César Llave

Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes

Plant virus infection involves the production of viral small RNAs (vsRNAs) with the potential to associate with distinct Argonaute (AGO)-containing silencing complexes and mediate diverse silencing effects on RNA and chromatin. We used multiplexed, high-throughput pyrosequencing to profile populations of vsRNAs from plants infected with viruses from different genera. Sense and antisense vsRNAs of 20 to 24 nucleotides (nts) spread throughout the entire viral genomes in an overlapping configuration; virtually all genomic nucleotide positions were represented in the data set. We present evidence to suggest that every genomic position could be a putative cleavage site for vsRNA formation, although viral genomes contain specific regions that serve as preferential sources of vsRNA production. Hotspots for vsRNAs of 21-, 22-, and 24-nt usually coincide in the same genomic regions, indicating similar target affinities among Dicer-like (DCL) enzymes. In the light of our results, the overall contribution of perfectly base paired double-stranded RNA and imperfectly base paired structures within single-stranded RNA to vsRNA formation is discussed. Our census of vsRNAs extends the current view of the distribution and composition of vsRNAs in virus-infected plants, and contributes to a better understanding of vsRNA biogenesis.

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.

Virus-derived small interfering RNAs at the core of plant-virus interactions.

Once a virus enters a cell, viral double-stranded RNA (dsRNA) is targeted by the RNA silencing machinery to initiate a cascade of regulatory events directed by viral small interfering RNAs (vsiRNAs). Recent genetic and functional studies along with the high-throughput sequencing of vsiRNAs have shed light on the genetic and structural requirements for virus targeting, the origins and compositions of vsiRNAs and their potential for controlling gene expression. The precise nature of the triggering molecules of virus-induced RNA silencing or the targeting constraints for viral genome recognition and processing represent outstanding questions that will be discussed in this review. The contribution of vsiRNAs to antiviral defense and host genome modifications has profound implications for our understanding of viral pathogenicity and host specificity in plants.

Identification of a novel microRNA (miRNA) from rice that targets an alternatively spliced transcript of the Nramp6 (Natural resistance‐associated macrophage protein 6) gene involved in pathogen resistance

Plants have evolved efficient defence mechanisms to defend themselves from pathogen attack. Although many studies have focused on the transcriptional regulation of defence responses, less is known about the involvement of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in plant immunity. This work investigates miRNAs that are regulated by elicitors from the blast fungus Magnaporthe oryzae in rice (Oryza sativa). Small RNA libraries were constructed from rice tissues and subjected to high-throughput sequencing for the identification of elicitor-responsive miRNAs. Target gene expression was examined by microarray analysis. Transgenic lines were used for the analysis of miRNA functioning in disease resistance. Elicitor treatment is accompanied by dynamic alterations in the expression of a significant number of miRNAs, including new members of annotated miRNAs. Novel miRNAs from rice are proposed. We report a new rice miRNA, osa-miR7695, which negatively regulates an alternatively spliced transcript of OsNramp6 (Natural resistance-associated macrophage protein 6). This novel miRNA experienced natural and domestication selection events during evolution, and its overexpression in rice confers pathogen resistance. This study highlights an miRNA-mediated regulation of OsNramp6 in disease resistance, whilst illustrating the existence of a novel regulatory network that integrates miRNA function and mRNA processing in plant immunity.

High-Throughput Sequencing, Characterization and Detection of New and Conserved Cucumber miRNAs

Micro RNAS (miRNAs) are a class of endogenous small non coding RNAs involved in the post-transcriptional regulation of gene expression. In plants, a great number of conserved and specific miRNAs, mainly arising from model species, have been identified to date. However less is known about the diversity of these regulatory RNAs in vegetal species with agricultural and/or horticultural importance. Here we report a combined approach of bioinformatics prediction, high-throughput sequencing data and molecular methods to analyze miRNAs populations in cucumber (Cucumis sativus) plants. A set of 19 conserved and 6 known but non-conserved miRNA families were found in our cucumber small RNA dataset. We also identified 7 (3 with their miRNA* strand) not previously described miRNAs, candidates to be cucumber-specific. To validate their description these new C. sativus miRNAs were detected by northern blot hybridization. Additionally, potential targets for most conserved and new miRNAs were identified in cucumber genome. In summary, in this study we have identified, by first time, conserved, known non-conserved and new miRNAs arising from an agronomically important species such as C. sativus. The detection of this complex population of regulatory small RNAs suggests that similarly to that observe in other plant species, cucumber miRNAs may possibly play an important role in diverse biological and metabolic processes.

High-throughput sequencing of Hop stunt viroid-derived small RNAs from cucumber leaves and phloem.

Small RNA (sRNA)-guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self-replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of viroid-derived sRNAs (vd-sRNAs) is usually associated with viroid infection. However, the pathways involved in the biogenesis of vd-sRNAs are largely unknown. Here, we analyse, by high-throughput pyrosequencing, the profiling of the Hop stunt viroid (HSVd) vd-sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd-sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd-sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd-sRNAs. Our sequence data suggest that viroid-derived double-stranded RNA functions as one of the main precursors of vd-sRNAs. Remarkably, phloem vd-sRNAs accumulated preferentially as 22-nucleotide species with a consensus sequence over-represented. This bias in size and sequence in the HSVd vd-sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd-sRNAs to the phloem tissue of infected cucumber plants.

MicroRNAs: more than a role in plant development?

SUMMARY Eukaryotic small RNA comprises several classes of 21-25 nucleotide non-coding RNA, of which microRNA (miRNA) has gained a great deal of attention because it is directly involved in controlling growth and development in plants. miRNAs are processed by the RNase III-like Dicer, although recent studies have implicated additional gene products in the step-wise maturation of miRNAs from their primary nuclear transcripts. They function as sequence-specific guides to trigger cleavage or translational repression of target mRNAs that have complementary sequences. Natural miRNA targets encode members of large families of transcription factors, which are collectively required for a number of developmental processes. In addition to developmental regulation, some miRNAs might be involved in specific physiological responses to several types of stresses, such as those induced by pathogen infections. Strikingly, the potyviruses, the largest group of plant RNA viruses, are able to interfere with miRNA-guided cleavage of multiple regulatory targets in plants, thus modulating gene expression of the host cell.

A meta-analysis reveals the commonalities and differences in Arabidopsis thaliana response to different viral pathogens.

Understanding the mechanisms by which plants trigger host defenses in response to viruses has been a challenging problem owing to the multiplicity of factors and complexity of interactions involved. The advent of genomic techniques, however, has opened the possibility to grasp a global picture of the interaction. Here, we used Arabidopsis thaliana to identify and compare genes that are differentially regulated upon infection with seven distinct (+)ssRNA and one ssDNA plant viruses. In the first approach, we established lists of genes differentially affected by each virus and compared their involvement in biological functions and metabolic processes. We found that phylogenetically related viruses significantly alter the expression of similar genes and that viruses naturally infecting Brassicaceae display a greater overlap in the plant response. In the second approach, virus-regulated genes were contextualized using models of transcriptional and protein-protein interaction networks of A. thaliana. Our results confirm that host cells undergo significant reprogramming of their transcriptome during infection, which is possibly a central requirement for the mounting of host defenses. We uncovered a general mode of action in which perturbations preferentially affect genes that are highly connected, central and organized in modules.

Analysis of the melon (Cucumis melo) small RNAome by high-throughput pyrosequencing

BackgroundMelon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21-24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon.ResultsWe constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analysed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans- acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs.ConclusionWe have discovered and analysed a large number of conserved and melon-specific sRNAs, including miRNAs and their potential target genes. This provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melon-virus interactions.

Silencing suppressor activity of the Tobacco rattle virus-encoded 16-kDa protein and interference with endogenous small RNA-guided regulatory pathways

Higher plants use RNA silencing as a defense mechanism against viral infections, but viruses may encode suppressor proteins that counteract these defenses. Several virus-encoded suppressors also exert an inhibitory effect on endogenous small RNA regulatory pathways. Here we characterized the Tobacco rattle virus-encoded 16-kDa (TRV-16K) protein as a suppressor that blocked local RNA silencing induced by single (s)- and double-stranded (ds) RNA, indicating that TRV-16K interfered with a step in the silencing pathway downstream of dsRNA formation. The suppressor activity of TRV-16K was severely compromised by moderate to high dosages of dsRNA inducer. When silencing was locally triggered by ssRNA or low levels of dsRNA, silencing suppression by TRV-16K was associated with reduced accumulation of silencing-related siRNAs. TRV-16K also prevented partially cell-to-cell movement and systemic propagation of silencing but not transitive amplification of RNA silencing. We showed that neither TRV nor TRV-16K caused a global deregulation of the microRNA-regulatory pathway in Arabidopsis, suggesting that interference with microRNA biology was not a prerequisite for TRV, and probably many other plant viruses, to develop systemic infections in plants.