Inter- and Intramolecular recombinations in the Cucumber Mosaic Virus genome related to adaptation to Alstroemeria

In four distinct alstroemeria-infecting cucumber mosaic virus (CMV) isolates, additional sequences of various lengths were present in the 3' nontranslated regions of their RNAs 2 and 3, apparently the result of intra- and intermolecular recombination events. Competition experiments revealed that these recombined RNA 2 and 3 segments increased the biological fitness of CMV in alstroemeri

The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells

The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is described here that NS3 is also active in mammalian cells. This activity is independent of the inducer molecule used. Using either synthetic siRNAs or a short hairpin RNA construct, NS3 was able to significantly suppress the RNAi-mediated silencing of luciferase expression in both monkey (Vero) and human (HEK293) cells. These results support the proposed mode of action of NS3 to act by sequestering siRNAs, the key molecules of the RNAi pathway conserved in all eukaryotes. The possible applications of this protein in modulating RNAi and investigating the proposed antiviral RNAi response in mammalian cell systems are discussed

Efficiency of Rz-1 based rhizomania resistance and molecular studies on BNYVV isolates from sugar beet cultivation in Geece

A survey was carried out to investigate the current situation concerning rhizomania disease incidence in sugar beet cultivation of Greece. A systematic field evaluation over locations and years revealed a consistent disease severity pattern according to favourable agroclimatic conditions and pointed to the so far effectiveness of the Rz1 gene-based resistance, as no major disease outbreaks were observed. Molecular analyses aiming at the characterization of the type and genetic diversity of the virus further confirmed the widespread occurrence of BNYVV in the country, as evidenced by RT-PCR amplification of all five known genomic molecules and nested-PCR assays. None of the isolates contained an RNA 5, typically found in pathotype P. On the basis of RFLP patterns, all BNYVV isolates analysed were classified as pathotype A. Sequence determination of the full-length RNA 3-encoded p25 protein, responsible for symptom development, revealed amino acid motifs ACHG/VCHG in the hypervariable region aa67–70. The presence of valine in position 67 did not appear associated with increased pathogenicity and resistance breaking properties, as earlier reported

Binding of small interfering RNA molecules is crucial for RNA interference suppressor activity of rice hoja blanca virus NS3 in plants

The NS3 protein of rice hoja blanca tenuivirus represents a viral suppressor of RNAi that sequesters small interfering (si)RNAs in vitro. To determine whether this siRNA binding property is the critical determinant for the suppressor activity of NS3, an alanine point mutational analysis was performed and the resulting mutant proteins were tested for both siRNA binding ability and RNAi suppressor activity in plants. Alanine substitutions of lysine residues at position 173-175 resulted in mutant proteins that lost both their affinity for siRNAs and their RNAi suppressor activity in planta. This indicates that siRNA binding of NS3 is indeed essential for the suppressor function of NS3 and that residues at position 173-175 are involved in the siRNA binding and suppressor activit

Tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum- and Golgi-derived pleomorphic membrane structures in plant cells

Tomato spotted wilt virus (TSWV) particles are spherical and enveloped, an uncommon feature among plant infecting viruses. Previous studies have shown that virus particle formation involves the enwrapment of ribonucleoproteins with viral glycoprotein containing Golgi stacks. In this study, the localization and behaviour of the viral glycoproteins Gn and Gc were analysed, upon transient expression in plant protoplasts. When separately expressed, Gc was solely observed in the endoplasmic reticulum (ER), whereas Gn was found both within the ER and Golgi membranes. Upon co-expression, both glycoproteins were found at ER-export sites and ultimately at the Golgi complex, confirming the ability of Gn to rescue Gc from the ER, possibly due to heterodimerization. Interestingly, both Gc and Gn were shown to induce the deformation of ER and Golgi membranes, respectively, also observed upon co-expression of the two glycoproteins. The behaviour of both glycoproteins within the plant cell and the phenomenon of membrane deformation are discussed in light of the natural process of viral infectio

A Primitive Non-symmetric 3-Class Association Scheme on 36 Elements with p111 = 0 Exists and is Unique

AbstractIn this paper we construct a primitive, non-symmetric 3-class association scheme with parameters v = 36, v1 = 7, p111 = 0 and p211 = 4 and show that such a scheme is determined by its parameters

Development of a locus-specific, co-dominant SCAR marker for assisted-selection of the Sw-5 (Tospovirus resistance) gene cluster in a wide range of tomato accessions

The best levels of broad-spectrum Tospovirus resistance reported in tomatoes thus far are conferred by the Sw-5 locus. This locus contains at least five paralogues (denoted Sw-5a through Sw-5e), of which Sw-5b represents the actual resistance gene. Here we evaluated a panel of seven PCR primer pairs matching different sequences within a genomic region spanning the Sw-5a and Sw-5b gene cluster. Primer efficiency evaluation was done employing tomato isolines with and without the Sw-5 locus. One primer pair produced a single and co-dominant polymorphism between susceptible and resistant isolines. Sequence analysis of these amplicons indicated that they were specific for the Sw-5 locus and their differences were due to insertions/deletions. The polymorphic SCAR amplicon encompass a conserved sequence of the promoter region of the functional Sw-5b gene, being located in the position -31 from its open reading frame. This primer pair was also evaluated in field assays and with a collection of accessions known to be either susceptible or resistant to tospoviruses. An almost complete correlation was found between resistance under greenhouse/field conditions and the presence of the marker. Therefore, this primer pair is a very useful tool in marker-assisted selection systems in a large range of tomato accessions

In vitro transcription of Tomato spotted wilt virus is independent of translation

Ongoing transcription in vitro of Tomato spotted wilt virus (TSWV) has previously been demonstrated to require the presence of reticulocyte lysate. This dependence was further investigated by testing the occurrence of transcription in the presence of two translation inhibitors: edeine, an inhibitor that still allows scanning of nascent mRNAs by the 40S ribosomal subunit, and cycloheximide, an inhibitor that completely blocks translation including ribosome scanning. Neither of these inhibitors blocked TSWV transcription initiation or elongation in vitro, as demonstrated by de novo-synthesized viral mRNAs with globin mRNA-derived leader sequences, suggesting that TSWV transcription in vitro requires the presence of (a component within) reticulocyte lysate, rather than a viral protein resulting from translation

Iris Yellow Spot Virus in the Netherlands: Occurence in Onion and Confirmation of Transmission by Thrips tabaci

Since its first detection in the Netherlands in 1992, Iris yellow spot virus (IYSV, genus Tospovirus) has been reported worldwide in Allium crops, in a few ornamentals and in a small number of weeds. After recent findings of IYSV in Alstroemeria and Eustoma in the Netherlands, a number of neighbouring onion fields were surveyed. In 2005 and 2006, only few infected plants were found with obvious symptoms of IYSV. In 2007, after sampling and testing small leaf samples with various types of damage, including small brown-yellow colored spots and spots with thrips feeding damage, a high percentage of plants were found with positive IYSV scores in ELISA. Infection by IYSV could be confirmed in most ELISA-positive samples by RT-PCR. Under laboratory conditions, evidence was obtained that Thrips tabaci acts as a vector for this virus. Acquisition of the IYSV from infected Datura stramonium plants resulted in virus uptake and replication in over 60% of the thrips exposed, as determined by Western blotting and immunolocalisation of the virus in the foregut, and in epithelial and midgut muscle cells using antibodies against both the viral N and NSs proteins. Successful transmission of IYSV to seedlings of Emilia sonchifolia was observed, however, only at low frequency under the laboratory conditions used

Phage display-selected single chain antibodies confer high levels of resistance against Tomato spotted wilt virus

Rational design of antibodies targeting essential viral proteins can complement the palette of antiviral resistance strategies. Here, stable and high expression of single-chain monoclonal antibodies targeting the nucleoprotein of the economically important plant virus Tomato spotted wilt virus, a protein that is involved in multiple steps in the viral infection cycle, is reported. High cytoplasmic expression levels of three selected phage display-derived anti-viral single-chain antibodies were established. Of these antibodies, two led to high levels of resistance against this plant virus. Protoplast experiments provided evidence that the two resistance-conferring antibodies may have a different mode of action and could be combined for higher durability of resistance in the fiel