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Brome mosaic virus (BMV) is generally thought to be of little economic importance to crops; consequently, there is little information about its impact on wheat production under field conditions. After repeated detection of BMV in Ohio wheat fields at incidences up to 25%, the virus was isolated, sequenced, characterized, and tested for its impact on soft red winter wheat (SRWW). The Ohio isolate of brome mosaic virus (BMV-OH) was found to be >99% identical to a BMV-Fescue isolate (accession no. DQ530423-25) and capable of systemically infecting multiple monocot and dicot species, including cowpea and soybean, in experimental inoculations. BMV-OH was used in field experiments during the 2016 and 2017 growing seasons to quantify its effect on SRWW grain yield and development when inoculated at Feekes 1, 5, 8, and 10 in two to four cultivars. Cultivar and timing of inoculation had statistically significant (P < 0.05) main and interaction effects on grain yield, wheat growth, and multiple components of yield. Compared with noninoculated controls, BMV-OH reduced grain yield by up to 61% when inoculated at Feekes 1 and by as much as 25, 36, and 31% for inoculations at Feekes 5, 8, and 10, respectively. The magnitude of the yield reduction varied among cultivars and was associated with reductions in grain size and weight or plant population. These findings suggest that BMV could impact wheat productivity in Ohio and will serve as the basis for more large-scale investigations of the effects of this virus in commercial fields.

Brome mosaic virus (BMV) (the Russian strain) infects monocot plants and has been studied extensively in barley and wheat. Here, we report BMV can systemically infect rice (Oryza sativa var. japonica), including cultivars in which the genomes have been determined. The BMV capsid protein can be found throughout the inoculated plants. However, infection in rice exhibits delayed symptom expression or no symptoms when compared with wheat (Triticum aestivum). The sequences of BMV RNAs isolated from rice did not reveal any nucleotide changes in RNA1 or RNA2, while RNA3 had only one synonymous nucleotide change from the inoculum sequence. Preparations of purified BMV virions contained RNA1 at a significantly reduced level relative to the other two RNAs. Analysis of BMV RNA replication in rice revealed that minus-strand RNA1 was replicated at a reduced rate when compared with RNA2. Thus, rice appears to either inhibit RNA1 replication or lacks a sufficient amount of a factor needed to support efficient RNA1 replication.

Cell-penetrating peptides (CPP) can translocate across the cell membrane and have been extensively studied for the delivery of proteins, nucleic acids, and therapeutics in mammalian cells. However, characterizations of CPP in plants have only recently been initiated. We showed that the intact virion and a recombinant capsid protein (CaP) from a plant-infecting nonenveloped icosahedral RNA virus, Brome mosaic virus (BMV), can penetrate the membranes of plant protoplasts but are trapped by the extracellular matrix. Furthermore, a 22-residue peptide derived from the N-terminal region of the CaP (CPNT) can enter barley protoplasts and cells of intact barley and Arabidopsis roots. An inhibitor of the macropinocytosis reduced CPNT entry, while treatment with NiCl2 changed the cellular localization of CPNT. CPNT increased uptake of the green flourescent protein (GFP) into the cell when covalently fused to GFP or when present in trans of GFP. The BMV CPNT overlaps with the sequence known to bind BMV RNA, and it can deliver BMV RNAs into cells, resulting in viral replication, as well as deliver double-stranded RNAs that can induce gene silencing.

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein–green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.

The natural variation of Arabidopsis thaliana in response to a bromovirus, Spring beauty latent virus (SBLV), was examined. Of 63 Arabidopsis accessions tested, all were susceptible when inoculated with SBLV, although there was a large degree of variation in symptom development. Most accessions, including Columbia (Col-0), were symptomless or developed only mild symptoms, but four accessions, including S96, showed severe symptoms of SBLV infection. Genetic analysis suggested that the difference in the responses of Col-0 and S96 to SBLV was controlled by a single semidominant locus. We have designated this locus SSB1 (symptom development by SBLV infection). By using genetic markers, SSB1 was mapped to chromosome IV. The patterns of distribution and accumulation of SBLV in sensitive accessions were similar to those in the insensitive accessions. In addition, symptom development in S96 by SBLV infection was critically interrupted by the presence of the NahG gene, which encodes salicylic acid (SA) hydroxylase. These data suggest that symptom development in A. thaliana controlled by SSB1 is independent of the efficiency of SBLV multiplication and is dependent on SA signaling.

Previously, we demonstrated that Broad bean mottle virus (BBMV), a member of the genus Bromovirus, could accumulate RNA 2-derived defective interfering (DI) RNAs during infection. In this work, we study how host and environmental factors affect the accumulation of DI RNAs. Serial passages of BBMV through selected plant species reveal that, with low-multiplicity inocula, some systemic hosts (Vicia faba, Nicotiana clevelandii, and N. tabacum cv. Samsum) support DI RNA accumulation after the first passage cycle but other hosts (Phaseolus vulgaris, Pisum sativum, and Glycine max) do not. However, several passages with the high-multiplicity inocula can generate DI RNAs in pea plants. Local lesion hosts (Chenopodium quinoa, C. amaranticolor, and C. murale) remain free of the DI RNA components. The size of the de novo-formed DI RNAs depends on the host and on environmental conditions. For instance, broad bean plants cultivated in a greenhouse or in a growth chamber at 20°C accumulated DI RNAs of 2.4 or 1.9 kb in size, respectively. A reverse trend was observed in pea plants. Lower temperatures greatly facilitated the formation of DI RNAs in broad bean and pea hosts after the first passage. The importance of these findings for the studies on DI RNAs are discussed.

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.

Nicotiana benthamiana plants expressing Brome mosaic virus (BMV) p2 protein complemented replication of RNAs1+3 but, surprisingly, supported little or no replication of RNA-2. Despite this, the p2 transgenic plants were able to support systemic migration of RNAs-1 and -3. Kinetic analyses showed identical degradation rates for RNAs-2 and -3, greatly detracting from the concept of an induction of an RNA-2-specific degradation system. Deletion analysis identified a 200-nucleotide sequence that may contribute to silencing in a context-specific manner. When (R)1 progeny of a severely silencing p2 transgenic line were tested for virus resistance, three different classes of reactions were observed. In class 1 and class 3 plants, the virus moved systemically and showed various extents of RNA-2 silencing. However, in class 2 plants, there was a stochastic onset of post-transcriptional silencing in the systemic leaves that was reminiscent of virus recovery. Plants showing recovery tended to have a greater number of transgene loci than did those exhibiting componentspecific silencing. The induction of silencing did not appear to be dependent solely on the combined steady state levels of the transgene and viral RNA. Some plants transformed with a p2 frameshift construct showed a complete silencing phenotype, but none showed RNA-2-specific silencing. While the relationship between the two types of silencing remains unclear, we speculate that our observations reflect early events in the induction of virus recovery.

We have recently shown that acquisition of a 6.4-kb, double-stranded (ds) RNA (M1) by hyphal anastomosis is associated with enhanced vigor and virulence, whereas its removal by hyphal tipping correlates with diminished virulence in the plant-pathogenic basidiomycete Rhizoctonia solani. The M1 dsRNA is not encapsidated by a typical nucleocapsid, has a circular and/or concatemeric form, and is associated with the mitochondrial and cytosolic fractions. M1 possesses six open reading frames (ORFs) the longest of which (ORF 2) is located on the (+) strand, and encodes a putative polypeptide consisting of 1,747 amino acids or 199.4 kDa. This polypeptide has a significant amino acid sequence similarity, including six conserved helicase domains and an ATP/GTP binding motif, with the 1A protein of broad bean mottle virus (BBMV) and other bromoviruses. ORF 5, which is located on the (-) strand of M1 and is complementary to a region of ORF 2, codes for a putative polypeptide that has a significant amino acid sequence similarity with the cytochrome c oxidase assembly factor. This complementarity provides direct evidence suggesting that the long-standing hypothesis of viruses evolving from cellular genes may be valid.

The coat protein (CP) open reading frame (ORF) of brome mosaic virus (BMV) has been mutated to study host-related CP functions in barley, a systemic host, and in Chenopodium hybridum L. which supports both local lesion formation and systemic spread of BMV. To test the role of the N-terminal region of CP, mutants C1 to C3, which synthesized the CP lacking first seven amino acids, and mutant D1, which had Trp 22 and Thr 23 replaced with Phe-Gly-Ser, were generated. C1 to C3 inhibited virus systemic spread in C. hybridum but not in barley while D1 only reduced virus accumulation in noninoculated leaves of C. hybridum. More internal CP regions were tested by mutation of Lys 63 to Leu (mutant SP3) and Lys 129 to Arg (mutant SP1). SP1 behaved similarly to C1 to C3 while SP3 similarly to D1. In addition, SP3 reduced concentrations of RNA3 and RNA4 in both hosts. Apparently, various CP regions differentially affect, either directly or indirectly, virus translocation in different hosts, suggesting both the CP and host factors to be important for virus spread. Larger deletions in the CP ORF (mutants BB4 and SX1) or a decrease of CP production by using a frameshift mutant C, inhibited virus systemic spread in both hosts, and delayed the appearance of smaller local lesions on C. hybridum. Thus, the CP is not required for cell-to-cell movement but is required for systemic translocation of BMV.

Cowpea chlorotic mottle virus (CCMV, bromovirus group) was isolated from Desmodium heterocarpon and Clitoria ternatea plants growing in experimental plots at the International Institute of Tropical Agriculture, Ibadan, Nigeria. It was identified by serology, particle morphology, pH sensitivity, and analyses of viral coat protein and nucleic acids. In reciprocal agar-gel diffusion tests, the CCMV-Nigerian isolate and the type CCMV strain were found to be serologically distinct. This is the first report of the occurrence of a virus resembling CCMV outside of the United States and Costa Rica

Full-length complementary DNA (cDNA) copies of genomic RNA1, RNA2, and RNA3 segments of cowpea chlorotic mottle virus (CCMV) strains D, N, and S were synthesized using polymerase chain reaction and were cloned downstream of a T7 RNA polymerase promoter. Mixtures of the homologous in vitro-transcribed RNAs produced typical CCMV symptoms when inoculated on soybean (cv. Bragg) and cowpea (cv. California Blackeye) plants. Using either gel-purified or in vitro-transcribed CCMV RNA components, the pseudorecombinants SSD, SSN, DDS, and NNS were constructed. The properties of these pseudorecombinants were tested by comparing the induced symptoms, virus concentration, and systemic spread. These studies revealed a direct involvement of RNA3 genetic information in necrotic lesion formation and in breakage of CCMV resistance in soybean. Exchanges between RNA1 or RNA2 components further affected the biological features of reassorted CCMV variants. The true nature of the pseudorecombinants was confirmed by reconstitution of the parental strains from the corresponding pseudorecombinant RNA components.

Spring beauty latent virus (SBLV) has been proposed as a new member of the bromovirus group. Four dsRNAs (M r 2.2, 2.0, 1.4, and 0.5× 10 6 ) with a profile similar to those of the bromoviruses were obtained when plant tissue infected with SBLV was analyzed. A fifth ds RNA (M r 1.2× 10 6 ) with nucleic acid similarity to the genomic RNA also was obtained. Molecular hybridization with cDNA to SBLV RNA did not show nucleic acid similarity between SBLV and four bromoviruses. Infectivity tests with SBLV ssRNAs indicated that it has a tripartite genome because RNAs 1, 2, and 3 were required for infectivity. When RNAs 1, 2, 36, and 4 were translated, 95, 48, and 23 kDa proteins were obtained (...)

Recherche de produits chimiques antiviraux ayant un large spectre d'activite contre les virus vegetaux

HOBBS, H. A., and J. P. FULTON. 1979. Beetle transmission of cowpea, chlorotic mottle virus. Phytopathology 69:255-256. Cowpea chlorotic mottle virus was transmitted by the bean leaf beetle, from and transmitted to beans. Levels were much lower when the virus was Cerotoma trifurcata, and by the spotted cucumber beetle, Diabrotica either acquired from or transmitted to cowpeas. Lespedeza cuneata, which undecimpunctata howardii. Three strains of the virus were transmitted at may be an overwintering host, was a poor source of virus for vectors. low levels. Levels of transmission were highest when virus was acquired Several viruses in the bromovirus group are transmitted by antiserum, using the Ouchterlony gel diffusion technique. Virus in leaffeeding beetles. Cowpea chlorotic mottle virus (CCMV) was beetles that had fed on infected plants was tested by severing one transmitted by the chrysomelid species Cerotoma trifurcata metathoracic leg and collecting in a capillary tube the hemolymph (Forster) and Diabrotica undecimpunctata howardii (Barber) (6); that exuded. Hemolymph was diluted approximately 1:300 in 0.01 the bean yellow stipple strain (BYSV) of CCMV by C. ruficornis M phosphate buffer, pH 7.2, and inoculated to young Monarch Olivier and D. balteata LeConte (2,3); and broad bean mottle virus cowpea plants. (BBMV) by D. undecimpunctata howardii, Acalymma trivittatum (Mannerheim), and Colaspis brunnea (F.) (7). Levels of transmission were moderate to low in all cases. Because CCMV is