Wheat streak mosaic virus coat protein is a host-specific long-distance transport determinant in oat.

Viral determinants involved in systemic infection of hosts by monocot-infecting plant viruses are poorly understood. Wheat streak mosaic virus (WSMV, genus Tritimovirus, family Potyviridae) exclusively infects monocotyledonous crops such as wheat, oat, barley, maize, triticale, and rye. Previously, we reported that WSMV CP amino acids 36-84 are expendable for systemic infection of wheat, maize, barley and rye. In this study, the role of coat protein (CP) in systemic infection of oat by WSMV was examined by using a series of viable deletion mutants. WSMV bearing deletions within or encompassing all of amino acids 36-57 efficiently infected oat, indicating that these amino acids are dispensable for systemic infection of oat. However, WSMV mutants lacking CP amino acids 58-84 or 85-100 failed to systemically infect oat. Furthermore, green fluorescent protein-tagged WSMV mutants lacking CP amino acids 58-100 elicited local foci in oat but failed to enter the vasculature. These data suggest that CP amino acids 58-100 are required for systemic infection of oat by WSMV by specifically facilitating virus long-distance transport in oat.

Crop-associated virus reduces the rooting depth of non-crop perennial native grass more than non-crop-associated virus with known viral suppressor of RNA silencing (VSR).

As agricultural acreage expanded and came to dominate landscapes across the world, viruses gained opportunities to move between crop and wild native plants. In the Midwestern USA, virus exchange currently occurs between widespread annual Poaceae crops and remnant native perennial prairie grasses now under consideration as bioenergy feedstocks. In this region, the common aphid species Rhopalosiphum padi L. (the bird cherry-oat aphid) transmits several virus species in the family Luteoviridae, including Barley yellow dwarf virus (BYDV-PAV, genus Luteovirus) and Cereal yellow dwarf virus (CYDV-RPV and -RPS, genus Polerovirus). The yellow dwarf virus (YDV) species in these two genera share genetic similarities in their 3'-ends, but diverge in the 5'-regions. Most notably, CYDVs encode a P0 viral suppressor of RNA silencing (VSR) absent in BYDV-PAV. Because BYDV-PAV has been reported more frequently in annual cereals and CYDVs in perennial non-crop grasses, we examine the hypothesis that the viruses' genetic differences reflect different affinities for crop and non-crop hosts. Specifically, we ask (i) whether CYDVs might persist within and affect a native non-crop grass more strongly than BYDV-PAV, on the grounds that the polerovirus VSR could better moderate the defenses of a well-defended perennial, and (ii) whether the opposite pattern of effects might occur in a less defended annual crop. Because previous work found that the VSR of CYDV-RPS possessed greater silencing suppressor efficiency than that of CYDV-RPV, we further explored (iii) whether a novel grass-associated CYDV-RPS isolate would influence a native non-crop grass more strongly than a comparable CYDV-RPV isolate. In growth chamber studies, we found support for this hypothesis: only grass-associated CYDV-RPS stunted the shoots and crowns of Panicum virgatum L. (switchgrass), a perennial native North American prairie grass, whereas crop-associated BYDV-PAV (and coinfection with BYDV-PAV and CYDV-RPS) most stunted annual Avena sativa L. (oats). These findings suggest that some of the diversity in grass-infecting Luteoviridae reflects viral capacity to modulate defenses in different host types. Intriguingly, while all virus treatments also reduced root production in both host species, only crop-associated BYDV-PAV (or co-infection) reduced rooting depths. Such root effects may increase host susceptibility to drought, and indicate that BYDV-PAV pathogenicity is determined by something other than a P0 VSR. These findings contribute to growing evidence that pathogenic crop-associated viruses may harm native species as well as crops. Critical next questions include the extent to which crop-associated selection pressures drive viral pathogenesis.

Genome-Wide Association Mapping of Barley Yellow Dwarf Virus Tolerance in Spring Oat (Avena sativa L.).

Barley yellow dwarf viruses (BYDVs) are responsible for the disease barley yellow dwarf (BYD) and affect many cereals including oat (Avena sativa L.). Until recently, the molecular marker technology in oat has not allowed for many marker-trait association studies to determine the genetic mechanisms for tolerance. A genome-wide association study (GWAS) was performed on 428 spring oat lines using a recently developed high-density oat single nucleotide polymorphism (SNP) array as well as a SNP-based consensus map. Marker-trait associations were performed using a Q-K mixed model approach to control for population structure and relatedness. Six significant SNP-trait associations representing two QTL were found on chromosomes 3C (Mrg17) and 18D (Mrg04). This is the first report of BYDV tolerance QTL on chromosome 3C (Mrg17) and 18D (Mrg04). Haplotypes using the two QTL were evaluated and distinct classes for tolerance were identified based on the number of favorable alleles. A large number of lines carrying both favorable alleles were observed in the panel.

Mild and severe cereal yellow dwarf viruses differ in silencing suppressor efficiency of the P0 protein.

Viral pathogenicity has often been correlated to the expression of the viral encoded-RNA silencing suppressor protein (SSP). The silencing suppressor activity of the P0 protein encoded by cereal yellow dwarf virus-RPV (CYDV-RPV) and -RPS (CYDV-RPS), two poleroviruses differing in their symptomatology was investigated. CYDV-RPV displays milder symptoms in oat and wheat whereas CYDV-RPS is responsible for more severe disease. We showed that both P0 proteins (P0(CY-RPV) and P0(CY-RPS)) were able to suppress local RNA silencing induced by either sense or inverted repeat transgenes in an Agrobacterium tumefaciens-mediated expression assay in Nicotiana benthamiana. P0(CY-RPS) displayed slightly higher activity. Systemic spread of the silencing signal was not impaired. Analysis of short-interfering RNA (siRNA) abundance revealed that accumulation of primary siRNA was not affected, but secondary siRNA levels were reduced by both CYDV P0 proteins, suggesting that they act downstream of siRNA production. Correlated with this finding we showed that both P0 proteins partially destabilized ARGONAUTE1. Finally both P0(CY-RPV) and P0(CY-RPS) interacted in yeast cells with ASK2, a component of an E3-ubiquitin ligase, with distinct affinities.

Environmental nutrient supply alters prevalence and weakens competitive interactions among coinfecting viruses.

The rates and ratios of environmental nutrient supplies can determine plant community composition. However, the effect of nutrient supplies on within-host microbial interactions is poorly understood. Resource competition is a promising theory for understanding microbial interactions, because microparasites require nitrogen (N) and phosphorus (P) for synthesis of macromolecules such as nucleic acids and proteins. To better understand the effects of nutrient supplies to hosts on pathogen interactions, we singly inoculated and coinoculated Avena sativa with two virus species, barley yellow dwarf virus-PAV (BYDV-PAV) and cereal yellow dwarf virus-RPV (CYDV-RPV). Host plants were grown across a factorial combination of N and P supply rates that created a gradient of N : P supply ratios, one being replicated at low and high nutrient supply. Nutrient supply affected prevalence and the interaction strength among viruses. P addition lowered CYDV-RPV prevalence. The two viruses had a distinct competitive hierarchy: the coinoculation of BYDV-PAV lowered CYDV-RPV infection rate, but the reverse was not true. This antagonistic interaction occurred at low nutrient supply rates and disappeared at high N supply rate. Given the global scale of human alterations of N and P cycles, these results suggest that elevated nutrient supply will increase risks of virus coinfection with likely effects on virus epidemiology, virulence and evolution.

Cocksfoot mottle virus coat protein is dispensable for the systemic infection.

BACKGROUND: The Sobemovirus genome consists of polycistronic single-stranded positive-sense RNA. The first ORF encodes P1, a suppressor of RNA silencing required for virus movement. The coat protein (CP) is expressed from the 3' proximal ORF3 via subgenomic RNA. In addition to its structural role, the CP of some sobemoviruses has been reported to be required for systemic movement and to interact with P1. The aim of this study was to analyse the role of Cocksfoot mottle virus (CfMV) CP in the suppression of RNA silencing and virus movement. METHODS: Agrobacterium-mediated transient expression method was used for testing CfMV CP capacity to suppress RNA silencing. CP substitution and deletion mutants were generated to examine the role of this protein in CfMV infection, using three host plants (oat, barley and wheat). The viral movement was characterised with CfMV expressing EGFP fused to the C-terminus of CP. RESULTS: In the current study we show that CfMV CP is an additional RNA silencing suppressor. Interestingly, we observed that all CP mutant viruses were able to infect the three tested host plants systemically, although usually with reduced accumulation. CfMV expressing EGFP was detected in epidermal and mesophyll cells of inoculated leaves. Although EGFP fluorescence was not detected in upper leaves, some plants displayed CfMV symptoms. Analysis of the upper leaves revealed that the viruses had lost the EGFP sequence and sometimes also most of the CP gene. CONCLUSIONS: The present study demonstrates that CfMV CP suppresses RNA silencing but, surprisingly, is dispensable for systemic movement. Thus, CfMV does not move as virion in the tested host plants. The composition of the movement RNP complex remains to be elucidated.

Coat protein expression strategy of oat blue dwarf virus.

Oat blue dwarf virus (OBDV) is a member of the genus Marafivirus whose genome encodes a 227 kDa polyprotein (p227) ostensibly processed post-translationally into its functional components. Encoded near the 3' terminus and coterminal with the p227 ORF are ORFs specifying major and minor capsid proteins (CP). Since the CP expression strategy of marafiviruses has not been thoroughly investigated, we produced a series of point mutants in the OBDV CP encoding gene and examined expression in protoplasts. Results support a model in which the 21 kDa major CP is the product of direct translation of a sgRNA, while the 24 kDa minor CP is a cleavage product derived from both the polyprotein and a larger ~26 kDa precursor translated directly from the sgRNA. Cleavage occurs at an LXG[G/A] motif conserved in many viruses that use papain-like proteases for polyprotein processing and protection against degradation via the ubiquitin-proteasome system.

Coat protein based molecular characterization of Barley yellow dwarf virus isolates identified on oat plants in Pakistan.

Barley yellow dwarf virus (BYDV) is a potential threat to the agriculture production. The amplified complete coat protein sequences of the isolate M07 and M12 were determined to be 597 bp and 603bp, respectively. M07 showed maximum nucleotide sequence identity of 87.6% (84.3% amino acid sequence identity) to a Chinese isolate of BYDV-PAV. Whereas, the isolate M12 showed maximum nucleotide sequence identity of 94.5% (94.0% amino acid sequence identity) to French isolate BYDV-PAV. Since more than 10 o/o differences, among the amino acid level of any gene product, is the sole criterion to discriminate between species within the family Luteoviridae, the isolate M07 that shows maximum of 84.3% (less than 90%) amino acid sequence identity with previously known Luteovirus species, is thus, recommended to be a distinct PAV species within the genus Luteovirus.

Genomic and proteomic analysis of Schizaphis graminum reveals cyclophilin proteins are involved in the transmission of cereal yellow dwarf virus.

Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are transmitted by aphid vectors. The identification of aphid genes and proteins mediating virus transmission is critical to develop agriculturally sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Two cyclophilin B proteins, S28 and S29, were identified previously in populations of Schizaphisgraminum that differed in their ability to transmit the RPV strain of Cereal yellow dwarf virus (CYDV-RPV). The presence of S29 was correlated with F2 genotypes that were efficient virus transmitters. The present study revealed the two proteins were isoforms, and a single amino acid change distinguished S28 and S29. The distribution of the two alleles was determined in 12 F2 genotypes segregating for CYDV-RPV transmission capacity and in 11 genetically independent, field-collected S. graminum biotypes. Transmission efficiency for CYDV-RPV was determined in all genotypes and biotypes. The S29 isoform was present in all genotypes or biotypes that efficiently transmit CYDV-RPV and more specifically in genotypes that efficiently transport virus across the hindgut. We confirmed a direct interaction between CYDV-RPV and both S28 and S29 using purified virus and bacterially expressed, his-tagged S28 and S29 proteins. Importantly, S29 failed to interact with a closely related virus that is transported across the aphid midgut. We tested for in vivo interactions using an aphid-virus co-immunoprecipitation strategy coupled with a bottom-up LC-MS/MS analysis using a Q Exactive mass spectrometer. This analysis enabled us to identify a third cyclophilin protein, cyclophilin A, interacting directly or in complex with purified CYDV-RPV. Taken together, these data provide evidence that both cyclophilin A and B interact with CYDV-RPV, and these interactions may be important but not sufficient to mediate virus transport from the hindgut lumen into the hemocoel.

Discovery and targeted LC-MS/MS of purified polerovirus reveals differences in the virus-host interactome associated with altered aphid transmission.

Circulative transmission of viruses in the Luteoviridae, such as cereal yellow dwarf virus (CYDV), requires a series of precisely orchestrated interactions between virus, plant, and aphid proteins. Natural selection has favored these viruses to be retained in the phloem to facilitate acquisition and transmission by aphids. We show that treatment of infected oat tissue homogenate with sodium sulfite reduces transmission of the purified virus by aphids. Transmission electron microscopy data indicated no gross change in virion morphology due to treatments. However, treated virions were not acquired by aphids through the hindgut epithelial cells and were not transmitted when injected directly into the hemocoel. Analysis of virus preparations using nanoflow liquid chromatography coupled to tandem mass spectrometry revealed a number of host plant proteins co-purifying with viruses, some of which were lost following sodium sulfite treatment. Using targeted mass spectrometry, we show data suggesting that several of the virus-associated host plant proteins accumulated to higher levels in aphids that were fed on CYDV-infected plants compared to healthy plants. We propose two hypotheses to explain these observations, and these are not mutually exclusive: (a) that sodium sulfite treatment disrupts critical virion-host protein interactions required for aphid transmission, or (b) that host infection with CYDV modulates phloem protein expression in a way that is favorable for virus uptake by aphids. Importantly, the genes coding for the plant proteins associated with virus may be examined as targets in breeding cereal crops for new modes of virus resistance that disrupt phloem-virus or aphid-virus interactions.

Cocksfoot mottle sobemovirus establishes infection through the phloem.

Cocksfoot mottle virus (CfMV) localization in oat plants was analyzed during three weeks post infection by immunohistochemical staining to follow its spread through different tissues. In early stages of infection, the virus was first detectable in phloem parenchyma and bundle sheath cells of inoculated leaves. Bundle sheath and phloem parenchyma were also the cell types where the virus was first detected in stems and systemic leaves of infected plants. In later stages of infection, CfMV spread also into the mesophyll surrounding vascular bundles and was seldom detected in xylem parenchyma of inoculated leaves. In systemic leaves, CfMV was not detected from xylem. Moreover, sometimes it was found from phloem only. In straw and roots, CfMV was detected both from phloem and xylem. According to our observations, CfMV predominantly moves through phloem, which makes the systemic movement of CfMV different from that of another monocot-infecting sobemovirus, Rice yellow mottle virus (RYMV).

Agrobacterium-mediated infection of whole plants by yellow dwarf viruses.

Barley yellow dwarf virus-PAV (BYDV-PAV) and cereal yellow dwarf virus-RPV (CYDV-RPV) are only transmitted between host plants by aphid vectors and not by mechanical transmission. This presents a severe limitation for the use of a reverse genetics approach to analyze the effects of mutations in these viruses on plant infection and aphid transmission. Here we describe the use of agroinfection to infect plants with BYDV-PAV and CYDV-RPV. The cDNAs corresponding to the complete RNA genomes of BYDV-PAV and CYDV-RPV were cloned into a binary vector under the control of the cauliflower mosaic virus 35S promoter and the nopaline synthase transcription termination signal. The self-cleaving ribozyme from hepatitis virus D was included to produce a transcript in planta with a 3' terminus identical to the natural viral RNA. ELISA and RT-PCR analysis showed that the replicons of BYDV-PAV and CYDV-RPV introduced by Agrobacterium into Nicotiana benthamiana and N. clevelandii gave rise to a local infection in the infiltrated mesophyll cells. After several weeks systemic infection of phloem tissue was detected, although no systemic symptoms were observed. Three heterologous virus silencing suppressors increased the efficiency of agroinfection and accumulation of BYDV-PAV and CYDV-RPV in the two Nicotiana species. The progeny viruses purified from infiltrated tissues were successfully transmitted to oat plants by aphids, and typical yellow dwarf symptoms were observed. This study reports the first agroinfection of eudicot plants using BYDV-PAV and CYDV-RPV.

Protein-RNA linkage and post-translational modifications of two sobemovirus VPgs.

Sobemoviruses possess a viral genome-linked protein (VPg) attached to the 5' end of viral RNA. VPg is processed from the viral polyprotein. In the current study, Cocksfoot mottle virus (CfMV) and Rice yellow mottle virus (RYMV) VPgs were purified from virions and analysed by mass spectrometry. The cleavage sites in the polyprotein and thereof the termini of VPg were experimentally proven. The lengths of the mature VPgs were determined to be 78 and 79 aa residues, respectively. The amino acid residues covalently linked to RNA in the two VPgs were, surprisingly, not conserved; it is a tyrosine at position 5 of CfMV VPg and serine at position 1 of RYMV VPg. Phosphorylations were identified in CfMV and RYMV VPgs with two positionally similar locations T20/S14 and S71/S72, respectively. RYMV VPg contains an additional phosphorylation site at S41.

Modeling the competition between viruses in a complex plant-pathogen system.

In this article, we propose a mathematical model that describes the competition between two plant virus strains (MAV and PAV) for both the host plant (oat) and their aphid vectors. We found that although PAV is transmitted by two aphids and MAV by only one, this fact, by itself, does not explain the complete replacement of MAV by PAV in New York State during the period from 1961 through 1976; an interpretation that is in agreement with the theories of A. G. Power. Also, although MAV wins the competition within aphids, we assumed that, in 1961, PAV mutated into a new variant such that this new variant was able to overcome MAV within the plants during a latent period. As shown below, this is sufficient to explain the swap of strains; that is, the dominant MAV was replaced by PAV, also in agreement with Power's expectations.

Reverse transcription loop-mediated isothermal amplification of DNA for detection of Barley yellow dwarf viruses in China.

A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Barley yellow dwarf viruses (BYDVs) was developed. In this procedure, three sets of four primers matching a total of six sequences of the coat protein or read-through protein genes of BYDVs - one each for three species, namely BYDV-GAV, -GPV and -PAV were synthesized for developing a specific and sensitive RT-LAMP assay for total RNA extracts from field-infected wheat plants in such a way that a loop could be formed and elongated during DNA amplification. RT-LAMP assays for each of three species of BYDV/CYDVs in China exhibited high specificity and could detect viral sequences in total RNA extracts from infected oat tissues at dilutions of 1 x 10(-5). All field samples collected from different regions of China showed the same result using both RT-LAMP and RT-PCR. This relatively simple and sensitive technique showed excellent potential with field-collected samples and for use in grassroots agencies in developing countries with limited resources.

First infectious clone of the propagatively transmitted Oat blue dwarf virus.

Oat blue dwarf virus (OBDV) is a small, phloem-limited marafivirus that replicates in its leafhopper vector. We have developed complete cDNA clones of OBDV from which infectious transcripts may be derived--the first such clones for any propagatively transmitted plant virus. Prior to clone construction, the reported sequences of the 5' and 3' ends were confirmed using 5' RACE, primer extension, and ligation-anchored PCR. Using vascular puncture of maize seeds with capped transcripts, multiple clones were shown to be infectious at an average rate of 24.3% (range 14-36%). Aster leafhoppers successfully transmitted OBDV to oats and barley after feeding on detached, infected maize leaves. Proteins and RNAs consistent in size with those expected in OBDV infection were detected in young leaves via western and northern blotting, respectively. One construct, pOBDV-2r, was designated as the reference clone. An infectious clone of OBDV will be valuable in examining the interaction of this virus with both its insect and plant hosts.

Stem-loop structure of Cocksfoot mottle virus RNA is indispensable for programmed -1 ribosomal frameshifting.

The -1 programmed ribosomal frameshifting (-1 PRF) mechanism utilized by many viruses is dependent on a heptanucleotide slippery sequence and a downstream secondary structure element. In the current study, the RNA structure downstream from the slippery site of cocksfoot mottle sobemovirus (CfMV) was proven to be a 12bp stem-loop with a single bulge and a tetranucleotide loop. Several deletion and insertion mutants with altered stem-loop structures were tested in wheat germ extract (WGE) for frameshifting efficiency. The impact of the same mutations on virus infectivity was tested in oat plants. Mutations shortening or destabilizing the stem region reduced significantly but did not abolish -1 PRF in WGE. The same mutations proved to be deleterious for virus infection. However, extending the loop region to seven nucleotides had no significant effect on frameshifting efficiency in WGE and did not hamper virus replication in infected leaves. This is the first report about the experimentally proven RNA secondary structure directing -1 PRF of sobemoviruses.

Soil-borne viruses affecting cereals: known for long but still a threat.

A number of furo- and bymoviruses, which are naturally transmitted by the ubiquitous soil-borne protozoon Polymyxa graminis are of great economic importance for autumn-sown wheat, barley, rye, triticale, and oats. The two barley-affecting bymoviruses, Barley yellow mosaic virus and Barley mild mosaic virus, are widespread in Europe and East Asia. Production of wheat, rye, and triticale is threatened by three related furoviruses and two bymoviruses. Soil-borne wheat mosaic virus mainly occurs in North America and Chinese wheat mosaic virus in Asia, whereas Soil-borne cereal mosaic virus predominates in Europe. The bymovirus Wheat yellow mosaic virus so far is restricted to Asia; Wheat spindle streak mosaic virus occurs in North America and Europe. Oat plants can be separately or mixed-infected by a bymo- and a furovirus, named Oat mosaic virus and Oat golden stripe virus, respectively. Because P. graminis cannot be controlled by chemicals and the viruses retain their infectivity in the resting spores for many years cultivation of resistant plants is the only means of preventing severe losses. The genetic variability of the known viruses and the sporadic reports of newly emerging pathogens, such as Aubian wheat mosaic virus, make continued resistance breeding and careful monitoring of field crops necessary.

Expression of the barley yellow dwarf virus-GAV movement protein and its detection in the infected and transgenic plants.

Movement proteins (MPs) that facilitate virus movement in the plants were identified in a number of plant viruses. In this study, full-length MP gene of the Chinese isolate Barley yellow dwarf virus-GAV (BYDV-GAV) was cloned and expressed in Escherichia coli. About 32% of the expressed MP was soluble providing the concentration of isopropyl-beta-D-galactopyranoside (IPTG), time of the induction, temperature and shaking speed were optimized. The soluble MP was purified using nickel-affinity column. Immune serum prepared against purified MP was used for the detection of MP in the BYDV-GAV infected leaves of oat and in the leaves of transgenic wheat plants expressing the full-length and truncated MP gene.