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Viruses rely completely on host translational machinery to produce the proteins encoded by their genes. Controlling translation initiation is important for gaining translational advantage in conflicts between the host and virus. The eukaryotic translation initiation factor 4E (eIF4E) has been reported to be hijacked by potyviruses for virus multiplication. The role of translation regulation in defence and anti‐defence between plants and viruses is not well understood. We report that the transcript level of eIF6 was markedly increased in turnip mosaic virus (TuMV)‐infected Nicotiana benthamiana. TuMV infection was impaired by overexpression of N. benthamiana eIF6 (NbeIF6) either transiently expressed in leaves or stably expressed in transgenic plants. Polysome profile assays showed that overexpression of NbeIF6 caused the accumulation of 40S and 60S ribosomal subunits, the reduction of polysomes, and also compromised TuMV UTR‐mediated translation, indicating a defence role for upregulated NbeIF6 during TuMV infection. However, the polysome profile in TuMV‐infected leaves was not identical to that in leaves overexpressing NbeIF6. Further analysis showed that TuMV NIb protein, the RNA‐dependent RNA polymerase, interacted with NbeIF6 and interfered with its effect on the ribosomal subunits, suggesting that NIb might have a counterdefence role. The results propose a possible regulatory mechanism at the translation level during plant–virus interaction. [ABSTRACT FROM AUTHOR]

Zucchini yellow mosaic virus (ZYMV) seriously damages cucurbits worldwide. Control of ZYMV by cross‐protection has been practised for decades, but selecting useful mild viruses is time‐consuming and laborious. Most attenuated potyviruses used for cross‐protection do not induce hypersensitive reaction (HR) in Chenopodium quinoa, a local lesion host Chenopodium quinoa. Here, severe ZYMV TW‐TN3 tagged with green fluorescent protein (GFP), designated ZG, was used for nitrous acid mutagenesis. From three trials, 11 mutants were identified from fluorescent spots without HR in inoculated C. quinoa leaves. Five mutants caused attenuated symptoms in squash plants. The genomic sequences of these five mutants revealed that most of the nonsynonymous changes were located in the HC‐Pro gene. The replacement of individual mutated HC‐Pros in the ZG backbone and an RNA silencing suppression (RSS) assay indicated that each mutated HC‐Pro is defective in RSS function and responsible for reduced virulence. Four mutants provided high degrees of protection (84%–100%) against severe virus TW‐TN3 in zucchini squash plants, with ZG 4‐10 being selected for removal of the GFP tag. After removal of the GFP gene, Z 4‐10 induced symptoms similar to ZG 4‐10 and still provided 100% protection against TW‐TN3 in squash, thus is considered not a genetically engineered mutant. Therefore, using a GFP reporter to select non‐HR mutants of ZYMV from C. quinoa leaves is an efficient way to obtain beneficial mild viruses for cross‐protection. This novel approach is being applied to other potyviruses. [ABSTRACT FROM AUTHOR]

Control of plant viruses by cross‐protection is limited by the availability of effective protective strains. Incorporation of an NIa‐protease processing site in the extreme N‐terminal region of the helper component protease (HC‐Pro) of turnip mosaic virus (TuMV) resulted in a mutant virus TuHNDI that induced highly attenuated symptoms. Recombination analysis verified that two variations, F7I mutation and amino acid 7‐upstream‐deletion, in HC‐Pro co‐determined TuHNDI attenuation. TuHNDI provided complete protection to Nicotiana benthamiana and Brassica campestris subsp. chinensis plants against infection by the severe parental strain. Aphid transmission tests revealed that TuHNDI was not aphid‐transmissible. An RNA silencing suppression (RSS) assay by agroinfiltration suggested the RSS‐defective nature of the mutant HC‐Pro. In the context (amino acids 3–17) encompassing the two variations of HC‐Pro, we uncovered an FWKG−α‐helix 1 (αH1) element that influenced the functions of aphid transmission and RSS, whose motifs were located far downstream. We further demonstrated that HC‐Pro F7 was a critical residue on αH1 for HC‐Pro functions and that reinstating αH1 in the RSS‐defective HC‐Pro of TuHNDI restored the protein's RSS function. Yeast two‐hybrid and bimolecular fluorescence complementation assays indicated the FWKG−αH1 element as an integral part of the HC‐Pro self‐interaction domain. The possibility of regulation of the mechanistically independent functions of RSS and aphid transmission by the FWKG−αH1 element is discussed. Extension of TuMV HC‐Pro FWKG−αH1 variations to another potyvirus, zucchini yellow mosaic virus, also generated nonaphid‐transmissible cross‐protective mutant viruses. Hence, the modification of the FWKG−αH1 element can generate effective attenuated viruses for the control of potyviruses by cross‐protection. [ABSTRACT FROM AUTHOR]

Summary: One up‐regulated host gene identified previously was found involved in the infection process of Bamboo mosaic virus (BaMV), a single‐stranded positive‐sense RNA virus. The full length cDNA of this gene was cloned by 5′ and 3′‐rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved really interesting new gene (RING) domain and a transmembrane domain. The gene might function as an ubiquitin E3 ligase. We designated this protein in Nicotiana benthamiana as ubiquitin E3 ligase containing RING domain 1 (NbUbE3R1). Further characterization by using Tobacco rattle virus‐based virus‐induced gene silencing (loss‐of‐function) revealed that increased BaMV accumulation was in both knockdown plants and protoplasts. The gene might have a defensive role in the replication step of BaMV infection. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed in N. benthamiana plants. The wild‐type NbUbE3R1‐orange fluorescent protein (NbUbE3R1‐OFP), NbUbE3R1/△TM‐OFP (removal of the transmembrane domain) and NbUbE3R1/mRING‐OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting the accumulation of BaMV. The common feature of these constructs was the intact substrate‐interacting domain. Yeast two‐hybrid and co‐immunoprecipitation experiments used to determine the possible viral‐encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an up‐regulated gene, NbUbE3R1 that plays a role in BaMV replication. [ABSTRACT FROM AUTHOR]

Summary: RNA viruses have very compact genomes and so provide a unique opportunity to study how evolution works to optimize the use of very limited genomic information. A widespread viral strategy to solve this issue concerning the coding space relies on the expression of proteins with multiple functions. Members of the family Potyviridae, the most abundant group of RNA viruses in plants, offer several attractive examples of viral factors which play roles in diverse infection‐related pathways. The Helper Component Proteinase (HCPro) is an essential and well‐characterized multitasking protein for which at least three independent functions have been described: (i) viral plant‐to‐plant transmission; (ii) polyprotein maturation; and (iii) RNA silencing suppression. Moreover, multitudes of host factors have been found to interact with HCPro. Intriguingly, most of these partners have not been ascribed to any of the HCPro roles during the infectious cycle, supporting the idea that this protein might play even more roles than those already established. In this comprehensive review, we attempt to summarize our current knowledge about HCPro and its already attributed and putative novel roles, and to discuss the similarities and differences regarding this factor in members of this important viral family. [ABSTRACT FROM AUTHOR]

Localized expression of genes in plants from T- DNAs delivered into plant cells by Agrobacterium tumefaciens is an important tool in plant research. The technique, known as agroinfiltration, provides fast, efficient ways to transiently express or silence a desired gene without resorting to the time-consuming, challenging stable transformation of the host, the use of less efficient means of delivery, such as bombardment, or the use of viral vectors, which multiply and spread within the host causing physiological alterations themselves. A drawback of the agroinfiltration technique is its temperature dependence: early studies have shown that temperatures above 29 °C are nonpermissive to tumour induction by the bacterium as a result of failure in pilus formation. However, research in plant sciences is interested in studying processes at these temperatures, above the 25 °C experimental standard, common to many host-environment and host-pathogen interactions in nature, and agroinfiltration is an excellent tool for this purpose. Here, we measured the efficiency of agroinfiltration for the expression of reporter genes in plants from T- DNAs at the nonpermissive temperature of 30 °C, either transiently or as part of viral amplicons, and envisaged procedures that allow and optimize its use for gene expression at this temperature. We applied this technical advance to assess the performance at 30 °C of two viral suppressors of silencing in agropatch assays [ Potato virus Y helper component proteinase ( HCPro) and Cucumber mosaic virus 2b protein] and, within the context of infection by a Potato virus X ( PVX) vector, also assessed indirectly their effect on the overall response of the host Nicotiana benthamiana to the virus. [ABSTRACT FROM AUTHOR]

The development and use of cultivars that are genetically resistant to viruses is an efficient strategy to tackle the problems of virus diseases. Over the past two decades, the model plant Arabidopsis thaliana has been documented as a host for a broad range of viral species, providing access to a large panel of resources and tools for the study of viral infection processes and resistance mechanisms. Exploration of its natural genetic diversity has revealed a wide range of genes conferring virus resistance. The molecular characterization of some of these genes has unveiled resistance mechanisms distinct from those described in crops. In these respects, Arabidopsis represents a rich and largely untapped source of new genes and mechanisms involved in virus resistance. Here, we review the current status of our knowledge concerning natural virus resistance in Arabidopsis. We also address the impact of environmental conditions on Arabidopsis-virus interactions and resistance mechanisms, and discuss the potential of applying the knowledge gained from the study of Arabidopsis natural diversity for crop improvement. [ABSTRACT FROM AUTHOR]

Sharka disease, caused by Plum pox virus ( PPV), is the most important viral disease affecting Prunus species. A major PPV resistance locus ( PPVres) has been mapped to the upper part of apricot ( Prunus armeniaca) linkage group 1. In this study, a physical map of the PPVres locus in the PPV-resistant cultivar 'Goldrich' was constructed. Bacterial artificial chromosome ( BAC) clones belonging to the resistant haplotype contig were sequenced using 454/ GS-FLX Titanium technology. Concurrently, the whole genome of seven apricot varieties (three PPV-resistant and four PPV-susceptible) and two PPV-susceptible apricot relatives ( P. sibirica var. davidiana and P. mume) were obtained using the Illumina- Hi Seq2000 platform. Single nucleotide polymorphisms ( SNPs) within the mapped interval, recorded from alignments against the peach genome, allowed us to narrow down the PPVres locus to a region of ∼196 kb. Searches for polymorphisms linked in coupling with the resistance led to the identification of 68 variants within 23 predicted transcripts according to peach genome annotation. Candidate resistance genes were ranked combining data from variant calling and predicted functions inferred from sequence homology. Together, the results suggest that members of a cluster of meprin and TRAF-C homology domain ( MATHd)-containing proteins are the most likely candidate genes for PPV resistance in apricot. Interestingly, MATHd proteins are hypothesized to control long-distance movement ( LDM) of potyviruses in Arabidopsis, and restriction for LDM is also a major component of PPV resistance in apricot. Although the PPV resistance gene(s) remains to be unambiguously identified, these results pave the way to the determination of the underlying mechanism and to the development of more accurate breeding strategies. [ABSTRACT FROM AUTHOR]

In the Potyvirus genus, the P1 protein is the first N-terminal product processed from the viral polyprotein, followed by the helper-component proteinase ( HCPro). In silencing suppression patch assays, we found that Potato virus Y ( PVY) HCPro expressed from a P1- HCPro sequence increased the accumulation of a reporter gene, whereas protein expressed from an HCPro sequence did not, even with P1 supplied in trans. This enhancing effect of P1 has been noted in other potyviruses, but has remained unexplained. We analysed the accumulation of PVY HCPro in infiltrated tissues and found that it was higher when expressed from P1- HCPro than from HCPro sequences. Co-expression of heterologous suppressors increased the steady-state level of mRNA expressed from the HCPro sequence, but not that of protein. This suggests that, in the absence of P1 upstream, either HCPro acquires a conformation that affects negatively its activity or stability, or that its translation is reduced. To test these options, we purified HCPro expressed in the presence or absence of upstream P1, and found no difference in purification pattern and final soluble state. By contrast, alteration of the Kozak context in the HCPro mRNA sequence to favour translation increased partially suppressor accumulation and activity. Furthermore, protein activity was not lower than in protein expressed from P1- HCPro sequences. Thus, a direct role for P1 on HCPro suppressor activity or stability, by influencing its conformation during translation, can be excluded. However, P1 could still have an indirect effect favouring HCPro accumulation. Our data highlight the relevance of cis-acting translational elements in the heterologous expression of HCPro. [ABSTRACT FROM AUTHOR]

SUMMARY Two tobacco vein necrosis (TVN) determinants, the residues K400 and E419, have been identified previously in the helper component-protease (HC-Pro) protein sequence of Potato virus Y (PVY). However, since their description, non-necrotic PVY isolates with both K400 and E419 necrotic determinants have been reported in the literature. This suggests the presence in the viral genome of other, as yet uncharacterized, TVN determinant(s). The identification of PVYN pathogenicity determinants was approached through the replacement of genomic regions of the necrotic PVYN-605 infectious clone by corresponding sequences from the non-necrotic PVYO-139 isolate. Series of PVYN/O chimeras and site-directed PVY mutants were constructed to test the involvement of different parts of the PVY genome (from nucleotide 421 to nucleotide 9629) in the induction of TVN symptoms. The analysis of both the genomic characteristics and biological properties of these mutants made it possible to highlight the involvement, in addition to residues K400 and E419, of the residue N339 of the HC-Pro protein and two regions in the cytoplasmic inclusion (CI) protein to nuclear inclusion protein a-protease (NIa-Pro) sequence (nucleotides 5496-5932 and 6233-6444) in the induction of vein necrosis in tobacco infected by PVY isolates. [ABSTRACT FROM AUTHOR]

Tomato leaf curl New Delhi virus (ToLCNDV) infection causes significant yield loss in tomato. The availability of a conventional tolerance source against this virus is limited in tomato. To understand the molecular mechanism of virus tolerance in tomato, the abundance of viral genomic replicative intermediate molecules and virus-directed short interfering RNAs (siRNAs) by the host plant in a naturally tolerant cultivar H-88-78-1 and a susceptible cultivar Punjab Chhuhara at different time points after agroinfection was studied. We report that less abundance of viral replicative intermediate in the tolerant cultivar may have a correlation with a relatively higher accumulation of virus-specific siRNAs. To study defence-related host gene expression in response to ToLCNDV infection, the suppression subtractive hybridization technique was used. A library was prepared from tolerant cultivar H-88-78-1 between ToLCNDV-inoculated and Agrobacterium mock-inoculated plants of this cultivar at 21 days post-inoculation (dpi). A total of 106 nonredundant transcripts was identified and classified into 12 different categories according to their putative functions. By reverse Northern analysis and quantitative real-time polymerase chain reaction (qRT-PCR), we identified the differential expression pattern of 106 transcripts, 34 of which were up-regulated (>2.5-fold induction). Of these, eight transcripts showed more than four fold induction. qRT-PCR analysis was carried out to obtain comparative expression profiling of these eight transcripts between Punjab Chhuhara and H-88-78-1 on ToLCNDV infection. The expression patterns of these transcripts showed a significant increase in differential expression in the tolerant cultivar, mostly at 14 and 21 dpi, in comparison with that in the susceptible cultivar, as analysed by qRT-PCR. The probable direct and indirect relationship of siRNA accumulation and up-regulated transcripts with the ToLCNDV tolerance mechanism is discussed. [ABSTRACT FROM AUTHOR]

The ubiquitin/26S proteasome system (UPS) plays a central role in plant protein degradation. Over the past few years, the importance of this pathway in plant–pathogen interactions has been increasingly highlighted. UPS is involved in almost every step of the defence mechanisms in plants, regardless of the type of pathogen. In addition to its proteolytic activities, UPS, through its 20S RNase activity, may be part of a still unknown antiviral defence pathway. Strikingly, UPS is not only a weapon used by plants to defend themselves, but also a target for some pathogens that have evolved mechanisms to inhibit and/or use this system for their own purposes. This article attempts to summarize the current knowledge on UPS involvement in plant–microbe interactions, a complex scheme that illustrates the never-ending arms race between hosts and microbes. [ABSTRACT FROM AUTHOR]

Soybean mosaic virus (SMV), a member of the genus Potyvirus, is transmitted by aphids in a non-persistent manner. It has been well documented that the helper component-proteinase (HC-Pro) plays a role as a ‘bridge’ between virion particles and aphid stylets in the aphid transmission of potyviruses. Several motifs, including the KITC and PTK motifs on HC-Pro and the DAG motif on the coat protein (CP), have been found to be involved in aphid transmission. Previously, we have shown strong interaction between SMV CP and HC-Pro in a yeast two-hybrid system (YTHS). In this report, we further analysed this CP–HC-Pro interaction based on YTHS and an in vivo binding assay to identify crucial amino acid residues for this interaction. Through this genetic approach, we identified two additional amino acid residues (H256 on CP and R455 on HC-Pro), as well as G12 on the DAG motif, crucial for the CP–HC-Pro interaction. We introduced mutations into the identified residues using an SMV infectious clone and showed that these mutations affected the efficiency of aphid transmission of SMV. We also investigated the involvement of the PTK and DAG motifs in the CP–HC-Pro interaction and aphid transmission of SMV. Our results support the concept that physical interaction between CP and HC-Pro is important for potyviral aphid transmission. Based on the combination of our current results with previous findings, the possibility that aphid transmission may be regulated by more complex molecular interactions than the simple involvement of HC-Pro as a bridge is discussed. [ABSTRACT FROM AUTHOR]

Taxonomy: Lettuce mosaic virus (LMV) belongs to the genus Potyvirus (type species Potato virus Y) in the family Potyviridae. Physical properties: The virion is filamentous, flexuous with a length of 750 nm and a width of 15 nm. The particles are made of a genomic RNA of 10 080 nucleotides, covalently linked to a viral-encoded protein (the VPg) at the 5′ end and with a 3′ poly A tail, and encapsidated in a single type of capsid protein. The molecular weight of the capsid protein subunit has been estimated electrophoretically to be 34 kDa and estimated from the amino acid sequence to be 31 kDa. Genome organization: The genome is expressed as a polyprotein of 3255 amino-acid residues, processed by three virus-specific proteinases into ten mature proteins. Hosts: LMV has a worldwide distribution and a relatively broad host range among several families. Weeds and ornamentals can act as local reservoirs for lettuce crops. In particular, many species within the family Asteraceae are susceptible to LMV, including cultivated and ornamental species such as common ( Lactuca sativa), prickly ( L. serriola) or wild ( L. virosa) lettuce, endive/escarole ( Cichorium endiva), safflower ( Carthamus tinctorius), starthistle ( Centaurea solstitialis), Cape daisy ( Osteospermum spp.) and gazania ( Gazania rigens). In addition, several species within the families Brassicaceae, Cucurbitaceae, Fabaceae, Solanaceae and Chenopodiaceae are natural or experimental hosts of LMV. Genetic control of resistance to LMV: The only resistance genes currently used to protect lettuce crops worldwide are the recessive genes mo11 and mo12 corresponding to mutant alleles of the gene encoding the translation initiation factor eIF4E in lettuce. It is believed that at least one intact copy of eIF4E must be present to ensure virus accumulation. Transmission: LMV is transmitted in a non-persistent manner by a high number of aphid species. Myzus persicae and Macrosiphum euphorbiae are particularly active in disseminating this virus in the fields. LMV is also seedborne in lettuce. The effectiveness of LMV transmission depends on the cultivar and the age of the seed carrier at the inoculation time. Symptoms: The characteristic symptoms on susceptible lettuce cultivars are dwarfism, mosaic, distortion and yellowing of the leaves with sometimes a much reduced heart of lettuce (failure to form heads). The differences in virus strains, cultivars and the physiological stage of the host at the moment of the attack cause different symptom severity: from a very slight discoloration of the veins to severe necrosis leading to the death of the plant. [ABSTRACT FROM AUTHOR]

Taxonomy: Zucchini yellow mosaic virus (ZYMV) is a member of genus Potyvirus, family Potyviridae. ZYMV is a positive-strand RNA virus. Physical properties: Virions are flexuous filaments of 680–730 nm in length and 11–13 nm in diameter, composed of about 2000 subunits of a single 31-kDa protein (calculated). The genome RNA size is 9.6 kb covalently linked to a viral-encoded protein (the VPg) at the 5′ end, and with a 3′ poly A tail. The 5′ end of the sequence is AU-rich (69%). Viral proteins: The genome is expressed as a polyprotein cleaved by three viral proteases and processed into ten putative mature proteins. The structural coat protein is processed from the carboxyl terminus of the polyprotein and is highly immunogenic. Host and symptoms: Natural and experimental infection has been reported mainly in the Cucurbitaceae. Experimental local lesion hosts include Chenopodium amaranticolour, C. quinoa and Gomphrena globosa. Some ZYMV strains cause symptomless infection as in Ranunculus sardous, Nicotiana benthamiana and Sesamum indicum. ZYMV causes stunting and major foliar deformation with dark green blisters and mosaics in cucurbit hosts, eventually developing a filamentous leaf phenotype. In general, symptoms are severe on cucurbit hosts and cause dramatic reductions in yields due to severe fruit deformation. The virus is present in all the plant tissues at relatively high concentrations ( c. 0.1 mg/mL of purified virus per 1 g fresh leaf tissue). The most suitable species for maintenance and purification is Cucurbita pepo. Transmission: ZYMV is efficiently transmitted by aphids in a non-persistent manner. The coat protein (CP) and the helper component-protease (HC-Pro) are required for aphid transmission, through the CP DAG motif and the HC-Pro KLSC and PTK motifs. Mechanical transmission is efficient both in the laboratory and naturally. Economic importance: ZYMV disease is a major constraint in the production of cucurbits world-wide. The virus can cause massive damage (to total loss) to cucurbit crops, and prevents the growth of some cucurbit crops in certain areas. Control of ZYMV requires the integration of conventional resistance and transgenic breeding along with cross-protection technologies. [ABSTRACT FROM AUTHOR]

Taxonomy: Black currant reversion virus (BRV) is the first identified mite-transmitted member of the genus Nepovirus (family Comoviridae). A few systematic studies have been performed to compare virus isolates from different geographical locations. Physical properties: Purified preparations contain two closely sedimenting centrifugal components (B and M for RNA1 and RNA2, respectively) at varying ratios, and occasionally a T component (for satellite RNA). The BRV capsids have a diameter of 27 nm and they are putatively composed of 60 copies of a single species of capsid (coat) protein assembled in an icosahedral lattice. Diluted plant sap loses its infectivity within 1 day at 20 °C and in 4-8 days at 4 °C. Hosts: The natural host range of BRV is limited; it infects black currant ( Ribes nigrum L.) and some related Ribes species. The transmission of the virus is by the eriophyid gall mite of black currant ( Cecidophyopsis ribis). A number of herbaceous plants can be infected experimentally. BRV is the agent of black currant reversion disease (BRD), which is economically the most significant virus disease in Ribes species. BRV and BRD occur widely in locations where black currant is cultivated commercially. [ABSTRACT FROM AUTHOR]