Belén Martínez-García

Structural and Genetic Requirements for the Biogenesis of Tobacco Rattle Virus-Derived Small Interfering RNAs

ABSTRACT In plants, small RNA-guided processes referred to as RNA silencing control gene expression and serve as an efficient antiviral mechanism. Plant viruses are inducers and targets of RNA silencing as infection involves the production of functional virus-derived small interfering RNAs (siRNAs). Here we investigate the structural and genetic components influencing the formation of Tobacco rattle virus (TRV)-derived siRNAs. TRV siRNAs are mostly 21 nucleotides in length and derive from positive and negative viral RNA strands, although TRV siRNAs of positive polarity are significantly more abundant. This asymmetry appears not to correlate with the presence of highly structured regions of single-stranded viral RNA. The Dicer-like enzyme DCL4, DCL3, or DCL2 targets, alone or in combination, viral templates to promote synthesis of siRNAs of both polarities from all regions of the viral genome. The heterogeneous distribution profile of TRV siRNAs reveals differential contributions throughout the TRV genome to siRNA formation. Indirect evidence suggests that DCL2 is responsible for production of a subset of siRNAs derived from the 3′ end region of TRV. TRV siRNA biogenesis and antiviral silencing are strongly dependent on the combined activity of the host-encoded RNA-dependent RNA polymerases RDR1, RDR2, and RDR6, thus providing evidence that perfectly complementary double-stranded RNA serves as a substrate for siRNA production. We conclude that the overall composition of viral siRNAs in TRV-infected plants reflects the combined action of several interconnected pathways involving different DCL and RDR activities.

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

BackgroundDouble-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.ResultsWe 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.ConclusionOur 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.

In vitro characterization of a miR-122-sensitive double-helical switch element in the 5′ region of hepatitis C virus RNA

It has been proposed that the hepatitis C virus (HCV) internal ribosome entry site (IRES) resides within a locked conformation, owing to annealing of its immediate flanking sequences. In this study, structure probing using Escherichia coli dsRNA-specific RNase III and other classical tools showed that this region switches to an open conformation triggered by the liver-specific microRNA, miR-122. This structural transition, observed in vitro, may be the mechanistic basis for the involvement of downstream IRES structural domain VI in translation, as well as providing a role of liver-specific miR-122 in HCV infection. In addition, the induced RNA switching at the 5′ untranslated region could ultimately represent a new mechanism of action of micro-RNAs.

Host-dependent differences during synergistic infection by Potyviruses with potato virus X.

SUMMARY A comparative analysis of the synergistic interaction between PVX and either PVY or TEV potyviruses was performed in Nicotiana benthamiana and N. tabacum plants. In each PVX/potyvirus combination, doubly infected plants developed much more severe symptoms than singly infected ones. However, while PVX accumulation increased in doubly infected N. tabacum plants compared with singly infected plants, the accumulation of PVX did not vary drastically in doubly infected N. benthamiana plants with respect to single infected ones. These findings suggest that the relationship between viral titre enhancement and synergism in PVX/potyvirus infections is host dependent. Since PVX and potyviruses contain suppressors of a plant antiviral defence system mediated by gene silencing, differences observed in the response of these two related hosts to PVX/potyvirus interactions might reflect the effect of these viruses on host specific antiviral defences.

A Single Amino Acid Mutation in the Plum pox virus Helper Component-Proteinase Gene Abolishes Both Synergistic and RNA Silencing Suppression Activities

ABSTRACT The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-proteinase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PVX recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombinant virus expressing a variant of PPV HC-Pro containing a single point mutation ( HCL(134)H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL(134)H mutant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally, we compared the effects on virus accumulation in N. benthamiana plants infected with either the PVX recombinant constructs or with native viruses in double infection experiments. PVX (+) and (-) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an increase in PVX pathology, compared with plants infected with PVX alone. This finding confirms that the enhancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro.

Chromatin regulates DNA torsional energy via topoisomerase II-mediated relaxation of positive supercoils.

Eukaryotic topoisomerases I (topo I) and II (topo II) relax the positive (+) and negative (−) DNA torsional stress (TS) generated ahead and behind the transcription machinery. It is unknown how this DNA relaxation activity is regulated and whether (+) and (−)TS are reduced at similar rates. Here, we used yeast circular minichromosomes to conduct the first comparative analysis of topo I and topo II activities in relaxing chromatin under (+) and (−)TS. We observed that, while topo I relaxed (+) and (−)TS with similar efficiency, topo II was more proficient and relaxed (+)TS more quickly than (−)TS. Accordingly, we found that the relaxation rate of (+)TS by endogenous topoisomerases largely surpassed that of (−)TS. We propose a model of how distinct conformations of chromatin under (+) and (−)TS may produce this unbalanced relaxation of DNA. We postulate that, while quick relaxation of (+)TS may facilitate the progression of RNA and DNA polymerases, slow relaxation of (−)TS may serve to favor DNA unwinding and other structural transitions at specific regions often required for genomic transactions.

Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage

By transporting one DNA double helix (T-segment) through a double-strand break in another (G-segment), topoisomerase II reduces fractions of DNA catenanes, knots and supercoils to below equilibrium values. How DNA segments are selected to simplify the equilibrium DNA topology is enigmatic, and the biological relevance of this activity is unclear. Here we examined the transit of the T-segment across the three gates of topoisomerase II (entry N-gate, DNA-gate and exit C-gate). Our experimental results uncovered that DNA transport probability is determined not only during the capture of a T-segment at the N-gate. When a captured T-segment has crossed the DNA-gate, it can backtrack to the N-gate instead of exiting by the C-gate. When such backtracking is precluded by locking the N-gate or by removing the C-gate, topoisomerase II no longer simplifies equilibrium DNA topology. Therefore, we conclude that the C-gate enables a post-DNA passage proofreading mechanism, which challenges the release of passed T-segments to either complete or cancel DNA transport. This proofreading activity not only clarifies how type-IIA topoisomerases simplify the equilibrium topology of DNA in free solution, but it may explain also why these enzymes are able to solve the topological constraints of intracellular DNA without randomly entangling adjacent chromosomal regions.

DNA Topology and Global Architecture of Point Centromeres

DNA is wrapped in a left-handed fashion around histone octasomes containing the centromeric histone H3 variant CENP-A. However, DNA topology studies have suggested that DNA is wrapped in a right-handed manner around the CENP-A nucleosome that occupies the yeast point centromere. Here, we determine the DNA linking number difference (ΔLk) stabilized by the yeast centromere and the contribution of the centromere determining elements (CDEI, CDEII, and CDEIII). We show that the intrinsic architecture of the yeast centromere stabilizes +0.6 units of ΔLk. This topology depends on the integrity of CDEII and CDEIII, but it is independent of cbf1 binding to CDEI and of the variable length of CDEII. These findings suggest that the interaction of the CBF3 complex with CDEIII and a distal CDEII segment configures a right-handed DNA loop that excludes CDEI. This loop is then occupied by a CENP-A histone complex, which does not have to be inherently right-handed.

Transient expression of homologous hairpin RNA interferes with PVY transmission by aphids

Hairpin RNAs have been used to confer resistance to viruses in plants through RNA silencing. However, it has not been demonstrated that RNA silencing was effective against inoculation by aphids of non-persistently transmitted viruses, the major route of plant virus spread in nature. As a proof-of-principle strategy, we made use of Agrobacterium tumefaciens to transiently express a hairpin RNA homologous to Potato virus Y (PVY) in plant tissues. A complete and specific interference with aphid transmission of PVY was achieved by inducers of RNA silencing, as demonstrated by specific siRNAs accumulation in agroinfiltrated tissues. To our knowledge, this is the first report of successful interference with non-persistent transmission of a plant virus using RNA interference.

Development and use of detection methods specific for Cucumber vein yellowing virus (CVYV)

Two methods for the detection of Cucumber vein yellowing virus (CVYV) on infected plants were developed, based on the information provided by cDNA clones covering the 3′-end of the genome of a Spanish isolate (CVYV-AILM). The sequenced portion of the CVYV-AILM genome showed a 96.6% aminoacid identity with that of a reported sequence of another CVYV isolate from Israel (Lecoq et al., 2000). The first detection method used a RNA specific probe for hybridization with nucleic acids extracted from infected plants. The probe was complementary to a portion of the CVYV genome including the C-terminal part of the NIb and most of the coat protein (CP) coding regions. The second detection method employed polyclonal antisera raised against recombinant viral CP expressed in bacteria. The specific antibodies were used to detect the presence of virus particles in plant extracts. Both procedures resulted in a highly specific detection of CVYV in plants infected with different isolates of the virus. No interference was observed with other cucurbit-infecting viruses. Sensitivities achieved were sufficient for routine diagnosis of the presence of the virus in plants.