentous virus was purified using CIM monolithic supports. The advantages of this new purification procedure make it an attractive method in serological diagnostic tool production, which requires purified viruses for the immunization step. Moreover, the outcome of this study could serve as starting point for the improvement of the purification methods of other important filamentous viruses. (C) 2012 Elsevier B.V. All rights reserved.

International audience; Miscanthus is a perennial C4-grass that has received much interest as a potential biomass crop in Europe. However, little is known about the consequences of its introduction in terms of impact on the local agroecosytem. In this context, laboratory experiments were conducted to investigate the potential colonization of this new exotic plant species by three of the main aphid pest species of common crops in Picardie, northern France. In host preference experiments, the two polyphagous aphid species studied, Aphis fabae (Scop) and Myzus persicae (Sulzer), exhibited an exclusive preference for their host plant, whereas the cereal specialist Rhopalosiphum padi (L.) showed no preference between its host plant and miscanthus. When assessed by electrical penetration graph technique, plant tissue probing activity by all three species always was characterized by pathway phases including potential drops that are typically associated to the transmission of noncirculative viruses. Phloem ingestion was observed in 5% of the polyphagous aphid individuals tested and in 20% of the R. padi tested. Aphids kept in clip-cages on miscanthus had a low survival rate and were unable to reproduce. These results demonstrate that miscanthus is not a suitable host for these three main aphid pest species but could act as a potential host for some viruses transmitted in a noncirculative manner and also in a circulative nonpropagative manner. The use of miscanthus as a barrier crop to limit the flow of aphid vectors and their phytoviruses is discussed.

Transmission of Olive mild mosaic virus (OMMV) is facilitated by Olpidium brassicae (Wor.) Dang. An OMMV mutant (OMMVL11) containing two changes in the coat protein (CP), asparagine to tyrosine at position 189 and alanine to threonine at position 216, has been shown not to be Olpidium brassicae-transmissible owing to inefficient attachment of virions to zoospores. In this study, these amino acid changes were separately introduced into the OMMV genome through site-directed mutagenesis, and the asparagine-to-tyrosine change was shown to be largely responsible for the loss of transmission. Analysis of the structure of OMMV CP by comparative modelling approaches showed that this change is located in the interior of the virus particle and the alanine-to-threonine change is exposed on the surface. The asparagine-to-tyrosine change may indirectly affect attachment via changes in the conformation of viral CP subunits, altering the receptor binding site and thus preventing binding to the fungal zoospore.

Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5′-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene., Author Summary Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is an interesting subviral RNA because it changes the green mosaic induced by CMV into a bright yellow mosaic in Nicotiana tabacum. The molecular basis underlying the induction of symptoms by viruses is not well understood, and this Y-sat-mediated modification of symptoms has been a long-standing mystery. In this study, we discovered the molecular mechanism involved in the Y-sat-induced yellowing. First, we showed that transgenic N. benthamiana plants that expressed the inverted-repeat sequence of Y-sat also developed a yellow phenotype, similar to the Y-sat-infected plants. Then, we found that tobacco magnesium protoporphyrin chelatase subunit I gene (ChlI, the key gene involved in chlorophyll synthesis) was downregulated in the transgenic plants and in the Y-sat-infected plants. We then identified a 22-nt long sequence that is complementary to the Y-sat including four G-U pairs in the ChlI mRNA. Finally, we demonstrated that a short interfering RNA (siRNA) derived from Y-sat specifically targeted and downregulated the ChlI mRNA, thus impairing the chlorophyll biosynthesis pathway. This discovery of the molecular basis of the symptom modification induced by Y-sat is the first demonstration that a subviral RNA can induce disease symptoms by regulating host gene expression through the RNA silencing machinery.

Soybean dwarf virus (SbDV), first identified as an agricultural problem in Japan, has emerged as a growing problem in the Midwestern United States. The majority of research on SbDV had been limited to four lab maintained strains from Japan. SbDV had been found in clover in the eastern United States, but these isolates rarely emerged into soybeans. These isolates were analyzed by multiplex PCR and sequencing, revealing that some were infections of both Y and D components, including a recombinant subisolate. Phylogenetic analyses for the US isolates revealed a broad diversity of SbDV, with selection pressure greater on the movement protein than the coat protein. The field isolates from the Eastern United States showed differences in symptoms, aphid transmission and host range, demonstrating that a study of field isolates is an important complement to laboratory maintained strains in understanding the biology and evolution of plant viruses. Published by Elsevier Inc. Virology

International audience; Control of grapevine virus and phytoplasma diseases is currently based on prophylactic measures and cultural practices. Certification programs aim to avoid the introduction of diseased grapevines into healthy vineyards, and cultural practices aim to reduce the populations of virus vectors to limit virus spread. These approaches however are of limited effectiveness. Additionally, there is no host resistance to viruses in grapevine identified so far. Genetic engineering provides new approaches to develop pathogen-resistant grapevines. Transgenic grapevines expressing virus-derived genes that can confer resistance have been obtained in several laboratories. The viral coat protein gene has been the most commonly used to engineer resistance. More recently, new approaches based on gene silencing, specifically triggering plant defence mechanisms, have opened new ways to engineer pathogen resistance in grapevines. The possibilities, benefits and advantages, but also the risks involved with the introduction of transgenic grapevines in the field, as well as their acceptability, are discussed in this review.

Virus-like particles, 27 nm in diameter, were observed in extracts of individual Varroa destructor mites and in sections of mite tissue. Application of a purification procedure resulted in virus preparations that were used to prepare an antiserum to detect the virus in individual mites. Immunohistology studies showed that the gastric caecae were heavily infected, whereas no immunostaining could be detected in other mite tissues or organs, like the salivary glands, brain, rectum or reproductive organs. By electron microscopy large aggregates of virus-like particles in para-crystalline lattices were found in cells of the gastric caecae. The particles, reminiscent to picorna-like viruses, occurred mainly in the cytoplasm, whereas some virus particles were sparsely scattered in vacuoles. Occasionally, particles were observed in membrane-bound vesicles or in long tubular membrane structures in the cytoplasm. The accumulation of the picorna-like virus particles in the cytoplasm and the presence of the virus in membrane structures give a strong indication that the virus replicates in the mite.

2 pages., The 15 articles included in this special issue of Virus Research were based on material presented at the Xth International Plant Virus Epidemiology Symposium

Tomato Yellow Leaf Curl Virus Disease incited by Tomato yellow leaf curl virus (TYLCV) causes huge losses in tomato production worldwide and is caused by different related begomovirus species. Breeding for TYLCV resistance has been based on the introgression of multiple resistance genes originating from several wild tomato species. In this study we have fine-mapped the widely used Solanum chilense–derived Ty-1 and Ty-3 genes by screening nearly 12,000 plants for recombination events and generating recombinant inbred lines. Multiple molecular markers were developed and used in combination with disease tests to fine-map the genes to a small genomic region (approximately 70 kb). Using a Tobacco Rattle Virus–Virus Induced Gene Silencing approach, the resistance gene was identified. It is shown that Ty-1 and Ty-3 are allelic and that they code for a RNA–dependent RNA polymerase (RDR) belonging to the RDRγ type, which has an atypical DFDGD motif in the catalytic domain. In contrast to the RDRα type, characterized by a catalytic DLDGD motif, no clear function has yet been described for the RDRγ type, and thus the Ty-1/Ty-3 gene unveils a completely new class of resistance gene. Although speculative, the resistance mechanism of Ty-1/Ty-3 and its specificity towards TYLCV are discussed in light of the function of the related RDRα class in the amplification of the RNAi response in plants and transcriptional silencing of geminiviruses in plants., Author Summary Tomato yellow leaf curl virus and related begomoviruses cause major economic damage to tomato production in tropical and subtropical regions around the world. Because cultivated tomato is inherently susceptible to these viruses, breeders have incorporated several resistance alleles from wild tomato relatives. Among these are the commercially important alleles, Ty-1 and Ty-3, which were introgressed from the wild tomato relative Solanum chilense. These genes were originally mapped to different regions on chromosome 6, but recent findings suggest they may rather be alleles of the same gene. Here, we describe the precise mapping of Ty-1 and Ty-3 to a common chromosomal region, and we show that Ty-1 and Ty-3 are alleles that code for an RNA–dependent RNA polymerase of a class for which no function had been described before. Thus, Ty-1/Ty-3 unveils a completely new class of resistance genes. These results will be useful to breeders who utilize these genes in their breeding programs, and further studies should shed new light on the mechanism by which this gene functions.

Kiwifruit (Actinidia deliciosa) was introduced to New Zealand more than one hundred years ago and the New Zealand-raised cv. Hayward is now the dominant cultivar grown worldwide. Further accessions of kiwifruit seed and scionwood have been sourced from China for research and breeding. In one importation consignment, the first virus naturally infecting kiwifruit, Apple stem grooving virus (ASGV), was identified following symptoms observed in quarantined plants (2003). Since that time a further 12 viruses have been identified in kiwifruit. We classify these 13 viruses into three groups. The first group comprises the non-specialist viruses and includes Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV) both members of the family Bromoviridae. The group also includes a further five viruses that appear to have limited effect on kiwifruit: two tobamoviruses, Ribgrass mosaic virus (RMV) and Turnip vein clearing virus (TVCV); a tombusvirus, Cucumber necrosis virus (CNV); a novel potexvirus; and Apple stem gooving virus (ASGV, genus Capillovirus). Most of the viruses classified in this first group are cosmopolitan and sometimes orchard weeds provide reservoirs for infection. The second group comprises the kiwifruit-adapted viruses. This group includes three novel viruses. i.e. two vitiviruses, Actinidia virus A (AcVA) and Actinidia virus B (AcVB), and a citrivirus closely related to Citrus leaf blotch virus (CLBV). In addition, preliminary evidence of a novel virus belonging to the Closteroviridae family has been obtained. The third group of viruses induces disease in kiwifruit. To date only two viruses have caused significant damage to kiwifruit within commercial orchards. In New Zealand, Cherry leaf roll virus (CLRV) has been detected on kiwifruit associated with symptoms including leaf spots, fruit malformation, reduction in yield, bark cracking and cane wilting. Pelargonium zonate spot virus (PZSV) has been detected in Italy associated with severe symptoms on leaves and fruit.