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 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.

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.

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.

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.

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.

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.

A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site.

Programmed ribosomal frameshifting allows one mRNA to encode regulate expression of, multiple open reading frames (ORFs). The polymerase encoded by ORF 2 of Barley yellow dwarf virus (BYDV) is expressed via minus one (-1) frameshifting from the overlapping ORF 1. Previously, this appeared to be mediated by a 116 nt RNA sequence that contains canonical -1 frameshift signals including a shifty heptanucleotide followed by a highly structured region. However, unlike known -1 frameshift signals, the reporter system required the zero frame stop codon and did not require a consensus shifty site for expression of the -1 ORF. In contrast, full-length viral RNA required a functional shifty site for frameshifting in wheat germ extract, while the stop codon was not required. Increasing translation initiation efficiency by addition of a 5' cap on the naturally uncapped viral RNA, decreased the frameshift rate. Unlike any other known RNA, a region four kilobases downstream of the frameshift site was required for frameshifting. This included an essential 55 base tract followed by a 179 base tract that contributed to full frameshifting. The effects of most mutations on frameshifting correlated with the ability of viral RNA to replicate in oat protoplasts, indicating that the wheat germ extract accurately reflected control of BYDV RNA translation in the infected cell. However, the overall frameshift rate appeared to be higher in infected cells, based on immunodetection of viral proteins. These findings show that use of short recoding sequences out of context in reporter constructs may overlook distant signals. Most importantly, the remarkably long-distance interaction reported here suggests the presence of a novel structure that can facilitate ribosomal frameshifting.

Satellite cereal yellow dwarf virus-RPV (satRPV) RNA requires a douXble hammerhead for self-cleavage and an alternative structure for replication.

The 110 nt hammerhead ribozyme in the satellite RNA of cereal yellow dwarf virus-RPV (satRPV RNA) folds into an alternative conformation that inhibits self-cleavage. This alternative structure comprises a pseudoknot with base-pairing between loop (L1) and a single-stranded bulge (L2a), which are located in hammerhead stems I and II, respectively. Mutations that disrupt this base-pairing, or otherwise cause the ribozyme to more closely resemble a canonical hammerhead, greatly increase self-cleavage. In a more natural multimeric sequence context containing the full-length satRPV RNA and two copies of the hammerhead, wild-type RNA cleaves much more efficiently than in the 110 nt context. Mutations in the upstream hammerhead, including a knock-out in the catalytic core, affect cleavage at the downstream cleavage site, indicating that multimers of satRPV RNA cleave via a double hammerhead. The double hammerhead includes base-pairing between two copies of the L1 sequence which extends stem I. Disruption of L1-L1 base-pairing slows cleavage of the multimer. L1-L2a base-pairing is required for efficient replication of satRPV RNA in oat protoplasts. Mutations that affect self-cleavage of the multimer do not correlate with replication efficiency, indicating that the ability to self-cleave is not a primary determinant of replication. We present a replication model in which multimeric satRPV RNA folds into alternative conformations that cannot form in the monomer. One potential metastable intermediate conformation involves L1-L2a base-pairing that may facilitate formation of the double hammerhead. However, we conclude that L1-L2a also performs some other essential function in the satRPV RNA replication cycle, because the L1-L2a base-pairing is more important than efficient self-cleavage for replication.

Maize and oat antixenosis and antibiosis against Delphacodes kuscheli (Homoptera: Delphacidae), vector of "Mal de Rio Cuarto" of maize in Argentina.

"Mal de Rio Cuarto" (MRC) is the most important virus disease of maize, Zea mays L., in Argentina. Several maize lines show different levels of resistance to MRC in the field; however, no studies have been conducted to investigate resistance mechanisms against its insect vector, Delphacodes kuscheli Fennah (Homoptera: Delphacidae). Oat, Avena spp., is the main overwintering host of D. kuscheli and main source of populations that infest maize. Although oat varieties resistant to the greenbug, Schizaphis graminum (Rondani) (Homoptera: Aphididae) are commercially available, their effect on D. kuscheli is unknown. We conducted laboratory experiments to test for the presence of antixenosis and antibiosis resistance mechanisms on six maize lines with different levels of field resistance to MRC, and seven commercial oat cultivars that include two S. graminum-resistant varieties. We did not find antibiotic effects of maize lines on D. kuscheli longevity and survivorship patterns, but we obtained antixenotic effects from the LP2 line (field moderate) due to reduced settling preference and feeding. Oat 'Bonaerense Payé and 'Suregrain INTA' showed both antixenosis and antibiosis, with significantly less settling preference, oviposition in the no-choice test, and reduced total fecundity in comparison with the other varieties studied. The S. graminum-resistant 'Boyera F. A.' and 'Tambera F. A.' did not showed a consistent pattern of resistance versus D. kuscheli across all experiments. Our results indicate the presence of potential sources of insect resistance in the maize lines and oat cultivars tested that may be used in MRC integrated pest management programs.

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.

A single nucleotide substitution in the alpha a gene confers oat pathogenicity to barley stripe mosaic virus strain ND18.

A 236-nucleotide region from the alpha a gene of strain CV42 (pathogenic to oat), when substituted for the homologous region in strain ND18 (nonpathogenic to oat), was shown previously to confer a near wild-type oat pathogenicity to this strain (Weiland and Edwards, 1994, Virology 201: 116-126). The data suggested that six amino acid substitutions in the alpha a gene were responsible for the differences in oat pathogenicity, and that threonine-724, encoded by CV42, might be a critical amino acid in determining pathogenicity of barley stripe mosaic virus (BSMV) to oat. In the present work, codons specifying T-724, I-764, and N-785 (encoded by CV42 RNA alpha) were substituted individually and in combination for those coding for P-724, T-764, and K-785 (encoded by ND18 RNA alpha), respectively, by site-directed mutagenesis. The core K-733, T-734, and K-736 positions (CV42) were substituted for Q-733, S-734, and Q-736 (ND18) as a single block. The results of inoculations with these mutants indicate that the C2261-->A2261 nucleotide substitution (P-724-->T-724) by itself is sufficient to enable strain ND18 to infect oat plants, although poorly. Additional substitution of CV42 codons into ND18 RNA alpha at the remaining five positions altered symptom type, decreased the timing of the appearance of symptoms, and increased the percentage of plants infected per inoculation. Nonetheless, all mutants accumulated to similar levels in inoculated oat protoplasts after a 24-h period. Using a recombinant RNA beta from which beta-glucuronidase could be expressed, results were obtained suggesting that the multiplication of strain ND18 and the nonpathogenic variants generated in the study was restricted in the inoculated leaf. The data indicate a potential pathway by which pathogenicity to oat evolved in BSMV.

Apoptotic cell death is a common response to pathogen attack in oats.

We have examined the characteristics of cell death induced by pathogen infection in oats with respect to following hallmark apoptotic features: DNA laddering, chromatin condensation, and electron microscopic-terminal deoxynucleotidyl transferase-mediated UTP end labeling positive response. A wide range of plant pathogens representing different levels of parasitism in susceptible and resistant interactions were used for the inocula, which include (i) an obligate parasite, Puccinia coronata f. sp. avenae (the crown rust fungus); (ii) a facultative biotroph parasite, Magnaporthe grisea (the blast fungus); (iii) pathogenic bacteria, Pseudomonas syringae pv. atropurpurea and P. syringae pv. coronafaciens (the halo or stripe blights of oats); and (iv) Ryegrass mottle virus. Surprisingly, any of the pathogens used induced most of the apoptotic features in oat cells at and around the infection sites, indicating that apoptotic cell death is a common phenomenon in oats during pathogen attack. The localization and the timing of apoptotic cell death during a course of infection were, however, quite different depending on the interactions (compatible or incompatible) and the pathogens (fungi, bacteria, or viruses). Possible roles of apoptotic cell death in the susceptible and resistant interactions are discussed.

Sequence analysis of wheat and oat furovirus capsid protein genes suggests that oat golden stripe virus is a strain of soil-borne wheat mosaic virus.

In northern blots, cDNA probes prepared to soil-borne wheat mosaic virus (SBWMV) RNA-1 and RNA-2 hybridized to RNA-1 and RNA-2, respectively, from a UK isolate of oat golden stripe virus (OGSV), as well as to their homologous RNAs. RT-PCR was used to amplify, clone and sequence a region of about 750 nucleotides spanning the capsid protein gene and part of the readthrough protein on RNA-2 from OGSV, a French isolate of SBWMV and two stable deletion mutants (Lab1 and Okl-7) of SBWMV isolates from Nebraska and Oklahoma respectively. There was very high (96.7-99.1%) nucleotide homology between all these sequences and the wild-type SBWMV sequences from Nebraska and Oklahoma. OGSV was more similar to SBWMV from France and Nebraska than were any of the isolates to SBWMV from Oklahoma. Of the few differences in the deduced amino acid sequences of the capsid proteins from the different isolates, OGSV differed from all SBWMV isolates only in one amino acid (isoleucine for valine at position 88). The high degree of similarity suggests that OGSV may best be classified as an oat strain of SBWMV.