Pea Enation Mosaic Virus Research Articles

Identification of a New Enamovirus Associated with Citrus Vein Enation Disease by Deep Sequencing of Small RNAs

To identify the causal agent of citrus vein enation disease, we examined by deep sequencing (Solexa-Illumina) the small RNA (sRNA) fraction from infected and healthy Etrog citron plants. Our results showed that virus-derived sRNAs (vsRNAs): (i) represent about 14.21% of the total sRNA population, (ii) are predominantly of 21 and 24 nucleotides with a biased distribution of their 5' nucleotide and with a clear prevalence of those of (+) polarity, and (iii) derive from all the viral genome, although a prominent hotspot is present at a 5'-proximal region. Contigs assembled from vsRNAs showed similarity with luteovirus sequences, particularly with Pea enation mosaic virus, the type member of the genus Enamovirus. The genomic RNA (gRNA) sequence of a new virus, provisionally named Citrus vein enation virus (CVEV), was completed and characterized. The CVEV gRNA was found to be single-stranded, positive-sense, with a size of 5,983 nucleotides and five open reading frames. Phylogenetic comparisons based on amino acid signatures of the RNA polymerase and the coat protein clearly classifies CVEV within the genus Enamovirus. Dot-blot hybridization and reverse transcription-polymerase chain reaction tests were developed to detect CVEV in plants affected by vein enation disease. CVEV detection by these methods has already been adopted for use in the Spanish citrus quarantine, sanitation, and certification programs.

The kissing-loop T-shaped structure translational enhancer of Pea enation mosaic virus can bind simultaneously to ribosomes and a 5' proximal hairpin.

The Pea enation mosaic virus (PEMV) 3' translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5'-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5' and 3' PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3' translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2'-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5' hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5' end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation.

Reactive oxygen and nitrogen species and hormone signalling in systemic infection of pea by Pea enation mosaic virus

The physiological responses of pea plants during 40 days of compatible interaction with Pea enation mosaic virus (PEMV) were evaluated. PEMV induces systemic changes in the concentration of phytohormones. At 5 days post inoculation (dpi), a simultaneous increase in abscisic acid (ABA) level and a decrease in salicylic acid (SA) level was observed, which is known to be involved in the suppression of hypersensitive reaction. In our pathosystem it preceded the virus presence in the systemic leaves. PEMV induces the accumulation of ABA, SA and Hsp70 and enhances peroxidase (POX) activity at 15 dpi, when it is already transmitted throughout the plant. The formation of enations was preceded by a local accumulation of nitric oxide, which was followed by the appearance of reactive oxygen species, mostly in the vicinity of leaf veins. Such heterogeneity suggests involvement of these molecules in the control of hyperplasia and tissue differentiation.  

A ribosome-binding, 3' translational enhancer has a T-shaped structure and engages in a long-distance RNA-RNA interaction.

Many plant RNA viruses contain elements in their 3' untranslated regions (3' UTRs) that enhance translation. The PTE (Panicum mosaic virus-like translational enhancer) of Pea enation mosaic virus (PEMV) binds to eukaryotic initiation factor 4E (eIF4E), but how this affects translation from the 5' end is unknown. We have discovered a three-way branched element just upstream of the PEMV PTE that engages in a long-distance kissing-loop interaction with a coding sequence hairpin that is critical for the translation of a reporter construct and the accumulation of the viral genome in vivo. Loss of the long-distance interaction was more detrimental than elimination of the adjacent PTE, indicating that the RNA-RNA interaction supports additional translation functions besides relocating the PTE to the 5' end. The branched element is predicted by molecular modeling and molecular dynamics to form a T-shaped structure (TSS) similar to the ribosome-binding TSS of Turnip crinkle virus (TCV). The PEMV element binds to plant 80S ribosomes with a K(d) (dissociation constant) of 0.52 μM and to 60S subunits with a K(d) of 0.30 μM. Unlike the TCV TSS, the PEMV element also binds 40S subunits (K(d), 0.36 μM). Mutations in the element that suppressed translation reduced either ribosome binding or the RNA-RNA interaction, suggesting that ribosome binding is important for function. This novel, multifunctional element is designated a kl-TSS (kissing-loop T-shaped structure) to distinguish it from the TCV TSS. The kl-TSS has sequence and structural features conserved with the upper portion of most PTE-type elements, which, with the exception of the PEMV PTE, can engage in similar long-distance RNA-RNA interactions.

The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0PE, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0PE has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0PE destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.

Photosynthetic alterations of pea leaves infected systemically by pea enation mosaic virus: A coordinated decrease in efficiencies of CO(2) assimilation and photosystem II photochemistry.

We have investigated photosynthetic changes of fully expanded pea leaves infected systemically by pea enation mosaic virus (PEMV) that often attacks legumes particularly in northern temperate regions. Atypical compatible virus-host interaction was monitored during 40 post-inoculation days (dpi). Aninitial PEMV-induced decrease in photosynthetic CO(2) assimilation was detected at 15dpi, when the virus appeared in the measured leaves. This decrease was not induced by stomata closure and corresponded with a decrease in the efficiency of photosystem II photochemistry (Φ(PSII)). Despite of a slight impairment of oxygen evolution at this stage, PSII function was not primarily responsible for the decrease in Φ(PSII). Chlorophyll fluorescence imaging revealed that Φ(PSII) started to decrease from the leaf tip to the base. More pronounced symptoms of PEMV disease appeared at later stages, when a typical mosaic and enations appeared in the infected leaves and oxidative damage of cell membranes was detected. From 30dpi, a degradation of photosynthetic pigments accelerated, stomata were closing and corresponding pronounced decline in CO(2) assimilation was observed. A concomitant photoprotective responses, i.e. an increase in non-photochemical quenching and accumulation of de-epoxidized xanthophylls, were also detected. Interestingly, alternative electron sinks in chloroplasts were not stimulated by PEMV infection, which is in contradiction to earlier reports dealing with virus-induced plant stresses. The presented results show that the PEMV-induced alterations in mature pea leaves accelerated leaf senescence during which a decrease in Φ(PSII) took place in coordinated manner with an inhibition of CO(2) assimilation.

Genomic characterization of pea enation mosaic virus-2 from the Pacific Northwestern USA.

Pea enation mosaic virus (PEMV) infects several legume crops, including chickpea (Cicer arietinum), faba bean (Vicia faba), lentil (Lens culinaris) and pea (Pisum sativum). The virus caused yield losses of food legumes in the Pacific Northwestern US during 1983, 1987 and 1990 [1]. Our recent surveys of pea and alfalfa fields in the states of Washington and Idaho, USA, have shown the prevalence of PEMV on pea. PEMV consists of a large (RNA-1 or PEMV-1) and a small (RNA-2 or PEMV-2) single-stranded positive-sense RNA, which are encapsidated separately into distinct isometric particles [2]. PEMV-2 is one of the seven distinct virus species in the genus Umbravirus. The other species of this genus are Carrot mottle virus (CMoV), Carrot mottle mimic virus (CMoMV), Groundnut rosette virus (GRV), Lettuce speckles mottle virus (LSMV), Tobacco mottle virus (TMoV) and Tobacco bushy top virus (TBTV) [3]. Members of this group lack the coat protein (CP) gene in their genomes and depend on a helper virus for survival [4]. PEMV-2 in association with PEMV-1 (genus Enamovirus), form a symbiotic bipartite virus complex referred to as PEMV. It has been suggested that RNA-1 and RNA-2 are essential for PEMV infection [4, 5]. The RNA-1 (5706 nucleotides) encodes five major ORFs (1-5) that have nucleotide and amino acid sequence similarities with subgroup II luteoviruses [5]. While PEMV-1 provides the necessary encapsidation and vector-transmission abilities for PEMV-2, the latter provides PEMV-1 with long-distance movement and mechanical transmission functions. As part of an ongoing project to determine the genetic diversity of PEMV, we recently characterized the genome of PEMV-1 from the Pacific Northwest [6]. So far, there are only two complete genomic sequences of PEMV-2 in GenBank (NC_003853 and AY714213). To better understand sequence diversity of PEMV-2, the genome structure and organization of two PEMV isolates, one from Idaho (PEMV-2-ID) and one from Washington (PEMV-2-WA), were determined in this study. The PEMV-2 genome (*4.2 kbp), like that of other umbraviruses, predominantly consists of four ORFs (1-4), which perform diverse functions [4, 7, 8]. RNA-2 lacks a polyadenylation signal at its 30 end and contains a large 50 genome-linked protein [9]. ORF-1, at the 50 end of the virus genome, initiates after a short, 20-nt non-coding region (NCR) and encodes a putative 33-kDa protein of unknown function. ORF-2 overlaps with ORF-1 at its 30 end and potentially encodes a protein of 65 kDa through a frameshift mechanism. ORF-2 contains sequence motifs characteristic of a viral RNA-dependent RNA-polymerase (RdRp). The presence of a polymerase cassette in RNA-2 also reveals its independent replicative capabilities. Also, due to the presence of an octanucleotide frameshift signal ‘‘GGATTTTT’’ immediately upstream of the stop codon of ORF-1, ORF-1, along with ORF-2, is expressed by a -1 frameshift mechanism as a minor translation product of 97 kDa [4]. ORFs 3 and 4 in the genome occur after a nonThe sequences described here were deposited in the GenBank database with the following accession numbers: JF713435 for PEMV2-WA and JF713436 for PEMV-2-ID.

Long-distance kissing loop interactions between a 3′ proximal Y-shaped structure and apical loops of 5′ hairpins enhance translation of Saguaro cactus virus

Circularization of cellular mRNAs is a key event prior to translation initiation. We report that efficient translation of Saguaro cactus virus (SCV) requires a 3′ translational enhancer (PTE) located partially in coding sequences. Unlike a similar PTE reported in the 3′ UTR of Pea enation mosaic virus that does not engage in an RNA:RNA interaction (Wang Z. et al., J. Biol. Chem. 284, 14189–14202, 2009), the SCV PTE participates in long distance RNA:RNA interactions with hairpins located in the p26 ORF and in the 5′ UTR of one subgenomic RNA. At least two additional RNA:RNA interactions are also present, one of which involves the p26 initiation codon. Similar PTE can be found in six additional carmoviruses that can putatively form long-distance interactions with 5′ hairpins located in comparable positions.

The Cap-Binding Translation Initiation Factor, eIF4E, Binds a Pseudoknot in a Viral Cap-Independent Translation Element

Eukaryotic initiation factor eIF4E performs a key early step in translation by specifically recognizing the m⁷GpppN cap structure at the 5' end of cellular mRNAs. Many viral mRNAs lack a 5' cap and thus bypass eIF4E. In contrast, we reported a cap-independent translation element (PTE) in Pea enation mosaic virus RNA2 that binds and requires eIF4E for translation initiation. To understand how this uncapped RNA is bound tightly by eIF4E, we employ SHAPE probing, phylogenetic comparisons with new PTEs discovered in panico- and carmoviruses, footprinting of the eIF4E binding site, and 3D RNA modeling using NAST, MC-Fold, and MC-Sym to predict a compact, 3D structure of the RNA. We propose that the cap-binding pocket of eIF4E clamps around a pseudoknot, placing a highly SHAPE-reactive guanosine in the pocket in place of the normal m⁷GpppN cap. This reveals a new mechanism of mRNA recognition by eIF4E.

Conditional facilitation of an aphid vector, Acyrthosiphon pisum, by the plant pathogen, pea enation mosaic virus.

Plant pathogens can induce symptoms that affect the performance of insect herbivores utilizing the same host plant. Previous studies examining the effects of infection of tic bean, Vicia faba L. (Fabales: Fabaceae), by pea enation mosaic virus (PEMV), an important disease of legume crops, indicated there were no changes in the growth and reproductive rate of its primary vector the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae). Here, we report the results of laboratory experiments investigating how A. pisum responded to PEMV infection of a different host plant, Pisum sativum L., at different stages of symptom development. Aphid growth rate was negatively related to the age of the host plant, but when they were introduced onto older plants with well-developed PEMV symptoms they exhibited a higher growth rate compared to those developing on uninfected plants of the same age. In choice tests using leaf discs A. pisum showed a strong preference for discs from PEMV-infected peas, probably in response to visual cues from the yellowed and mottled infected leaves. When adults were crowded onto leaves using clip-cages they produced more winged progeny on PEMV-infected plants. The results indicate that PEMV produces symptoms in the host plant that can enhance the performance of A. pisum as a vector, modify the production of winged progeny and affect their spatial distribution. The findings provide further evidence that some insect vector/plant pathogen interactions could be regarded as mutualistic rather than commensal when certain conditions regarding the age, stage of infection and species of host plant are met.

Molecular characterization of pea enation mosaic virus and bean leafroll virus from the Pacific Northwest, USA.

The family Luteoviridae consists of eight viruses assigned to three different genera, Luteovirus, Polerovirus and Enamovirus. The complete genomic sequences of pea enation mosaic virus (genus Enamovirus) and bean leafroll virus (genus Luteovirus) from the Pacific Northwest, USA, were determined. Annotation, sequence comparisons, and phylogenetic analysis of selected genes together with those of known polero- and enamoviruses were conducted.

First Report of a Nanovirus Disease of Pea in Germany.

During the growing season of 2009, a disease consisting of leaf rolling, top yellows, and plant stunting affected pea (Pisum sativum) in fields near Aschersleben, Saxony-Anhalt, Germany. Samples from symptomatic plants collected in July 2009 were analyzed at the JKI in Braunschweig for infections by various legume viruses by ELISA, immunoelectron microscopy, and transmission assays by sap and aphids. Of 23 samples, 9 were shown to contain Pea enation mosaic virus and three samples each contained Bean leafroll virus and Soybean dwarf virus. From two further samples that had tested negative for the aforementioned viruses, we succeeded in transferring a disease agent to faba bean (Vicia faba) seedlings by giving 50 to 100 individuals of the pea aphid (Acyrthosiphon pisum) acquisition and inoculation access feedings each of ~48 h. Following vector transmission, the agent caused severe yellowing and stunting in pea and faba bean, sometimes followed by necrosis. Attempts at mechanical transmission of the agent failed, and isolation of double-stranded RNA from infected tissue was not successful. Therefore, we considered the possible presence of a nanovirus (4). When using polyclonal antibodies (PAbs) against Faba bean necrotic yellows virus (FBNYV) for double-antibody sandwich (DAS)-ELISA analysis of the two isolates of the disease agent we observed weak but clearly positive reactions. To confirm these weak DAS-ELISA reactions, we used all available monoclonal antibodies (MAbs) raised against FBNYV (1) and faba bean necrotic stunt virus (FBNSV) (3) individually in triple-antibody sandwich (TAS)-ELISA in combination with the FBNYV PAbs for plate coating. Six of 26 MAbs reacted from weak to strong with the two pea isolates, with MAbs FBNYV-3-1F7 and FBNSV-5-1G8 giving the strongest reactions and none of the MAbs giving a differential reaction with the two pea isolates. Employing rolling circle amplification of total DNA extracted from symptomatic leaves of one of the pea isolates yielded a substantial amount of high molecular weight DNA, whereas little or no amplification occurred when using DNA from noninoculated pea leaves. Restriction of the amplified DNA in a nanovirus iteron-specific manner by AatII endonuclease yielded a predominant and abundant product of ~1 kb (3). Sequence comparisons of eight cloned DNAs of 1,002 nucleotides long unequivocally identified them as complete DNA-R component of a new member of the genus Nanovirus (2,4). Its DNA-R sequence (GenBank No. GU553134) is nearly equidistant from the DNA-R sequences of FBNYV (Y11405), FBNSV (GQ150778), Milk vetch dwarf virus (MDV) (AB027511) and Subterranean clover stunt virus (SCSV) (AJ290434), sharing with them respective sequence identities of 79, 78, 79, and 73%. Moreover, it is more distinct from the DNA-R sequences of FBNYV, FBNSV, and MDV than the three latter are from each other (86 to 91%). This together with the serological data relating to the capsid protein properties of this virus strongly suggest that it is distinct from the hitherto described nanoviruses FBNYV, MDV, FBNSV, and SCSV. Therefore, we propose the name pea necrotic yellow dwarf virus (PNYDV) for this new nanovirus naturally infecting pea in Germany.

A peptide that binds the pea aphid gut impedes entry of Pea enation mosaic virus into the aphid hemocoel.

Development of ways to block virus transmission by aphids could lead to novel and broad-spectrum means of controlling plant viruses. Viruses in the Luteoviridae enhanced are obligately transmitted by aphids in a persistent manner that requires virion accumulation in the aphid hemocoel. To enter the hemocoel, the virion must bind and traverse the aphid gut epithelium. By screening a phage display library, we identified a 12-residue gut binding peptide (GBP3.1) that binds to the midgut and hindgut of the pea aphid Acyrthosiphon pisum. Binding was confirmed by labeling the aphid gut with a GBP3.1-green fluorescent protein fusion. GBP3.1 reduced uptake of Pea enation mosaic virus (Luteoviridae) from the pea aphid gut into the hemocoel. GBP3.1 also bound to the gut epithelia of the green peach aphid and the soybean aphid. These results suggest a novel strategy for inhibiting plant virus transmission by at least three major aphid pest species.

Identification of novel sources of resistance to Pea enation mosaic virus in chickpea germplasm

Chickpea can be seriously affected by Pea enation mosaic virus (PEMV) in the Pacific Northwest region of the USA and other areas of the world. Use of pesticides to manage PEMV vector transmission is ineffective and PEMV‐resistant chickpeas have not been identified. Therefore, the Cicer core collection consisting of 499 wild Plant Introduction (PI) accessions and improved cultivars representing 25 countries and two chickpea phenotypes (Desi and Kabuli) was evaluated for resistance to PEMV based on a 1 to 5 scale where 1 = symptomless and 5 = severe mosaic and stunting. Eight accessions were considered resistant to PEMV based on mean disease severity values equal to or less than 1·9 in repeated greenhouse tests. All of the resistant accessions to PEMV were of the Desi type and all originated from Iran or India. PI 450763 was the only Kabuli accession that demonstrated tolerance to PEMV. PEMV resistance was not detected in commercial chickpea cultivars or advanced breeding lines currently grown in the USA and Canada. This is the first report of chickpea germplasm with resistance to PEMV.

Transcriptomic analysis of intestinal genes following acquisition of pea enation mosaic virus by the pea aphid Acyrthosiphon pisum

Viruses in the family Luteoviridae are strictly transmitted by aphids in a non-propagative, circulative and persistent mode. Virions ingested by aphids successively cross the gut and the accessory salivary gland epithelia before being released, together with saliva, into the plant vasculature. Virion transport through aphid cells occurs by a transcytosis mechanism. This study conducted a transcriptomic analysis of intestinal genes of the pea aphid Acyrthosiphon pisum following uptake of pea enation mosaic virus. Among the 7166 transcripts analysed, 128 were significantly regulated (105 genes downregulated and 23 upregulated). Of these genes, 5 % were involved in intracellular trafficking, endocytosis and signal transduction, three important steps in the internalization and transport of virions. The limited levels of downregulation (maximum of 3.45-fold) and upregulation (maximum of 1.37-fold) suggest that the virus hijacks a constitutive endocytosis-exocytosis mechanism without heavily perturbing cell metabolism. Although limited to about 20 % of the pea aphid genes, this work represents the first large-scale analysis of aphid gene regulation following virus acquisition. A better knowledge of this virus-vector interaction will be possible only when tools representing the complete genomic capacity of the aphid become available.

Analysis of the accumulation of Pea enation mosaic virus genomes in seed tissues and lack of evidence for seed transmission in pea (Pisum sativum).

Pea enation mosaic virus (PEMV) is an important virus disease of pea. International movement of commercial pea cultivars and germplasm can be problematic due to uncertainty about seed transmission of the viruses responsible for the disease. Whether PEMV is seedborne was assessed by collecting developing seed from infected plants and determining the relative concentrations of the PEMV-1 and PEMV-2 viral genomes using quantitative real-time reverse-transcription polymerase chain reaction. The relative accumulation of PEMV-1 and PEMV-2 was approximately 1,240 and 13,000 times higher, respectively, in leaf than in embryo tissues. Accumulation of PEMV-1 and PEMV-2 RNA was also significantly higher in pod walls and seed coats than in cotyledons or embryo axes. No evidence was obtained for seed transmission of PEMV in pea. Although PEMV-1 and PEMV-2 genomic RNAs were found in developing seed, no PEMV symptoms were observed in the field on more than 50,000 plants from seed derived from PEMV-infected source plants. These data demonstrate that PEMV is seedborne in pea but do not support a previous report that PEMV is seed transmitted. Absence of seed transmission may result from the low abundance of PEMV viral genomes in embryo tissue.

Towards universal detection of <i>Luteoviridae</i>

Luteoviridae is a plant virus family of 26 species which causes yield losses in cereals potatoes and other economically important crops worldwide Accurate detection is an important component of controlling the spread of luteoviruses and reducing yield losses PCRbased detection is often the method of choice for Luteoviridae as it is more sensitive than serological methods which frequently fail to detect infection due to the low concentration of luteoviruses in plants Since the currently available luteovirus primers are mostly speciesspecific and work under different PCR conditions universal primers are desirable Seven primers were tested that were designed to target the most conserved regions of the Luteoviridae genomes and that possess high homology to over 75 of Luteoviridae species Thirty luteovirus isolates representing 15 species were obtained from New Zealand and overseas these included all five species from the Luteovirus genus eight of the nine species from the Polerovirus genus (except CYDV RPS) PEMV1 (the only species from the Enamovirus genus) and CtRLV (which is not assigned to a genus) Between them the three combinations of primers detected all 13 of the Luteovirus and Polerovirus species tested

Structure of a Viral Cap-independent Translation Element That Functions via High Affinity Binding to the eIF4E Subunit of eIF4F

RNAs of many positive strand RNA viruses lack a 5' cap structure and instead rely on cap-independent translation elements (CITEs) to facilitate efficient translation initiation. The mechanisms by which these RNAs recruit ribosomes are poorly understood, and for many viruses the CITE is unknown. Here we identify the first CITE of an umbravirus in the 3'-untranslated region of pea enation mosaic virus RNA 2. Chemical and enzymatic probing of the approximately 100-nucleotide PEMV RNA 2 CITE (PTE), and mutagenesis revealed that it forms a long, bulged helix that branches into two short stem-loops, with a possible pseudoknot interaction between a C-rich bulge at the branch point and a G-rich bulge in the main helix. The PTE inhibited translation in trans, and addition of eIF4F, but not eIFiso4F, restored translation. Filter binding assays revealed that the PTE binds eIF4F and its eIF4E subunit with high affinity. Tight binding required an intact cap-binding pocket in eIF4E. Among many PTE mutants, there was a strong correlation between PTE-eIF4E binding affinity and ability to stimulate cap-independent translation. We conclude that the PTE recruits eIF4F by binding eIF4E. The PTE represents a different class of translation enhancer element, as defined by its structure and ability to bind eIF4E in the absence of an m(7)G cap.

Effect of sowing date and straw mulch on virus incidence and aphid infestation in organically grown faba beans (Vicia faba)

AbstractThe effect of sowing date on aphid infestation and the incidence of aphid‐transmitted viruses were investigated in organically managed, small‐scale field experiments with two faba bean cultivars over 3 years (2002–04). As an additional factor, straw mulch was applied in 2 of the 3 years shortly before the start of vector activity in May. Virus incidence was determined using enzyme‐linked immunosorbent assay and immunoelectron microscopy. Aphid flight activity was monitored using standard yellow water traps. Bean colonising aphids were assessed throughout the vegetation period by counting the number of plants infested with Acyrthosiphon pisum, Megoura viciae and Aphis fabae. Pea enation mosaic virus and bean yellow mosaic virus were the most abundant aphid‐transmitted viruses, being detected in 22–54% and 9–69%, respectively, of the total number of virus‐infected plants analysed per year. Further aphid‐transmitted viruses found in faba bean were bean leaf roll virus, beet western yellows virus, clover yellow vein virus (in 2002) and soybean dwarf virus (in 2004). A. pisum was the predominant aphid species colonising faba bean plants. Early sowing compared with late sowing led to a significant reduction of the total virus incidence in faba bean in all 3 years. However, significantly decreased levels of A. pisum colonisation as a result of early sowing were observed only in 1 year and one cultivar. Irrespective of sowing date, straw mulching had no significant effects on virus incidence and aphid colonisation. Compared with late sowing, early sowing significantly increased bean yield in all 3 years and kernel weight in 2 years, whereas straw mulching had no effect on yield.

The readthrough domain of pea enation mosaic virus coat protein is not essential for virus stability in the hemolymph of the pea aphid.

A fraction of the coat protein (CP) subunits in virions of members of the family Luteoviridae contain a C-terminal extension called the readthrough domain (RTD). The RTD is necessary for persistent aphid transmission, but its role is unknown. It has been reported to be required for virion stability in the hemolymph. Here, we tested whether this was the case for pea enation mosaic virus (PEMV) virions in the pea aphid (Acyrthosiphon pisum) using RNA1Delta, a natural deletion mutant lacking the middle portion of the RTD ORF, and CPDeltaRTD, in which the entire RTD ORF was deleted. In infected plants, RNA1Delta virions were as abundant and stable as wild-type (WT) virions, while CPDeltaRTD virions were unstable. No RTD of any size was translated from artificial subgenomic mRNA of CPDeltaRTD or RNA1Delta in vitro. Thus, only the major CP was present in the mutant virions. Using real-time RT-PCR to detect virion RNA, no significant differences in the concentration or stability of WT and RNA1Delta virions were detected in the aphid hemolymph at much longer times than are necessary for virus transmission. Thus, the RTD is not necessary for stability of PEMV RNA in the aphid hemolymph, and it must play another role in aphid transmission.