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Item does not contain fulltext The phenotypic manifestations of disease induced by viruses and subviral infectious entities are the result of complex molecular interactions between host and viral factors. The viral determinants of the diseased phenotype have traditionally been sought at the level of structural or non-structural proteins. However, the discovery of RNA silencing mechanisms has led to speculations that determinants of the diseased phenotype are caused by viral nucleic acid sequences in addition to proteins. RNA silencing is a gene regulation mechanism conserved within eukaryotic kingdoms (with the exception of some yeast species), and in plants and insects it also functions as an antiviral mechanism. Non-coding RNAs of viral origin, ranging in size from 21 to 24 nucleotides (viral small interfering RNAs, vsiRNAs) accumulate in virus-infected tissues and organs, in some cases to comparable levels as the entire complement of host-encoded small interfering RNAs. Upon incorporation into RNA-induced silencing complexes, vsiRNAs can mediate cleavage or induce translational inhibition of nucleic acid targets in a sequence-specific manner. This review focuses on recent findings that suggest an increased complexity of small RNA-based interactions between virus and host. We mainly address plant viruses, but where applicable discuss insect viruses as well. Prominence is given to studies that have indisputably demonstrated that vsiRNAs determine diseased phenotype by either carrying sequence determinants or, indirectly, by altering host-gene regulatory pathways. Results from these studies suggest biotechnological applications, which are also discussed.

"Virus infections are accompanied by massive production of viral small interfering RNAs due to the plant antiviral RNA silencing machinery. In addition, plant viruses with RNA genome infecting Arabidopsis were recently shown to trigger the production of virus-activated (va) endogenous siRNAs, that in turn drive a cis-silencing of gene expression. Our studies aimed to carry out a functional characterization of va-siRNAs in the case of crop plants infected by plant viruses having DNA genome. By NGS and bioinformatics, we have characterized small (s)RNA populations in Brassica rapa and B. napus infected by Cauliflower Mosaic Virus (CaMV), a plant pararetrovirus (double-stranded DNA genome). We found that CaMV infections were accompanied by the production of endogenous sRNA of 21- and 22-nucleotide in length, bona fide va-siRNA, from diverse protein-coding genes (CDS). A large proportion of the CDS generating va-siRNAs encode several components of the photosynthetic machinery. Accordingly, we revealed a significant down-regulation of transcripts generating va-siRNA, likely due to cis-silencing of gene expression.va-siRNAs were also found in a different plant pathosystem, i.e. Solanum lycopersicum-Tomato Leaf Curl Yellowing Sardinia Virus (TYLCSV), a plant virus with single-stranded DNA genome. Similarly to CaMV infections, some va-siRNAs in tomato explained cleavages of several mRNAs annotated as defense-related proteins and components of the photosynthetic pathway. However, in tomato functional va-siRNAs appeared to drive trans-silencing of gene expression.Accordingly to RNA virus infections, here we confirm that either CaMV or TYLCSV infections triggered va-siRNA. va-siRNAs were from the different class of genes depending on the taxonomic family to which the host belongs. Several components of the photosynthetic machinery were main targets of va-siRNAs thus revealing unifying qualitative features among plants infected from viruses of either RNA or of DNA genomes. Outputs of these data explain part of the phenotype plasticity in plants. Virus infections are accompanied by massive production of viral small interfering RNAs due to the plant antiviral RNA silencing machinery. In addition, plant viruses with RNA genome infecting Arabidopsis were recently shown to trigger the production of virus-activated (va) endogenous siRNAs, that in turn drive a cis-silencing of gene expression. Our studies aimed to carry out a functional characterization of va-siRNAs in the case of crop plants infected by plant viruses having DNA genome. By NGS and bioinformatics, we have characterized small (s)RNA populations in Brassica rapa and B. napus infected by Cauliflower Mosaic Virus (CaMV), a plant pararetrovirus (double-stranded DNA genome). We found that CaMV infections were accompanied by the production of endogenous sRNA of 21- and 22-nucleotide in length, bona fide va-siRNA, from diverse protein-coding genes (CDS). A large proportion of the CDS generating va-siRNAs encode several components of the photosynthetic machinery. Accordingly, we revealed a significant down-regulation of transcripts generating va-siRNA, likely due to cis-silencing of gene expression.va-siRNAs were also found in a different plant pathosystem, i.e. Solanum lycopersicum-Tomato Leaf Curl Yellowing Sardinia Virus (TYLCSV), a plant virus with single-stranded DNA genome. Similarly to CaMV infections, some va-siRNAs in tomato explained cleavages of several mRNAs annotated as defense-related proteins and components of the photosynthetic pathway. However, in tomato functional va-siRNAs appeared to drive trans-silencing of gene expression.Accordingly to RNA virus infections, here we confirm that either CaMV or TYLCSV infections triggered va-siRNA. va-siRNAs were from the different class of genes depending on the taxonomic family to which the host belongs. Several components of the photosynthetic machinery were main targets of va-siRNAs thus revealing unifying qualitative features among plants infected from viruses of either RNA or of DNA genomes. Outputs of these data explain part of the phenotype plasticity in plants. "

"siRNAs are the central players of RNAi in plants, fungi, insects, nematodes and mammals. Plant DCLs are encoded by multigenic families and fall into four clades containing orthologs of DCL1, DCL2, DCL3 and DCL4 genes originally discovered in Arabidopsis thaliana (At) and conserved in all plants, including Nicotiana benthamiana (Nb). In At, DCL1 primarily generates 21-22 nt miRNAs, while DCL4, DCL2 and DCL3 mainly generate 21, 22 and 24 nt endogenous siRNAs, respectively. Upon viral infection, all four DCLs generate viral siRNAs: DCL4 and DCL2 produce 21 and 22 nt siRNAs in defense against cytoplasmic RNA viruses, while DCL3 and, less efficiently, DCL1 produce 24 and 21 nt siRNAs in defense against nuclear DNA viruses. The application of siRNAs for crop protection is still limited, mainly because of poor knowledge about the biogenesis and function of distinct siRNA size-classes. A HA-tagged version of theNb DCL4 (HANbDCL4) was cloned under the control of the copper-inducible promoter CUP1, transformed into Saccharomyces cerevisiaeYPH499 cells and successfully expressed. The protein expression was detected by Western blot analysis and varied along with the growth temperature. Differential centrifugation of protein extracts was used to separate a membrane-enriched and soluble cytoplasmic fractions. Western blot analysis showed that HANbDCL4 was mostly associated to the cell membrane fraction. In addition, it was only partially removed after 0.1 M Na2CO3or 1 M KCl treatments. The partial association of HANbDCL4 to cell membranes was confirmed by immunofluorescence analysis of yeast cells, in which the expressed HANbDCL4 was mostly detected close to the nuclear membrane. HANbDCL4 was expressed in yeast cells together with the cymbidium ringspot virus defective interfering (DI) RNA and RNA dependent RNA polymerase (p92). Robust DI RNA replication was not affected by HANbDCL4 expression. By small RNA-seq preliminary evidences were obtained that HANbDCL4 is functional in generating sRNAs of viral origin when expressed in the tombusvirus-S. cerevisiae heterologous system."

In response to a viral infection, the plant's RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, a very fewof these siRNAs actually interfere with viral replication. A reliable approach to identify these immunologically effective siRNAs ( esiRNAs) and to define the characteristics underlying their activity has not been available so far. Here, we develop a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a virtual full protection of plants against a massive subsequent infection in transient applications. We find that the functionality of esiRNAs depends crucially on two properties: the binding affinity to Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional esiRNAs could be of great benefit for all RNA silencing-based plant protection measures against viruses and other pathogens.

A massive and diverse population of short non-coding (s)RNAs accumulates in plant tissues. sRNAs drive RNA silencing-based gene repression. Besides their role in tissue and organ development, differentiation and in the maintenance of genome integrity, sRNA play crucial roles in host response to a wide range of environmental conditions, including biotic stresses. By image data augmentation technique, we have selected Brassica rapa (turnip) and B. napus (canola) plant tissues infected by cauliflower mosaic virus (CaMV) showing mosaic and yellowing symptoms. sRNA populations associated to CaMV-infected tissues of the two crops were characterized by sequencing and bioinformatics and compared with those from the model pathosystem CaMV-Arabidopsis. We revealed orthologues genes producing endogenous sRNAs shared out among turnip, canola and Arabidopsis. In the three plant species, the majority of these siRNAs were from protein coding genes encoding components of the photosynthetic machinery, such as LHCB1.3, LHCB1.4, LHCA1, and FBA1. By quantitative real time PCR we revealed a significant down-regulation of transcripts generating the endogenous RNA in the two crops. Moreover, by in vitro assays we have proved the functionality of selected sRNAs in cleaving the corresponding RNA targets upon incorporation into specific core proteins of the class of "Argonautes". The results herein suggested that the generation of sRNA regulators from specific gene clusters of the photosynthetic machinery is an unifying qualitative feature among plant species of the Brassicaceae family under biotic stress.

SiRNAs are the central players of RNAi in plants, fungi, insects, nematodes and mammals. Plant DCLs are encoded by multigenic families and fall into four clades containing orthologs of DCL1, DCL2, DCL3 and DCL4 genes originally discovered in Arabidopsis thaliana (At) and conserved in all plants, including Nicotiana benthamiana (Nb). In At, DCL1 primarily generates 21-22 nt miRNAs, while DCL4, DCL2 and DCL3 mainly generate 21, 22 and 24 nt endogenous siRNAs, respectively. Upon viral infection, all four DCLs generate viral siRNAs: DCL4 and DCL2 produce 21 and 22 nt siRNAs in defense against cytoplasmic RNA viruses, while DCL3 and, less efficiently, DCL1 produce 24 and 21 nt siRNAs in defense against nuclear DNA viruses. The application of siRNAs for crop protection is still limited, mainly because of poor knowledge about the biogenesis and function of distinct siRNA size-classes. A HA-tagged version of the Nb DCL4 (HANbDCL4) was cloned under the control of the cupper inducible promoter CUP1, transformed into Saccharomyces cerevisiae YPH499 cells and successfully expressed. The protein expression was detected by Western blot analysis and varied along with the growth temperature. Differential centrifugation of protein extracts was used to separate a membrane-enriched and a soluble cytoplasmic fractions. Western blot analysis showed that HANbDCL4 was mostly associated to the cell membrane fraction. In addition, it was only partially removed after 0.1 M Na2CO3 or 1 M KCl treatments. The partial association of HANbDCL4 to cell membranes was confirmed by immunofluorescence analysis of yeast cells, in which the expressed HANbDCL4 was mostly detected close to the nuclear membrane. HANbDCL4 was expressed in yeast cells together with the cymbidium ringspot virus defective interfering (DI) RNA and RNA dependent RNA polymerase (p92). Robust DI RNA replication was not affected by HANbDCL4 expression. By small RNA-seq preliminar evidences were obtained that HANbDCL4 is functional in generating sRNAs of viral origin when expressed in the tombusvirus-S. cerevisiae heterologous system.

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.

Serine hydroxymethyltransferase (SHMT) is an ubiquitous enzyme present in prokaryotes and eukaryotes, including bacteria, yeasts, plants, animals and humans. SHMT is essential for cellular one-carbon and folate metabolisms, and it is involved in pathogenesis phenomena: in humans, mutations in SHMT have been associated to a wide range of disease development, 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.

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.

This volume explores the use of viral metagenomics to diagnose known viruses for plant and food production, human and animal health, and identifying viral vectors like insects. The chapters in this book cover topics, such as sRNAs isolation from tissues of grapevines and woody plants, high-resolution screening of arthropod and plant viral communities, identifying new pathogens in fish, detecting viruses in mycorrhizal fungi and their orchid host, and insect virus discovery through metagenomic and cell culture-based approaches. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and comprehensive, Viral Metagenomics: Methods and Protocols is a valuable resource for researchers and specialists who are interested in learning more about this evolving field.

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.

Nell'ambito dei Programmi di Sviluppo Rurale, P.S.R. Puglia 2007/2013, destinati al miglioramento del Sistema Regionale della biodiversità (Reg. CE n. 1698/2005, PSR Puglia 2007/2013, Misura 214 - Azione 4 Sub-azione a; e successivo P.S.R. Puglia 2014-2020, Misura 10 pagamenti-agro-climatico- ambientali, Art. 28 Reg. UE n. 1305/2013 Sottomisura 10.2) è stato finanziato il progetto: "Recupero, caratterizzazione, salvaguardia e valorizzazione di leguminose e cereali da granella e foraggio in Puglia" (SaVeGraINPuglia). Questo volume include schede descrittive delle leguminose, cereali e foraggere reperite in Puglia acquisite in regione e caratterizzate con approccio multidisciplinare nell'ambito del proggetto SaVeGraINPuglia.

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.

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.

Epigenetics describes phenomena associated with changes in gene expression that occur without apparent modification in genomic sequence. In plants, it explains at least in part phenotypic differences between genetically identical plants under environmental stimuli. In this manuscript, we describe and discuss studies carried out by the scientific community regarding epigenetic phe- nomena that are likely linked to plant-pathogen interaction.

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.

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.

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)

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'.

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.

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.

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 degli organismi 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.

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-specific manner. 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 by vsiRNAs. 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 apparently account for cleavage, suggesting that only a low percentage of vsiRNAs are involved in the antiviral response.

Nelle piante, i piccoli RNA interferenti regolatori (miRNAs e siRNAs) di lunghezza di 21-24 nucleotidi sono prodotti da specifiche ribonucleasi (DICER) aventi come substrato RNA precursori a doppia elica. I miRNA ed i siRNAs di origine endogena, tipicamente attivano specifici complessi proteici e riconoscono, per complementarieta, Le molecole di mRNAs bersaglio provocandone una sotto-regolazione post-trascrizionale. Gli RNA a doppia elica di origine virale, sono altresi substrato dei DICERs ed inducono la produzione siRNAs virali (vsiRNAs). I vsiRNAs possono guidare la disattivazione degli RNA virali analogamente alIa regolazione post-trascrizionale mediata dei miRNAs. Durante il periodo di collaborazione bilaterale CNR-MTA 2010-2012, sono stati congiuntamente investigati alcuni aspetti molecolari regolatori del silenziamento genico dell'RNA I coinvolti nello sviluppo e differenziazione delle piante e nella difesa antivirale.?

Background: 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.