In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

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In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens.

Double-stranded RNA (dsRNA) has been shown to play a key role as an inducer of different interference phenomena occurring in both the plant and animal kingdoms. Here, we show that dsRNA derived from viral sequences can interfere with virus infection in a sequence-specific manner by directly delivering dsRNA to leaf cells either by mechanical inoculation or via an Agrobacterium-mediated transient-expression assay. We have successfully interfered with the infection of plants by three viruses belonging to the tobamovirus, potyvirus, and alfamovirus groups, demonstrating the reliability of the approach. We suggest that the effect mediated by dsRNA in plant virus infection resembles the analogous phenomenon of RNA interference observed in animals. The interference observed is sequence specific, is dose dependent, and is triggered by dsRNA but not singlestranded RNA. Our results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of posttranscriptional gene silencing (PTGS) in natural virus infections, triggering the initiation step of PTGS that targets viral RNA for degradation. Gene sequences derived from different plant viruses have been introduced into a wide variety of plant species to produce transgenic plants protected against virus infection. In a number of cases, it is known that the mechanism of resistance is a posttranscriptional, RNA-mediated process that targets both the viral RNA and the transgene mRNA for degradation in a sequence-specific manner (11, 19). RNA-mediated virus resistance is a manifestation of posttranscriptional gene silencing (PTGS), a more general phenomenon which was first described as a coordinated and reciprocal inactivation of host gene and transgenes encoding the same sense RNA (reviewed in references 33 and 41). More recently, three components in the dynamics of PTGS have been proposed: initiation, propa

https://jvi.asm.org/content/jvi/75/24/12288.full.pdf

Double-Stranded RNA-Mediated Interference with Plant Virus Infection

Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. We have previously shown and patented that mechanical inoculation of in vitro-transcribed dsRNA derived from viral sequences specifically prevents virus infection in plants. The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour. We have developed an in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria, with a view to providing a practical control of virus diseases in plants. Partially purified bacterial dsRNAs promoted specific interference with the infection in plants by two viruses belonging to the tobamovirus and potyvirus groups. Furthermore, we have demonstrated that easy to obtain, crude extracts of bacterially expressed dsRNAs are equally effective protecting plants against virus infections when sprayed onto plant surfaces by a simple procedure. Virus infectivity was significantly abolished when plants were sprayed with French Press lysates several days before virus inoculation. Our approach provides an alternative to genetic transformation of plant species with dsRNA-expressing constructs capable to interfere with plant viruses. The main advantage of this mode of dsRNA production is its simplicity and its extremely low cost compared with the requirements for regenerating transgenic plants. This approach provides a reliable and potential tool, not only for plant protection against virus diseases, but also for the study of gene silencing mechanisms in plant virus infections.

/pdf/crude-extracts-of-bacterially-expressed-dsrna-can-be-used-to-4e3v28yim5.pdf

Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections

The genome of Ob, a tobamovirus that overcomes the N gene-mediated hypersensitive response (HR), was cloned as a cDNA, and its nucleotide sequence was determined. The genomic organization of Ob is similar to that of other tobamoviruses, consisting of 6506 nucleotides and containing at least four open reading frames. These open reading frames encode a 126-kD polypeptide with a 183-kD readthrough product, a 30.6-kD movement protein, and an 18-kD coat protein. A bacteriophage T7 promoter sequence was fused to the full-length cDNA clone to obtain infectious RNA transcripts. These transcripts, when inoculated onto tobacco plants, induced disease symptoms indistinguishable from plants inoculated with Ob viral RNA. To determine which viral factor is responsible for the resistance-breaking character of Ob, a recombinant virus was constructed in which the movement protein gene of tobacco mosaic virus was replaced with that of Ob. Cultivar Xanthi NN tobacco plants infected with this virus responded with an HR, indicating that the Ob movement protein alone does not act to overcome the N gene-mediated response. Following mutagenesis of the infectious Ob cDNA clone with hydroxylamine, populations of transcripts from the mutagenized DNA were inoculated onto Xanthi NN tobacco, and a variant that induced the HR was identified. The mutant was analyzed and found to contain a single nucleotide change in the 126-kD gene. Recreating the mutation in the Ob cDNA clone by site-directed mutagenesis resulted in a virus that caused symptoms identical to the chemically induced mutant.

Analysis of a tobacco mosaic virus strain capable of overcoming N gene-mediated resistance.

The entire genomic RNA of a Spanish isolate of pepper mild mottle virus (PMMV-S), a resistance-breaking virus in pepper, was cloned and sequenced and shown to be similar to other tobamoviruses in its genomic organization. It consisted of 6357 nucleotides (nt) and contained four open reading frames (ORFs) which encode a 126K protein and a readthrough 183K protein (nt 70 to 4908), a 28K protein (nt 4909 to 5682) and a 17.5K coat protein (nt 5685 to 6158). This is the first tobamovirus in which none of the ORFs overlap. Both its nucleic acid and predicted protein sequences were compared with the previously determined sequences of other tobamoviruses. The variations and similarities found and their relationship with the pathogenicity of this virus are discussed.

Nucleotide sequence of the genomic RNA of pepper mild mottle virus, a resistance-breaking tobamovirus in pepper

During a four-year (1982–1985) survey of plant viruses infecting pepper cultivars grown under plastic in the Southeastern region of Spain, a tobamovirus was found to be the major disease agent of this crop. The virus produces slight or no symptoms on the leaves, but causes chlorotic mottling, malformation and reduction in size with occasional necrosis on the fruits and was able to infect all commercial pepper cultivars tested, including those resistant to other tobamoviruses, causing a catastrophic disease. The biological and serological characterization of the virus showed that it is very similar to pepper mild mottle virus (PMMV) (Wetteret al. 1984) and therefore we have termed it as Spanish strain of PMMV (PMMV-S). The need of grouping all the so-called “pepper strains” of tobacco mosaic virus (TMV) as a new distinct member of the tobamovirus group with the name of PMMV is emphasized.

A Tobamovirus Causing Heavy Losses in Protected Pepper Crops in Spain

A Spanish strain of pepper mild mottle virus (PMMV-S) (Alonsoet al 1989) can be differentiated from the Italian PMMV (Wetteret al 1984) by the responses of Capsicum spp with resistance genes to tobamoviruses, by radioimmunoassay and by the electrophoretic mobility of their viral particles Moreover, the analysis of the electrophoretic mobility of the viral particles in agarose gels and that of the viral coat proteins in polyacrylamide-urea gels are reliable and rapid techniques for distinguishing PMMV isolates from other members of the tobamovirus group and thus can be used for diagnostic purposes These results support the proposal of grouping these pepper viruses, including the “pepper strains” of TMV, as a new tobamovirus subgroup (PARES 1985, BETTI et al 1987, WETTER et al 1987, ALONSO et al 1989)



ZusammenfassungCharakterisierung eines spanischen Stammes des Pepper Mild Mottle Virus (PMMV-S) und dessen Verwandtschaft mit anderen Tobamoviren



Ein spanischer Stamm des Pepper Mild Mottle Virus (PMMV-S) kann an Hand der Reaktionen von Capsicum spp mit Tobamovirenresistenzgenen, durch Radioimmunoassay und die elek-trophoretische Mobilitat dessen Viruspartikeln von dem italienischen PMMV unterschieden werden Auserdem sind die Analysen der elektrophoretischen Mobilitat der Viru spartikeln in Agarosegelen, und der der viralen Hullproteinen in Polyacrylamid-Ureagelen zuverlassige und schnelle Methoden, utn PMMV-lsolate von anderen Mitgliedern der Tobatnovirusgruppe zu unterscheiden und konnen daher auch fur diagnostische Zwecke benutzt werden Diese Ergebnisse unterstiitzen den Vorschlag, das diesc Paprika-Viren, auch die “pepper strains” des TMV, in einer neuen Sub-Gruppe des Tobamovirus zusammengestellt werden sollten

Characterization of a Spanish strain of pepper mild mottle virus (PMMV-S) and its relationship to other tobamoviruses.

Resistance in Capsicum spp. against the tobamoviruses

Tobacco mosaic virus pepper strains (TMV-ps) are becoming an important economic problem in protected pepper crops all over the world (1) since they are able to overcome all known genetic resistances to the main TMV so far found in Capsicum spp. (2). Inoculation of tobacco protoplasts with RNA of different well known TMV by electroporation has been recently accomplished (3,4,5). In order to iniciate studies on the mechanisms of pathogenesis and resistance to TMV-ps infections, RNA of a Spanish TMV-ps has been used to electroporate tobacco mesophyll protoplasts with a Bio-Rad Gene Pulser apparatus. Optimal electroporation conditions for protoplasts infection are reported here.

B. Wicke

Papers

Nucleotide sequence of the genomic RNA of pepper mild mottle virus, a resistance-breaking tobamovirus in pepper

A Tobamovirus Causing Heavy Losses in Protected Pepper Crops in Spain

Characterization of a Spanish strain of pepper mild mottle virus (PMMV-S) and its relationship to other tobamoviruses.

Resistance in Capsicum spp. against the tobamoviruses

Infection of Tobacco Protoplasts with RNA of a Pepper Strain of Tobacco Mosaic Virus by Electroporation