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Alfalfa mosaic virus (AMV) is a worldwide distributed virus that has a very wide host range and causes significant crop losses of many economically important crops, including potato ( Solanum tuberosum L.). In this study, the antiviral activity of Bacillus licheniformis strain POT1 against AMV on potato plants was evaluated. The dual foliar application of culture filtrate (CF), 24 h before and after AMV-inoculation, was the most effective treatment that showed 86.79% reduction of the viral accumulation level and improvement of different growth parameters. Moreover, HPLC analysis showed that a 20 polyphenolic compound was accumulated with a total amount of 7,218.86 and 1606.49 mg/kg in POT1-treated and non-treated plants, respectively. Additionally, the transcriptional analysis of thirteen genes controlling the phenylpropanoid, chlorogenic acid and flavonoid biosynthetic pathways revealed that most of the studied genes were induced after POT1 treatments. The stronger expression level of F3H , the key enzyme in flavonoid biosynthesis in plants, (588.133-fold) and AN2 , anthocyanin 2 transcription factor, (97.005-fold) suggested that the accumulation flavonoid, especially anthocyanin, might play significant roles in plant defense against viral infection. Gas chromatography-mass spectrometry (GC-MS) analysis showed that pyrrolo[1,2-a]pyrazine-1,4-dione is the major compound in CF ethyl acetate extract, that is suggesting it acts as elicitor molecules for induction of systemic acquired resistance in potato plants. To our knowledge, this is the first study of biological control of AMV mediated by PGPR in potato plants.

Prune dwarf virus (PDV) is an important viral pathogen of plum, sweet cherry, peach, and many herbaceous test plants. Although PDV has been intensively investigated, mainly in the context of phylogenetic relationship of its genes and proteins, many gaps exist in our knowledge about the mechanism of intercellular transport of this virus. The aim of this work was to investigate alterations in cellular organelles and the cell-to-cell transport of PDV in Cucumis sativus cv. Polan at ultrastructural level. To analyze the role of viral proteins in local transport, double-immunogold assays were applied to localize PDV coat protein (CP) and movement protein (MP). We observe structural changes in chloroplasts, mitochondria, and cellular membranes. We prove that PDV is transported as viral particles via MP-generated tubular structures through plasmodesmata. Moreover, the computer-run 3D modeling reveals structural resemblances between MPs of PDV and of Alfalfa mosaic virus (AMV), implying similarities of transport mechanisms for both viruses.

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T0 and T1 generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T4 generation in an agronomically elite ‘Grasslands Sustain’ genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T4 generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.

On entering a host cell, positive-strand RNA virus genomes have to serve as messenger for the translation of viral proteins. Efficient translation of cellular messengers requires interactions between initiation factors bound to the 5′-cap structure and the poly(A) binding protein bound to the 3′-poly(A) tail. Initiation of infection with the tripartite RNA genomes of alfalfa mosaic virus (AMV) and viruses from the genus Ilarvirus requires binding of a few molecules of coat protein (CP) to the 3′ end of the nonpolyadenylated viral RNAs. Moreover, infection with the genomic RNAs can be initiated by addition of the subgenomic messenger for CP, RNA 4. We report here that extension of the AMV RNAs with a poly(A) tail of 40 to 80 A-residues permitted initiation of infection independently of CP or RNA 4 in the inoculum. Specifically, polyadenylation of RNA 1 relieved an apparent bottleneck in the translation of the viral RNAs. Translation of RNA 4 in plant protoplasts was autocatalytically stimulated by its encoded CP. Mutations that interfered with CP binding to the 3′ end of viral RNAs reduced translation of RNA 4 to undetectable levels. Possibly, CP of AMV and ilarviruses stimulates translation of viral RNAs by acting as a functional analogue of poly(A) binding protein or other cellular proteins.

RNA 3 of alfalfa mosaic virus (AMV) encodes the 5'-proximal movement protein (MP) gene and the 3'-proximal coat protein (CP) gene which is expressed from a subgenomic RNA. Several strategies were explored to use this RNA as a vector for expression of the green fluorescent protein (GFP) in Nicotiana tabaccum plants expressing the viral polymerase proteins P1 and P2 (P12 plants). Insertion of a subgenomic promoter (sgp)-GFP cassette between the CP gene and the 3'-untranslated region (UTR) interfered with RNA accumulation in protoplasts, indicating that cis-acting sequences required for replication were disrupted. When GFP was fused to the N-terminus of MP or CP, the chimeric RNAs accumulated in protoplasts but cell-to-cell movement in plants was blocked. Insertion of a GFP-sgp cassette immediately upstream of the CP gene caused a hypersensitive host response. However, insertion of a GFP-sgp cassette upstream of the MP gene did not affect symptom formation and yielded a vector that expressed GFP in inoculated but not in the systemic leaves of both P12 tobacco and non-transgenic N. benthamina plants. When the size of the GFP gene was reduced from 700 to 300 nucleotides, virus infection was observed in the non-inoculated leaves. Analysis of the progeny of some chimera revealed novel data on replication, encapsidation and recombination of AMV RNA 3.

In non-transgenic host plants and protoplasts alfalfa mosaic virus displays a strong need for coat protein when starting an infection cycle. The \"protection model\" states that the three viral RNAs must have a few coat protein subunits at their 3' termini in order to protect them in the host cell against degradation by 3'- to- 5' exoribonucleases [Neeleman L, Van der Vossen EAG, Bol JF (1993) Virology 196: 883-887]. We demonstrated that the naked genome RNAs are slightly infectious, if the inoculation is done at very high concentrations, or if it is preceded by an additional inoculation with the RNAs 1 and 2 (encoding subunits for the viral RNA polymerase). This could mean that the necessity for protection by coat protein is lost if the RNAs in large quantities can overcome the activity of the degrading enzymes, or are protected by association with the RNA polymerase, respectively. However, after having tested in protoplasts the survival of separately preinoculated naked RNA 1 during several hours before RNA 2 was inoculated, on the one hand, or of simultaneously inoculated RNAs 1 and 2, with cycloheximide in the medium during the first hours after inoculation, on the other hand, we had to conclude that the viral genome RNAs are quite stable in the cell in the absence of coat protein or RNA polymerase, respectively. This invalidates the protection model. Accommodation of the above findings by our published \"messenger release model\" for genome activation [Houwing CJ, Jaspars EMJ (1993) Biochimie 75: 617-621] is discussed.

A virus-based vector was used for the transient expression of the alfalfa mosaic virus coat protein (CP) gene in protoplasts and plants. The accumulation of wild-type CP conferred strong protection against subsequent alfalfa mosaic virus infection, enabling the efficacy of CP mutants to be determined without developing transgenic plants. Expression of the CP mRNA alone without CP accumulation conferred weaker protection against infection. The activity of the N-terminal mutant CPs in protection did not correlate with their activities in genome activation. The activity of a C-terminal mutant suggested that encapsidation did not have a role in protection. Our results indicate that interaction of the CP with alfalfa mosaic virus RNA is not important in protection, thereby leaving open the possibility that interactions with host factors lead to protection.

The role of flagella and motility in the attachment of the foodborne pathogen Listeria monocytogenes to various surfaces is mixed with some systems requiring flagella for an interaction and others needing only motility for cells to get to the surface. In nature this bacterium is a saprophyte and contaminated produce is an avenue for infection. Previous studies have documented the ability of this organism to attach to and colonize plant tissue. Motility mutants were generated in three wild type strains of L. monocytogenes by deleting either flaA, the gene encoding flagellin, or motAB, genes encoding part of the flagellar motor, and tested for both the ability to colonize sprouts and for the fitness of that colonization. The motAB mutants were not affected in the colonization of alfalfa, radish, and broccoli sprouts; however, some of the flaA mutants showed reduced colonization ability. The best colonizing wild type strain was reduced in colonization on all three sprout types as a result of a flaA deletion. A mutant in another background was only affected on alfalfa. The third, a poor alfalfa colonizer was not affected in colonization ability by any of the deletions. Fitness of colonization was measured in experiments of competition between mixtures of mutant and parent strains on sprouts. Here the flaA and motAB mutants of the three strain backgrounds were impaired in fitness of colonization of alfalfa and radish sprouts, and one strain background showed reduced fitness of both mutant types on broccoli sprouts. Together these data indicate a role for flagella for some strains to physically colonize some plants, while the fitness of that colonization is positively affected by motility in almost all cases.

We engineered a 21-mer peptide representing amino acids 170–190 of the respiratory syncytial virus (RSV) G protein as a fusion with the Alfalfa mosaic virus (AlMV) coat protein (CP), produced recombinant AlMV particles presenting this peptide (VMR-RSV) on their surfaces and tested the immunogenicity in vitro in human dendritic cells and in vivo in non-human primates. Significant pathogen-specific immune responses were generated in both systems: (i) human dendritic cells armed with VMR-RSV generated vigorous CD4+ and CD8+ T cell responses; (ii) non-human primates that received these particles responded by mounting strong cellular and humoral immune responses. This approach may validate the use of a novel RSV vaccine delivery vehicle in humans.

In the family Bromoviridae, a mixture of the three genomic RNAs of bromo-, cucumo-, and oleaviruses is infectious as such, whereas the RNAs of alfamo- and ilarviruses require binding of a few molecules of coat protein (CP) to the 3′ end to initiate infection. Most studies on the early function of CP have been done on the alfamovirus Alfalfa mosaic virus (AMV). The 3′ 112 nucleotides of AMV RNAs can adopt two different conformations. One conformer consists of a tRNA-like structure that, together with an upstream hairpin, is required for minus-strand promoter activity. The other conformer consists of four hairpins interspersed by AUGC-sequences and represents a strong binding site for CP. Binding of CP to this conformer enhances the translational efficiency of viral RNAs in vivo 40-fold and blocks viral minus-strand RNA synthesis in vitro. AMV CP is proposed to initiate infection by mimicking the function of the poly(A)-binding protein.