Plant viruses modify gene expression in infected tissues by altering the micro (mi)RNA-mediated regulation of genes. Among conserved miRNA targets there are transcripts coding for transcription factors, RNA silencing core, and disease-resistance proteins. Paralogs in these gene families are widely present in plant genomes and are known to respond differently to miRNA-mediated regulation during plant virus infections. Using genome-wide approaches applied to Solanum lycopersicum infected by a nuclear-replicating virus, we highlighted miRNA-mediated cleavage events that could not be revealed in virus-free systems. Among them we confirmed miR6024 targeting and cleavage of RX-coiled-coil (RX-CC), nucleotide binding site (NBS), leucine-rich (LRR) mRNA. Cleavage of paralogs was associated with short indels close to the target sites, indicating a general functional significance of indels in fine-tuning gene expression in plant-virus interaction. miR6024-mediated cleavage, uniquely in virus-infected tissues, triggers the production of several 21-22 nt secondary siRNAs. These secondary siRNAs, rather than being involved in the cascade regulation of other NBS-LRR paralogs, explained cleavages of several mRNAs annotated as defence-related proteins and components of the photosynthetic machinery. Outputs of these data explain part of the phenotype plasticity in plants, including the appearance of yellowing symptoms in the viral pathosystem.

Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5'-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene

Many viral suppressors (VSRs) counteract antiviral RNA silencing, a central component of the plant's immune response by sequestration of virus-derived antiviral small interfering RNAs (siRNAs). Here, we addressed how VSRs affect the activities of cellular microRNAs (miRNAs) during a viral infection by characterizing the interactions of two unrelated VSRs, the Tombusvirus p19 and the Cucumovirus 2b, with miRNA 162 (miR162), miR168, and miR403. These miRNAs regulate the expression of the important silencing factors Dicer-like protein 1 (DCL1) and Argonaute proteins 1 and 2 (AGO1 and AGO2), respectively. Interestingly, while the two VSRs showed similar binding profiles, the miRNAs were bound with significantly different affinities, for example, with the affinity of miR162 greatly exceeding that of miR168. In vitro silencing experiments revealed that p19 and 2b affect miRNA-mediated silencing of the DCL1, AGO1, and AGO2 mRNAs in strict accordance with the VSR's miRNA-binding profiles. In Tombusvirus-infected plants, the miRNA-binding behavior of p19 closely corresponded to that in vitro Most importantly, in contrast to controls with a ?p19 virus, infections with wild-type (wt) virus led to changes of the levels of the miRNA-targeted mRNAs, and these changes correlated with the miRNA-binding preferences of p19. This was observed exclusively in the early stage of infection when viral genomes are proposed to be susceptible to silencing and viral siRNA (vsiRNA) concentrations are low. Accordingly, our study suggests that differential binding of miRNAs by VSRs is a widespread viral mechanism to coordinately modulate cellular gene expression and the antiviral immune response during infection initiation.IMPORTANCE Plant viruses manipulate their hosts in various ways. Viral suppressor proteins (VSRs) interfere with the plant's immune response by sequestering small, antivirally acting vsiRNAs, which are processed from viral RNAs during the plant's RNA-silencing response. Here, we examined the effects of VSRs on cellular microRNAs (miRNAs), which show a high degree of similarity with vsiRNAs. Binding experiments with two unrelated VSRs and three important regulatory miRNAs revealed that the proteins exhibit similar miRNA-binding profiles but bind different miRNAs at considerably different affinities. Most interestingly, experiments in plants showed that in the early infection phase, the Tombusvirus VSR p19 modulates the activity of these miRNAs on their target mRNAs very differently and that this differential regulation strictly correlates with the binding affinities of p19 for the respective miRNAs. Our data suggest that VSRs may specifically control plant gene expression and the early immune response by differential sequestration of miRNAs.

AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.

RNA silencing is a defense mechanism exploited by plants against viruses. Upon infection, viral genomes and their transcripts are processed by Dicer-like (DCL) ribonucleases into viral small interfering RNAs (vsRNAs) of 21-24 nucleotides that further guide silencing of viral transcripts. To get an insight into the molecular interaction between tomato and the monopartite phloem-limited begomovirus tomato yellow leaf curl Sardinia virus (TYLCSV), a pathogen inducing a devastating disease of tomato in the Mediterranean region, we characterized by deep sequencing the vsRNA population in virus-infected tomato plants, using a Solexa/Illumina platform. TYLCSV-sRNAs spanned the entire viral genome but were discontinuously distributed throughout it, with a prevalence from the transcribed regions. TYLCSV-sRNAs were mainly 21-22 nucleotides in length and their polarity was asymmetrically distributed along the genome. The most abundant vsRNAs originated from a narrow region overlapping the Rep/C4 genes and from a broader region including the end of the V2 and the beginning of the coat protein genes. Deep sequencing results were validated by different hybridization techniques. Comparisons with the data available on vsRNAs for other begomoviruses highlighted both similarities and differences. Host-derived RNA species cross-reacting with a portion of the viral genome corresponding to the most abundant vsRNAs hotspot were detected. Bioinformatics analyses were carried out to investigate the nature of these host molecules.

Over the past years deep sequencing experiments have opened novel doors to ?reconstruct viral populations in a high-throughput and cost-effective manner. Currently a ?substantial number of studies have been performed which employ Next Generation Sequencing (NGS) techniques to either analyse known viruses by means of a reference-guided approach or to discover novel viruses using a de novo-based strategy. Taking ?advantage of the well-known Cymbidium ringspot virus we have carried out a comparison of ?different bioinformatics tools to reconstruct the viral genome based on 21-27 nt short (s)RNA sequencing with the aim to identify the most efficient pipeline. The same approach was ?applied to a population of plants constituting an ancient variety of Cicer arietinum with red ?seeds. Among the discovered viruses, we describe the presence of a Tobamovirus referring to the Tomato mottle mosaic virus (NC_022230), which was not yet observed on C. arietinum nor revealed in Europe and a viroid referring to Hop stunt viroid (NC_001351.1) never reported in chickpea. Notably, a reference sequence guided approach appeared the most efficient in such kind of investigation. Instead, the de novo assembly reached a non-appreciable coverage although the most prominent viral species could still be identified. ?Advantages and limitations of viral metagenomics analysis using sRNAs are discussed.

Plant viruses modify gene expression in infected tissues by altering the micro (mi)RNA-mediated regulation of genes. Among conserved miRNA targets there are transcripts codifying for transcription factors, RNA silencing core and disease resistance proteins. Paralogs in these gene families are widely present in plant genomes and are known to respond differently to miRNA-mediated regulation during plant virus infections. Using genomic-wide approaches applied to Solanum lycopersicum infected by a nuclear-replicating virus we highlighted miRNA-mediated cleavage events that could not be revealed in virus-free systems: among them, we confirmed targeting of one paralogous Argonaute1, seven transcriptional factors from five different families cleaved by miR156, miR160, miR166, miR169 and miR172 and one RX-Coiled-coil (RX-CC), nucleotide binding (NBS) mRNA cleaved by miR6024. Interestingly, in most cases short indels close to the target sites discriminated cleavage of duplicates, indicating a functional significance of indels in fine tuning gene expression in plant-virus interaction. The same analysis was extended to Cicer arietinum plants and we could confirm that indels in close vicinity to the miRNA target sites of duplicated genes could discriminate miRNA-mediated post-transcriptional control of genes. Furthermore, we show that the miR6024 target site overlap to some extent to that of miR482 in the NBS-LRR. miR6024-mediated cleavage, uniquely in virus-infected tissues, triggers the production of several unique 21nt secondary siRNAs. These secondary siRNAs explained cleavages of several mRNAs annotated as involved in plant-pathogen interaction and appearance of viral symptoms.

Virus infections are accompanied by massive amount of viral (v) short-interfering (si)RNAs, due to the plant antiviral RNA silencing machinery. Recently, virus-activated (va) endogenous siRNAs have been also found associated to viral infections and are believed to play key roles in plant-virus interaction. Cauliflower mosaic virus (CaMV) is one of the important pathogens infecting members of Brassicaceae family with economic impact. Here we describe a functional characterization of CaMV v-siRNAs in either infected turnip (Brassica rapa), a sensitive host to CaMV, or canola (Brassica napus) as semisensitive host. We found that vsiRNA derive from all the viral genome, thus in contrast with what previously observed in the model plant Arabidopsis. Importantly, the vsiRNA distribution did not differ between turnip and canola, thus suggesting the different sensitivity of the two hosts to CaMV infection is not due to the sole production of vsiRNAs. Indeed, here we showed CaMV infections were accompanied by the production of endogenous sRNAs associated to diverse genomic regions, likely CaMV-triggered vasiRNAs. CaMV-triggered vasiRNAs are 21-24 nucleotide in length and are associated to either coding genes or non-coding loci with a 2-to-1 ratio between sense/antisense referred to the orientation of the gene. Also we found some endogenous sRNAs that were produced only in mock-inoculated plants but not in infected types. Notably, most of the coding genes derived vasiRNAs of both CaMV-infected turnip/canola, were from genes involved at different steps of the photosynthesis process. On the other hands, most of non-coding derived vasiRNAs were from 18s rRNA and 28s rRNA.

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a widely spread virus affecting Vitis spp. Although it establishes a compatible viral interaction in V. vinifera without the development of phenotypic alterations, it can occur as distinct variants that showed different symptoms on diverse Vitis species. In the present work, we analyzed the changes induced by GRSPaV in Vitis vinifera cv 'Bosco', an Italian white-grape variety, by combining agronomic, physiological and molecular approaches, in order to provide global information about the effects of GRSPaV. This virus induces a moderate decrease in physiological efficiency, yield performance and berry sugar content, associated to several transcriptomic alterations. Transcript changes were assessed by microarray analysis in petiole, leaf and berry samples collected at véraison, and by quantitative real-time RT-PCR. Global gene expression analyses showed some unexpected responses in grapevine never reported before for other plant-virus interactions, such as the increase in transcription of genes involved in photosynthesis and CO2 fixation associated to a moderate decrease in photosynthesis rate, the decrease of some defence mechanisms and overlapping responses to drought and salinity stresses. Basing on these last considerations, we hypothesized an interaction between GRSPaV-grapevine-drought and we subjected GRSPaV-free and infected plants to a drought treatment under controlled conditions. The virus changes grapevine responses to drought: GRSPaV-infected plants show higher stomatal opening, grow more than GRSPaV-free plants under water stress condition, and are able to extract more water from the soil. Furthermore, we prepared 4 libraries of small RNAs (GRSPaV-free and infected plants upon well watered and stress conditions), which were sequenced by Illumina technology. We observed a surprising profile of miRNAs associated with the presence of GRSPaV in plants upon water stress treatment. Viruses are obliged infectious entities and several studies have already showed that they may provide beneficial effects to the host in particular environmental conditions. We hypothesized that the long co-existence between grapevine and GRSPaV resulted in the evolution of a form of mutual adaptation between the virus and its host.

Virus-derived short interfering RNAs (vsiRNAs) isolated from grapevine V. vinifera Pinot Noir clone ENTAV115 were analyzed by high-throughput sequencing using the Illumina Solexa platform. We identified andcharacterized vsiRNAs derived from grapevine field plants naturally infected with different viruses belongingto the genera Foveavirus, Maculavirus, Marafivirus and Nepovirus. These vsiRNAs were mainly of 21 and 22nucleotides (nt) in size and were discontinuously distributed throughout Grapevine rupestris stem-pittingassociated virus (GRSPaV) and Grapevine fleck virus (GFkV) genomic RNAs. Among the studied viruses, GRSPaVand GFkV vsiRNAs had a 52 terminal nucleotide bias, which differed from that described for experimental viralinfections in Arabidopsis thaliana. VsiRNAs were found to originate from both genomic and antigenomicGRSPaV RNA strands, whereas with the grapevine tymoviruses GFkV and Grapevine Red Globe associatedvirus (GRGV), the large majority derived from the antigenomic viral strand, a feature never observed in otherplant–virus interactions.

Tomato yellow leaf curl Sardinia virus (TYLCSV) is a Begomovirus with a genome consisting of a circular single-stranded DNA molecule ca. 2800 bases in length. Its bidirectional promoter drives the generation of viral RNA transcripts coding for viral proteins required for its entire life cycle in the host plant. The RNA silencing machinery recognises the viral transcripts as foreign RNAs, thus leading on to the production of viral small interfering RNAs (v-siRNAs), likely responsible for v-siRNA-mediated antiviral defense. A cDNA library of small RNAs was generated from tissues of TYLCSV-infected tomato plants and sequenced on Solexa/Illumina sequencing platform. The subset of v-siRNAs was therefore identified; the major size classes of TYLCSV-derived siRNAs were 21 and 22 nt species spanning the entire viral genome but being discontinuously distributed throughout it. The most abundant v-siRNAs are from CP and C4 genes, whereas those from the intergenic region are poorly represented. Moreover, the majority of v-siRNAs are of sense polarity suggesting that they are from folded single-stranded viral transcript, similarly to what observed in the case of other v-siRNAs from positive stranded RNA plant viruses. The possible mechanism/s of v-siRNA biogenesis and their role in antiviral plant response will be also discussed.

Serine hydroxymethyltransferase (SHMT) is an ubiquitous enzyme present in prokaryotes and eukaryotes, includingbacteria, yeasts, plants, animals and humans. SHMT is essential for cellular one-carbon and folate metabolisms, and it isinvolved in pathogenesis phenomena: in humans, mutations in SHMT have been associated to a wide range of diseasedevelopment, whereas in plants, specific versions of SHMT can determine resistance to phytoparasitic nematodes (PPNs).PPNs cause economically important crop losses worldwide. PPNs acquire folate from their diet at developing feeding sites in the root apparatus. Modifications of the plant's folate pathway lead to a nutritional deficiency that starves the nematode and causes the degeneration the feeding cells.In model systems such as Glycine max L. infected by PPNs, the role of SHMT in host-nematode interaction mediated is further supported by the characterization of two resistance loci, i.e. Rgh1 and Rgh4 and the identification of functional SNPs. Accessions of Solanum Lycopersicum L., and Pisum sativum L., known to be resistant to PPNs have not yet described at the SHMT loci. Therefore, we have amplified and sequenced Rgh1 in such accessions in order to explore the presence of functional SNPs described in gmRhg1. The Rgh1 sequences carry amino-acidic sequence typical of the susceptible gmRhg1, both in resistant tomato and in pea, i.e. SNP130 (R130P) and SNP358 (Y358N). We have extended this analysis to tolerant accessions of Cicer arietinum L. to PPNs and we have confirmed what it was already known in tomato and pea.However, expression analysis in roots and shoots revealed significant up-regulation of caRgh1 exclusively in PPNs-infectedplants. The analysis suggests that a different not-fully characterized mechanism of resistance exists in host-PPN interactions.

In plants, RNA silencing is a surveillance mechanism against invading viruses. It involves the production of virus-derived small interfering RNAs (vsiRNAs), which guide the RNA-induced silencing complex (RISC) to inactivate viruses. vsiRNAs may also promote the silencing of host mRNAs in a sequence-specificmanner. In this work, vsiRNAs derived from two grapevineinfecting viruses (Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus) were selected from cDNA libraries of short RNAs and were cross-referenced with the remnants of both cleaved host transcripts and viral RNAs from a degradome dataset. We identified dozens of host transcripts targeted byvsiRNAs. Among them, several encode putative proteins involved in ribosome biogenesis and in biotic and abiotic stresses. Moreover, we identified vsiRNAs which explain the cleavage sites in viral genomes. A consistent fraction of vsiRNAs did not apparentlyaccount for cleavage, suggesting that only a low percentage of vsiRNAs are involved in the antiviral response.

In plants, RNA silencing is a surveillance mechanism against invading viruses. It involves the production of virus-derived small interfering RNAs (vsiRNAs), which guide the RNA-induced silencing complex (RISC) to inactivate viruses. Moreover, vsiRNAs may promote silencing of host mRNAs in a sequence-specific manner. To date, little is known about the efficiency of these vsiRNAs in guiding effector complexes to cleave target viral sequences or host transcripts. We have identified vsiRNAs derived from two grapevine-infecting viruses (Grapevine fleck virus and Rupestris stem pitting-associated virus) using the Illumina platform for deep sequencing. To better understand the role of vsiRNAs, a degradome analysis of viral RNA 5'-ends was analysed; it can identify the remnants of cleaved host transcripts and viral RNA targets. By integrating vsiRNA and degradome data, we have identified ca. 50 host transcripts targeted by vsiRNAs including. Among them, several encode putative proteins involved in ribosome biogenesis and in biotic and abiotic stresses and in other cellular pathways. Moreover, we have identified vsiRNAs which explains cleavage sites in viral genomes. A consistent fraction of vsiRNAs did not account for cleavage sites, suggesting that relatively few vsiRNAs are involved in the antiviral response. Aspects of vsiRNA-driven RNA targeting are discussed

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a virus that infects grapevines and establishes a compatible interaction in Vitis vinifera without the development of macroscopic phenotypic alterations. Some unexpected responses are induced in V. vinifera by GRSPaV, including overlapping responses to drought and salinity stress. In the frame of a CNR project (Progetto Premiale AQUA), we hypothesised an interaction between GRSPaV and drought and we subjected GRSPaV-free and infected plants to water stress under controlled conditions. By investigating ecophysiological parameters, we show that GRSPaV activates in V. vinifera a physiological state that induces resilience to drought. Indeed, infected plants under controlled water stress conditions show (i) a high rate of photosynthesis and stomatal conductance; (ii) low hydraulic resistance to water transport; and (iii) more growth than GRSPaV-free plants. The molecular basis of these virus-grapevine-drought interactions is still poorly understood; however, it is reasonable to hypothesise an involvement of RNA silencing, which is a natural defence against invading viruses in plants. Micro(mi)RNAs have fundamental roles in plant development and adaptation to stresses through post transcriptional control of several physiological pathway intermediates. The analysis of four libraries of small RNAs (i.e., from GRSPaV-free and infected plants, both in well watered and stress conditions) allowed us to highlight some known miRNAs and novel miRNA candidates that could be correlated with the physiological modifications in plants upon water stress treatment. Targets of known (i.e. miR396, miR164, miR156, miR3633) and novel miRNAs (miRC121, miRC129, miRs409712_2) annotated as players in drought stress adaptations were indeed validated. The relevance of miRNAs in the interaction between GRSPaV infection and water stress is not defined only by their tissue abundance but mainly by their activity on targeted mRNAs. In infected plants, the high photosynthesis reported above was accompanied by higher stomatal conductance, which was also linked to modifications in leaf morphology (high stomatal density and cell number). In infected grapevines, we observed the regulation of several miRNAs and their targets involved in leaf development (miR156, miR164, miR319, miR394, miR396). The grapevine is a woody plant that has been cultivated and selected together with its viruses for centuries. Our data support the idea of mutual adaptation between GRSPaV and grapevine, resulting in beneficial effects for the host under water stress conditions. Therefore, GRSPaV might represent the first example of a plant virus that is more appropriately defined by the categories of 'conditional mutualism' and 'beneficial virus' proposed by Roossinck (2011).

Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most prevalent viruses that infects grapevines and belongs to the genus Foveavirus, in the Betaflexiviridae family. We have shown previously that plants of V. vinifera cv Bosco infected by the GRSPaV-1 sequence variant exerted a very moderate decrease in physiological efficiency and yield. Importantly, this response overlapped with molecular responses to water and salinity stress. These considerations prompted us to investigate possible interactions between GRSPaV and abiotic stress. The molecular basis of these interactions is still poorly understood; however, it is reasonable to hypothesise an involvement of RNA silencing, which is a natural defence against invading viruses in plants. Both miRNAs and siRNAs are involved in several developmental processes in defence responses to biotic and abiotic stresses and in genome stability maintenance, as reviewed in many studies. We analysed the ecophysiological and molecular interactions between GRSPaV infection and drought in grapevine. During the progressive drying of soil, the stomatal conductance (gs) and net photosynthesis (Pn) of both infected and GRSPaV-free sets of plants decreased in response to water stress; however, this response was delayed in infected plants Prolonged drought conditions are known to reduce or block growth. However, in both irrigated and water stress conditions, infected plants showed a greater leaf area, height, and stem growth diameter compared to GRSPaV-free plants. In the small endogenous RNAs libraries produced from leaves of infected and GRSPaV-free plants under WW and SWS, we identified members of almost all known miRNAs and 25 novel miRNA candidates. The analysis of the data revealed that the accumulation of some known miRNAs was clearly associated with GRSPaV infection. In addition, when GRSPaV-infected plants were subjected to water stress, the ratio of miRNA accumulation between infected and GRSPaV-free grapevines changed significantly. Interestingly, this study clearly shows that the sanitary status of the plants may play a determinant role in miRNA regulation during abiotic stress. Our data support the idea of mutual adaptation between GRSPaV and grapevine, resulting in beneficial effects for the host under water stress conditions. Therefore, GRSPaV might represent the first example of a plant virus that is more appropriately defined by the categories of 'conditional mutualism' and 'beneficial virus'.

The plant Argonaute 1 protein (AGO1) is a central functional component of the posttranscriptional regulation of gene expression and the RNA silencing based antiviral defence..By genomic and molecular approaches we here reveal the presence of two homeologs of the AGO1-like gene in Nicotiana benthamiana, NbAGO1-1H and NbAGO1-1L. Both homeologs retain the capacity to transcribe mRNAs, which mainly differ in one 18-nucleotides insertion/deletion (indel). The indel does not modify the frame of the open reading frame, and it is located 8 nucleotides upstream of the target site of a micro RNA, miR168, which is an important modulator of AGO1 expression. We demonstrate that there is a differential accumulation of the two NbAGO1-1 homeolog mRNAs at conditions where miR168 is up-regulated such as during a Tombusvirus infection. The data reported suggest that the indel affects the miR168-guided regulation of NbAGO1 mRNA. The two AGO1 homeologs show full functionality in reconstituted, catalytically active RNA induced silencing complexes following the incorporation of siRNAs. Virus-Induced Gene Silencing experiments suggest a specific involvement of the NbAGO1 homeologs in symptom development. The results provide an example of the diversity of miRNA target regions in NbAGO1 homeolog genes, which has important implications for improving resilience measures of the plant during viral infections.

The Argonaute1 (ago1) gene was first discovered in Arabidopsis thaliana and further described trough plant mutants that showed pleiotropic developmental anomalies of all plant organs. In fact, most miRNAs are incorporated into AGO1 and guide the RNA Induced Silencing Complex (RISC) to its mRNA target through sequence complementarity; as a result, the mRNA translation is regulated by endonucleolytic cleavage or other yet incompletely characterized mechanisms. AGO1 homeostasis is in part coordinated through a feedback mechanism. Thus, miR168-guided cleavage of ago1 mRNA was demonstrated to ensure an optimal balance of miRNA steady-state levels for plant development and in response to environmental stimuli (rev. in [1]). Beside its role in PTGS and in concert with other proteins of the Argonaute clade, AGO1 plays a key role in the plant RNA silencing-based antiviral defence: similar to miRNAs, viral-derived (v)siRNAs are incorporated into AGO1-containing RISC leading to inactivation of viral RNAs by cleavage (rev. in [2] and [3]).Here we show that N. benthamiana possesses two ago1-1 homeolog genes, which differ mainly by one 18-nucleotide long insertion/deletion (indel). The indel is located close to the miR168 target site and our data suggests that its presence considerably affects the miR168-guided post-transcriptional regulation of ago1 mRNA. The indel effect is here highlighted during a viral infection, which increases miR168 accumulation. The two NbAGO1 homeologs show full functionality in reconstituted, catalytically active RISC following the incorporation of siRNAs. Moreover, Virus-Induced Gene Silencing (VIGS) experiments suggest a specific, redundant involvement of the ago1 homeologs in susceptibility to the viral infection but a divergent involvement in symptom development. The expression of two types of Nbago1-1 mRNAs is proposed to represent a case of diversity and of evolutionary adaptation to improve resilience measures of the plant during viral infections or other stresses accompanied by miR168 up-regulation. (the participation of VP to sfb648 is in part supported by project SaVeGrainIN- Puglia Reg. (CE) 1698/2005)

A common challenge in pathogen discovery by deep sequencing approaches is to recognize viral or subviral pathogens in samples of diseased tissue that share no significant homology with a known pathogen. Here we report a homology-independent approach for discovering viroids, a distinct class of free circular RNA subviral pathogens that encode no protein and are known to infect plants only. Our approach involves analyzing the sequences of the total small RNAs of the infected plants obtained by deep sequencing with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Viroid infection triggers production of viroid-derived overlapping siRNAs that cover the entire genome with high densities. PFOR retains viroid-specific siRNAs for genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, which is synthesized from circular or multimeric repeated-se- quence templates during viroid replication. We show that viroids from the two known families are readily identified and their full- length sequences assembled by PFOR from small RNAs sequenced from infected plants. PFOR analysis of a grapevine library further identified a viroid-like circular RNA 375 nt long that shared no sig- nificant sequence homology with known molecules and encoded active hammerhead ribozymes in RNAs of both plus and minus polarities, which presumably self-cleave to release monomer from multimeric replicative intermediates. A potential application of the homology-independent approach for viroid discovery in plant and animal species where RNA replication triggers the biogenesis of siRNAs is discussed.

In plants, microRNAs (miRNAs) comprise one of three classes of small RNAs regulating gene expression at post-transcriptional level. Many plant miRNAs are conserved and play a role in development, abiotic stress responses or pathogen responses. However, some miRNAs have been found only in certain species. Here, we use deep sequencing, computational and molecular methods to identify, profile and describe conserved and non-conserved miRNAs in four grapevine (Vitis vinifera) tissues. 24 conserved miRNA families were identified in all four tissues and 26 known but non-conserved miRNAs were also found. In addition to known miRNAs we also found 21 new grapevine specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. Furthermore, 21 other plausible miRNAs candidates have been described. We have found that many known and new miRNAs showed tissues-specific expression. Finally, 112 target mRNAs of known and 44 target mRNAs of new grapevine specific miRNAs were identified by genomic scale high-throughput sequencing of miRNA cleaved mRNAs.

Gli approcci metagenomici consentono oggi di tipizzare e valorizzare il patrimonio genetico autoctono attraverso la caratterizzazione dettagliata e concisa della variabilità in loci di particolare interesse. Fondamentale è peraltro la necessità di rivelare e studiare la biodiversità sia intesa come valorizzazione delle risorse genetiche di colture mediterranee sia come interazioni tra ospite, endofiti, agenti di controllo e agenti nocivi (virus e nematodi) per una spiccata sostenibilità ambientale della protezione e della conservazione del germoplasma autoctono. Il presente studio pone l'attenzione sulle varietà antiche: piante adattate alle condizioni climatiche di un dato territorio e che contribuiscono incisivamente al mantenimento della sua biodiversità. Inoltre, tra gli agenti patogeni, i nematodi fitoparassiti sono responsabili di ingenti perdite di produzione agricole a causa della limitata disponibilità di efficaci mezzi di lotta. L'identificazione di geni di resistenza a questi patogeni resta il luogo privilegiato del loro controllo e del miglioramento genetico delle varietà ad essi attualmente suscettibili.Nel presente lavoro si ricorre alla metagenomica al fine di verificare l'esistenza in varietà locali di Cicer arietinum di un meccanismo di resistenza a nematodi cisticoli precedentemente individuato in soia. La metodologia prevede l'individuazione di SNPs funzionali attraverso un approccio NGS (next generation sequence)-assisted. Oligonucleotidi degenerati e contenenti specifici barcodes sono stati opportunamente disegnati e validati sulla varietà locale "Cece rosso di Gioia del Colle" (provincia di Bari). Al campionamento in campo rappresentativo della popolazione, segue l'estrazione dell'RNA totale, la sintesi del cDNA e la generazione delle librerie di ampliconi sequenziabili mediante piattaforma Illumina. L'analisi della variabilità ai loci sequenziati consente di rivelare la presenza o l'assenza della resistenza ai nematodi cisticoli nell'ambito della popolazione di piante. L'esatta individuazione dell'eventuale individuo resistente è attualmente ancora in fase di studio.La metodologia ivi presentata contiene aspetti innovativi, versatili, ad un costo tale da poter essere applicata su materiale proveniente da diverse aree geografiche ed in vivaio, in campo, in serra. Sfrutta inoltre la potenzialità delle NGS di analizzare contemporaneamente un numero molto elevato di plantule consentendo di individuare l'organismo resistente anche se questo è rappresentato in bassa percentuale all'interno di un pool. Lo strumento potrebbe anche generare interesse nelle aziende sementiere e vivai per la selezione genetica NGS-assisted, per individuare semi che produrranno piante resistenti a nematodi fitopatogeni o ad altri patogeni che hanno un elevato impatto sull'erosione di varietà antiche.

L'articolo illustra gli obiettivi ed i risultati più salienti relativi al PSR Regione Puglia progetto SaVeGraINPuglia.

Genetic engineering for improvement of the recalcitrant crop chickpea (Cicer arietinum L.) was largely restricted by the lack of an efficient regeneration system. In vitro regeneration in two Egyptian chickpea varieties, Giza 531 and Giza 4 was achieved by direct organogenesis. A variety of embryo explants and different types and concentrations of growth regulators were investigated for maximum efficiency of shoot and root regeneration. Embryo axes with the adjacent part of cotyledon proved to be the most promising type of explant for shooting and rooting responses. 6-Benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were found to induce the highest percentages of shoot initiation and root formation, respectively. Although the Giza 531 variety produced a better response than the Giza 4 for shoot formation, it displayed lower performance for root induction. It would be rewarding if this optimized regeneration protocol paved the way toward the genetic improvement of the Egyptian chickpea.

Plant viruses modify gene expression in infected tissues by altering the micro (mi)RNA-mediated regulation of genes. Among conserved miRNA targets there are transcripts coding for transcription factors, RNA silencing core and disease resistance proteins. Paralogs in these gene families are widely present in plant genomes and are known to respond differently to miRNA-mediated regulation during plant virus infections. Using genome-wide approaches applied to Solanum lycopersicum infected by a nuclear-replicating virus we highlighted miRNA-mediated cleavage events that could not be revealed in virus-free systems: among them, we confirmed the targeting of one of the two Argonaute1 paralogs, seven transcriptional factors from five different families cleaved by miR156, miR160, miR166, miR169 and miR172 and one RX-Coiled-coil (RX-CC), nucleotide binding (NBS), leucine rich (LRR) mRNA cleaved by miR6024. Interestingly, in most cases short indels close to the target sites discriminated cleavage of duplicates, indicating a functional significance of short indels in fine-tuning gene expression in plant-virus interaction. miR6024-mediated cleavage, uniquely in virus-infected tissues, triggers the production of several unique 21nt secondary siRNAs. These secondary siRNAs, rather than being involved in the cascade regulation of other NBS-LRR paralogs, explained cleavages of several mRNAs annotated as defence related proteins and components of the photosynthetic machinery. Outputs of these data explain part of the phenotype plasticity in plants, including the appearance of yellowing symptoms in the viral pathosystem.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the post-transcriptional control of several pathway intermediates, thus playing pivotal roles in plant growth, development and response to biotic and abiotic stresses. In recent years, the grapevine genome release, small(s)-RNAseq and degradome-RNAseq together has allowed the discovery and characterisation of many miRNA species, thus rendering the discovery of additional miRNAs difficult and uncertain. Taking advantage of the miRNA responsiveness to stresses and the availability of virus-free Vitis vinifera plants and those infected only by a latent virus, we have analysed grapevines subjected to drought in greenhouse conditions. The sRNA-seq and other sequence-specific molecular analyses have allowed us to characterise conserved miRNA expression profiles in association with specific ecophysiological parameters. In addition, we here report 12 novel grapevine-specific miRNA candidatesand describe their expression profile. We show that latent viral infection can influence the miRNA profiles of V. vinifera in response to drought. Moreover, study of eco-physiological parameters showed that photosynthetic rate, stomatal conductance and hydraulic resistance to water transport were significantly influenced by drought and viral infection. Although no unequivocal cause-effect explanation could be attributed to each miRNA target, their contribution to the drought response is discussed.

The consistent identification of Fig badnavirus 1 (FBaV-1) in the totality of samples from mosaic-affected fig trees from New Zealand (Minafra et al., 2012) and Italy (unpublished informa- tion), prompted the examination of additional samples from adult fig trees with mosaic symptoms from Italy (10), France (1), Greece (3), Albania (1), Spain (1), Portugal (1), England (1), Hungary (3), Montenegro (1), Lebanon (19), Syria (11), Tunisia (15), Algeria (2), Turkey (4), South Africa (1), Mexico (1), Cuba (1), and Aus- tralia (1). Additional samples from Italy consisting of volunteer symptomless seedlings (20) and symptomless potted plants de- rived from explants of mosaic-affected trees subjected to heat therapy (4), brought to over 100 the total number of sources ana- lyzed. Total RNA was silica-extracted from cortical scrapings and subjected to RT-PCR using the primers P1s: 5'-GCT GAT CAC AAG AGG CAT GA-3' and P1as: 5'-TCC TTG TTT CCA CGT TCC TT-3' designed on the sequence deposited in GenBank un- der the accession No. JF411989. A product with the expected size (214 bp) was amplified from all samples, regardless of the geo- graphical origin (18 countries) and the type of source (adult trees, seedlings, heat-treated plants). These results confirm what appears to be an amazing and unique association of FBaV-1 with Ficus car- ica. The extent of the association is such (100%) and involves trees from such a wide range of geographical origins, that makes it plausible, as in the case of other pararetroviruses (Staginnus and Richert-Pöggeler, 2006), the suggestion that FBaV-1 may be inte- grated in the fig genome; a likelihood further supported by the virus presence in volunteer fig seedlings.

Il silenziamento dell'RNA è un meccanismo al quale studi recenti attribuiscono un ruolo fondamentale nella regolazione dell'espressione genica a livello sia trascrizionale sia posttrascrizionale. Si basa sul riconoscimento di tipo sequenza specifico del gene o dell'RNA messaggero da parte di piccole molecole di RNA (sRNA) presenti nella maggior parte degliorganismi eucariotici. Queste classi di molecole mediano un gran numero di diverse funzioni e, nelle piante, è ampiamente dimostrato un ruolo chiave nell'interazione ospite-patogeno, con conseguente effetto sull'espressione di fattori specifici dell'ospite coinvolti nella soppressione o nell'attivazione dei meccanismi di difesa della pianta. In questa nota si descrivono le tipologie di sRNA presenti in pianta e la loro funzione biologica, con particolare riferimento a quella di naturale difesa antivirale. Spazio viene anche dato alle applicazioni biotecnologiche che sono diventate oggetto di brevetto negli ultimi anni, in seguito alla scoperta dei meccanismi di silenziamento genico.

Next-generation sequencing has opened the door to the reconstruction of viral populations and examination of the composition of mutant spectra in infected cells, tissues, and host organisms. In this chapter we present details on the use of the Shannon entropy method to estimate the site-specific nucleotide relative variability of turnip crinkle virus, a positive (+) stranded RNA plant virus, in a large dataset of short RNAs of Cicer arietinum L., a natural reservoir of the virus. We propose this method as a viral metagenomics tool to provide a more detailed description of the viral quasispecies in infected plant tissue. Viral replicative fitness relates to an optimal composition of variants that provide the molecular basis of virus behavior in the complex environment of natural infections. A complete description of viral quasispecies may have implications in determining fitness landscapes for host-virus coexistence and help to design specific diagnostic protocols and antiviral strategies.

A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.

RNA silencing in plants and insects can function as a defence mechanism against invading viruses. RNA silencing-based antiviral defence entails the production of virus-derived small interfering RNAs which guide specific antiviral effector complexes to inactivate viral genomes. As a response to this defence system, viruses have evolved viral suppressors of RNA silencing (VSRs) to overcome the host defence. VSRs can act on various steps of the different silencing pathways. Viral infection can have a profound impact on the host endogenous RNA silencing regulatory pathways; alterations of endogenous short RNA expression profile and gene expression are often associated with viral infections and their symptoms. Here we discuss our current understanding of the main steps of RNA-silencing responses to viral invasion in plants and the effects of VSRs on endogenous pathways. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.

Cicer arietinum L. (chickpea) is the world's fourth most widely grown pulse. Chickpea seeds are a primary source of dietary protein for humans, and chickpea cultivation contributes to biological nitrogen fixation in the soil, given its symbiotic relationship with rhizobia. Therefore, chickpea cultivation plays a pivotal role in innovative sustainable models of agro-ecosystems inserted in crop rotation in arid and semi-arid environments for soil improvement and the reduction of chemical inputs. Indeed, the arid and semi-arid tropical zones of Africa and Asia have been primary areas of cultivation and diversification. Yet, nowadays, chickpea is gaining prominence in Canada, Australia and South America where it constitutes a main ingredient in vegetarian and vegan diets. Viruses and plant parasitic nematodes (PPNs) have been considered to be of minor and local impact in primary areas of cultivation. However, the introduction of chickpea in new environments exposes the crop to these biotic stresses, compromising its yields. The adoption of high-throughput genomic technologies, including genome and transcriptome sequencing projects by the chickpea research community, has provided major insights into genome evolution as well as genomic architecture and domestication. This review summarizes the major viruses and PPNs that affect chickpea cultivation worldwide. We also present an overview of the current state of chickpea genomics. Accordingly, we explore the opportunities that genomics, post-genomics and novel editing biotechnologies are offering in order to understand chickpea diseases and stress tolerance and to design innovative control strategies.

The begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) induces a devastating disease of tomato crops in the Mediterranean region. TYLCSV has a 2.7 kb circular single-stranded DNA genome that includes a bidirectional promoter driving the synthesis of viral RNA transcripts. These are recognised by the plant RNA silencing machinery as foreign RNAs that generate viral small interfering RNAs (v-siRNAs). Methods: A cDNA library of small RNAs was obtained from TYLCSV-infected tomato plants and sequenced on Solexa/Illumina platform. The subset of v-siRNAs was identified and characterized. Results: TYLCSV-derived siRNAs, mainly of 21-22 nt, spanned the entire viral genome but were discontinuously distributed throughout it. Viral-siRNAs from the non-coding region were poorly represented; the most abundant v-siRNAs were from the coat protein and the Rep/C4 genes and were mainly of sense polarity. The major hot spots were experimentally validated by reverse oligo dot blot analysis. Conclusions: The first high-resolution v-siRNA map for TYLCSV is reported. The mechanism/s of v-siRNA biogenesis and their role in antiviral plant response are discussed.