Mixed viral infections are common in globe artichoke (Cynara cardunculus var. scolymus). Despite lack ofevident symptoms, production losses are relevant. A rapid and accurate detection is advisable for selection and marketingof plant propagation material.Objectives: Our aim was the evaluation of a suspension microbeads array system for simultaneous detection ofArtichoke latent potyvirus (ArLV), Artichoke Italian latent nepovirus (AILV), Artichoke mottled crinkle tombusvirus (AMCV),Tomato spotted wilt tospovirus (TSWV), Turnip mosaic potyvirus (TuMV), Cucumber mosaic cucumovirus (CMV), Potatovirus X (PVX), Tobacco mosaic tobamovirus (TMV) and Pelargonium zonate spot anulavirus (PZSV).Methods: For PCR, oligonucleotides were designed from conserved regions in the viral genomes, using an Illumina´sAssay Design Tool. The actin gene of C. cardunculus was used as control. The microbeads-based procedure started witha first PCR target amplification. A further target-specific primer extension (TSPE), holding a unique 5' motif, was thenperformed. During this step biotinylated dCTPs were incorporated into the extension products. These were then linked tothe holographic microbeads bearing an oligo complementary to the 5' motif. Probe labelling was performed withstreptavidin-Alexa-647, for final scan using an Illumina BeadXpress Reader.Conclusions: Multiplex fluorescent microbeads assays allowed the contemporary detection of several viruses in a singlesample. This highly flexible microarray approach used a suspension of barcoded microbeads bearing specific sequencemotifs. It favoured the tailoring of user defined applications. The method is also suitable for identification of SNPs in viralstrains, within a unique host phenotype.

Plant response to environmental stresses and pathogen attacks involves several biologicalprocesses that require fine and precise regulation at transcriptional and post-transcriptional levels.MicroRNAs (miRNAs) are small RNAs (sRNAs) widely diffused in animals and plants andimplicated in post-transcriptional regulation of gene transcripts. Identification of differentiallyexpressed miRNAs, following infection, may provide an insight into the processes involved insignalling and defence of plants against pathogens.In order to characterize artichoke miRNAs differentially expressed during fungus or virusinfection, we sequenced five sRNA libraries obtained from artichoke using Illumina technology.Libraries were obtained from leaves and roots of control plants, and from plants infected withTomato spotted wilt virus (TSWV), or Verticillium dahliae.After removing low quality reads and adapter sequences, all artichoke libraries wereannotated according to small noncoding RNAs contained in Rfam, and all previously known plantmiRNAs extracted from the miRNA Registry Database (miRBase Release 18).Change in miRNA read counts between infected and non infected artichoke tissues wasrecorded and used to select putative infection-responsive miRNAs. Differential expression ofmiRNAs was validated by quantitative real-time PCR (qPCR).Artichoke miRNA precursors were identified and their fold-back structure was predictedusing Mfold program. Artichoke miRNA target genes were also identified and characterizedaccording to the homologous Arabidopsis proteins.In conclusion, miRNAs involved in the response to virus or fungus infection were detectedand validated in artichoke plant tissues.

The biological and molecular characterization of a Potato virus Y (PVY) isolate, denoted PVY(C)-to, associated with a necrotic phenotype of tomato plants grown in the province of Foggia (Apulia, southern Italy) is reported. The fully sequenced PVY(C)-to genome consists of 9,691 nucleotides and is 94,8% similar to that of PVY LYE84.2, an isolate of the PVY(C) strain group. Using programs in RDP package, a putative recombination breakpoint was identified at approximately nucleotide position 2056 to 2632, corresponding to the HC-Pro/P3 coding region. The event seems to represent an introgression of a PVY strain group sequence and, in particular, of PVY-OZ, which is a non recombinant isolate of the 0 lineage. From this analysis PVY(C)-to appeared to be a recombinant isolate. The virus has a very weak infectivity in pepper and possesses a CP coding region characterised by a PVY(C2) restrictotype. Our results seem to support the hypothesis that PVY(C)-to is a recombinant isolate of the PVY(C2) strain group.

The biological and molecular characterization is reported of a Watermelon mosaic virus isolate,denoted WMV-Le, associated with a necrotic phenotype of watermelon plants grown in the Provinces ofLecce and Taranto (Apulia, southern Italy). The fully sequencedWMV-Le genome consists of 10,045 nucleotidesand is 99.1% similar to that of WMV-C05-270, a French isolate from melon of the WMV moleculargroup 3. Using recombination detection programRDP3, putative recombination breakpoints were identifiedclose to nucleotide positions 42 to 1892, covering the 5? UTR/P1/HC-Pro region. The event represents theinsertion of a sequence fragment of an isolate similar to WMV-FBR04-37 in the background of an isolate similarto WMV-FMF00-LL1. The field symptomatology was reproduced in watermelon plants grown in an experimental greenhouse but the virus induced severe symptoms also in Cucumis sativus, C. melo, Cucurbita maxima and C. pepo.

Obiettivo del progetto "Recupero, caratterizzazione, Salvaguardia e Valorizzazione di leguminose e cereali daGranella e foraggio IN Puglia" - SaVeGraINPuglia (Misura 214 azione 4 sub.a, PSR 2007-2013) sezione cerealicola è la preservazione delle risorse genetiche cerealicole nei luoghi dove da almeno 80 anni sono state tradizionalmentecoltivate e dove sono ancora oggi variamente distribuite negli 11 ambiti di paesaggio inclusi nel Piano PaesaggisticoTerritoriale Regionale (PPTR). A tal fine sono state sinergicamente avviate azioni volte al reperimento di informazionistoriche, raccolta di antiche risorse genetiche vegetali locali, allevamento delle stesse al fine di procedere alla lorocaratterizzazione morfologica e genetica, risanamento, conservazione in situ nei luoghi dove sono state individuate.Le informazioni storiche sono state reperite in biblioteche inserite in ciascuno dei sei poli regionali afferenti alSistema Bibliotecario Nazionale (SBN), in biblioteche regionali, collaborando con i Gruppi di Azione Locale,associazioni culturali, esperti di settore e raccogliendo testimonianze di anziani agricoltori che hanno memoria delletecniche colturali, proverbi, ricette, usi rituali relativi alle risorse.Sono stati raccolti circa 78 campioni ed i siti di reperimento georeferenziati. I campioni sono stati catalogati econservati ex situ a basse temperature o mediante propagazione. Nell'ambito territoriale del Gargano, Monti Dauni, AltaMurgia, Murgia dei Trulli ed Arco Jonico Tarantino sono stati reperiti grani duri (Triticum durum desf.) e grani teneri(Triticum aestivum L.) e tra quest'ultimi in prevalenza la Bianchetta. Nel Tavoliere salentino e Salento delle Serre,prevalentemente orzo (Hordeum vulgare L.) ed avena (Avena sativa L.). In tutta la regione e in particolare nel Salento,prevale come coltura cerealicola di antica tradizione il grano duro Senatore Cappelli. Una sezione dei campioni è statasottoposta a risanamento fitosanitario e caratterizzazione morfologica, genetica, tecnologica, nutrizionale. Lacaratterizzazione morfologica e la propagazione a fini conservativi sono state eseguite in campi catalogoopportunamente predisposti al fine di procedere alle verifiche di alcuni caratteri. Per sei risorse cerealicole è statoinoltre possibile completare l'intero ciclo di azioni previste dal progetto, inclusa la conservazione in situ pressol'azienda detentrice della risorsa. Per tutte sono state predisposte schede di segnalazione, storiche e di caratterizzazionenonché schede digitali seguendo le indicazioni riportate nel Piano Nazionale sulla Biodiversità di interesse Agricolo(PNBA) e nei descrittori dell'International Board for Plant Genetic Resources (IBPGR).L'integrazione dei dati rilevati nell'ambito di ciascuna attività di reperimento, risanamento, caratterizzazione,conservazione in situ ed ex situ, ha messo in luce un possibile modello di conservazione dinamica dei cereali.

Bacteria isolated from soil and rhizosphere samples collected in Peru from Andean crops were tested in vitro and in vivo to determine their potential as plant growth promoters and their ability to induce systemic resistance to Alternaria alternata in tomato plants. The isolates were identified by sequencing their 16S ribosomal RNA gene. Test for phosphate solubilization, and indolacetic acid were also carried out, together with in vitro antagonism assays in dual cultures towards the plant pathogens Fusarium solani, A. alternata and Curvularia lunata. The three most promising isolates (Pa15, Ps155, Ps168) belonged to the genus Pseudomonas. Further assays were carried out with tomato plants to assess their plant protection effect towards A. alternata and as growth promoters. Inoculation of tomato seeds with all isolates significantly enhanced seed germination, plantlets emergence and plant development. Bacterial inoculation also reduce damage level caused by A. alternata. The expression levels of three tomato genes involved in the jasmonate (AOS), ethylene responsive (ERF-2) and pathogenesis related (PR-P2) pathways were determined in plants challenged with A. alternata, alone or with each bacterial isolate, respectively. Results showed that at 24 hours after infection, in absence of the pathogen, the expression level of the tested genes was very low. The presence of A. alternata alone and in combination with bacteria increased the transcripts of all genes. Data showed a potential of best performing isolate Ps168 to sustain tomato plants nutrition and activate defense-related genes for protection by pathogenic fungi.

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-Fny”2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVYSON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

L'istituto di Bioscienze e BioRisorse del Consiglio Nazionale delle Ricerche (CNR-IBBR) ha coordinatole attività dei venti partner del progetto provvedendo alle verifiche scientifiche ed amministrativesuddivise secondo le differenti macro-tipologie di risorse raccolte ed oggetto del programmaapprovato: leguminose, cereali e foraggere. L'attività di coordinamento scientifico si è avvalsadell'ausilio di tre aziende esterne che hanno agevolato, con i loro servizi, il raggiungimento degliobiettivi definiti nell'ambito delle attività di indagini storiche e bibliografiche, rilievi, raccolta eprima caratterizzazione, caratterizzazione morfologica, raccolta dati relativi agli agricoltori perla conservazione in situ, immissione dati nella banca dati di progetto, supporto agli utenti per laimmissione dati. Per le attività di coordinamento relativa alla gestione amministrativa e alla verificacontabile amministrativa ci si è avvalsi di una società e, per la gestione dati AGEA, di un consulenteesterno che si è occupato della immissione dati sul portale. E' stato infine affidato ad una aziendalocale la gestione dei tre eventi programmati nell'ambito del progetto e che hanno visto convolti tuttii partner.L'organigramma interno è servito a definire, nell'ambito delle due macro-tipologie leguminose ecereali, le aree d'intervento del personale del CNR-IBBR di Bari. Sono stati pertanto creati gruppi adhoc per indagini storiche, esplorazione del territorio, conservazione, caratterizzazione, risanamento,immissione dati, gestione informatica, segreteria e gestione amministrativa.

Relative abundance of host siRNAs, and their distribution in different size classes, was severely altered at 21 dpi and, at a lesser extent, at 30 dpi, as compared to healthy plants. MicroRNAs (miRNAs) accounted for a 2.8% of total reads mapping to the tomato genome in mock-inoculated plants, and this figure raised up to 8.5% in infected plants at 21 dpi. Fifty-seven miRNA species showed at least a two-fold increase and 56 at least a two-fold decrease in infected vs. healthy plants at 21 dpi, and most of them were similarly altered at both timepoints. MiRNA target genes, whose expression regulation was shown or predicted to be differentially modulated in infected plants, belong to specific functional categories involving transcription factors, kinases and genes with oxidoreductase activity, which may partially explain the disease symptoms induced by the virus. Abundant secondary siRNAs (e.g. phasiRNAs), depending on an upstream small RNA trigger and subsequent RDR and DCL activities, were induced by virus infection and shown to be biologically active by driving cleavage of pathogen-responsive genes, such as receptor-like kinases (RLKs).

RNA silencing (RS) is a conserved eukaryotic mechanism acting as anti-viral immune systemin plants. Small interfering RNAs (siRNAs) are RS effectors accumulating in virus-infected tissuesas immune system components, providing target specificity for post-transcriptional degradation ofinvading RNAs. Virus-derived siRNAs (vsiRNAs, 21-24 nt) are abundant in infected plants, withcertain regions ("hot spots") more represented than others.Two isolates of Potato virus Y (PVY), PVYC-to and PVY-SON41, yield very differentdisease phenotypes on tomato (Solanum lycopersicum), the former inducing leaf distortions, thelatter mild symptoms. We applied in silico and molecular approaches to identify PVY vsiRNAssuppressing host mRNAs by sequence complementarity and RS-based suppression, inducingdysfunctional processes upon infection. Aim was to explore differential expression of host-targetingvsiRNA, as responsible of isolates differences.A computational pipeline retrieved 21nt vsiRNAs from PVY isolates, complementary topredicted mRNAs (Solgenomics, ITAG2.3). Two PVYC-to genome regions showed specificsecondary structures absent in PVY-SON41, accounting for vsiRNAs accumulation. Two tomatotranscript lists were obtained, perfectly or imperfectly (one or two mismatches) complementary tovsiRNA computed for either PVY isolates, putative targets of vsiRNA-driven RS suppression.Targets common to both isolates were discarded, leaving a gene list representing only PVYC-totargets. Quantitative RT-PCR of transcription factors (e.g. NAC, MYB, TCP, HD-ZIP, MADS-boxfamilies) active in leaf development and morphogenesis showed differential expression uponPVYC-to and PVY-SON41 infections. Lower isolate-specific mRNA accumulation suggested RSdrivenpost-transcriptional regulation, confirming a link between symptoms and vsiRNAs-mediatedRS. Results from vsiRNAs deep sequencing from infected leaves are also discussed.

Lo studio delle varietà locali di "Fagioli di Sarconi" di interesse per il progetto VAL.BIO.LUC., finalizzato all'identificazione di genotipi superiori, necessita ad integrazione della loro valutazione agronomica, di dati sulle caratteristiche nutrizionali e tecnologiche della granella. Nell'ambito del progetto VAL.BIO.LUC., l'IBBR di Bari si è occupato di valutare sul materiale fornito dall'azienda Belisario di Sarconi (PZ) partner del progetto, i seguenti parametri:tra i macro-costituenti è stato determinato il contenuto in proteine grezze poiché per il fagiolo nell'ambito di questa tipologia, questo è il parametro con maggiore impatto sul valore nutrizionale del seme;tra i parametri legati alla fase di ammollo, preliminare al consumo, sono stati determinati l'indice di idratazione (HI), l'indice di rigonfiamento (SI), la percentuale in tegumento e il tempo di cottura;tra i fattori antinutrizionali è stato quantificato il contenuto in acido fitico. Inoltre, è stato condotto un saggio per verificare lo stato fitosanitario dei "Fagioli di Sarconi". Sono stati presi in considerazione due virus molto importanti spesso presenti in Phaseolus vulgaris L.: il virus del mosaico giallo del fagiolo (Bean yellow mosaic virus BYMV, Potyvirus) trasmissibile da afidi e il virus del mosaico comune del fagiolo (Bean common mosaic virus BCMV, Potyvirus), trasmesso per seme.

Non-coding small RNAs (sRNAs) regulate gene expression in plant development and stress response. Different sRNA, in particular microRNAs (miRNAs) are principal mediators of the plant immune system. Roots endophytic bacteria and fungi affect crops performance acting as key soil factors underpinning ecosystem functioning and determining the properties of associated biota. Objectives We aimed at identifying plant miRNAs involved in the tri-trophic interaction of a beneficial bacterium (Pseudomonas sp.) and Solanum lycopersicum challenged with the pathogenic fungus Alternaria alternata, through comparative analyses carried out in different plant-microorganism associations.Methods sRNA profiling was performed on 3-week-old plants of S. lycopersicum plants treated with Pseudomonas sp. (Ps155) or challenged with the pathogen, alone or in combination, or non-treated as control. RNA extraction and library construction was carried out in two biological replicates per treatment and sequenced by Illumina technology. The sRNA libraries were aligned to the tomato genome (release 2.40). Reads (100% matching) were mapped onto the miRBase release 21.0 to identify known miRNAs. Differential miRNA expression (DE) analysis was based on DESeq2.Conclusions 334 known tomato miRNAs were identified in the sRNA libraries, of which near 50% were shared across libraries. The four treatments were evaluated in pairwise comparisons to gain evidence for their DE among treatments. Statistical analyses highlighted 27 differential regulated miRNAs. These DE targeted different tomato genes including transcription and growth-regulating factors based on computational target prediction. Data provide a comparative analysis of changes in the small RNA profiles and the putative gene targets they control.

Molti cereali originari del bacino del Mediterraneo e coltivati in Italia mostrano un'ampia gamma di popolazioni locali ed ecotipi, altamente adattate al loro ambiente (landraces). Esse possono essere caratterizzate attraverso i loro fenotipi, morfotipi e le proprietà organolettiche. La resa e qualità di queste colture è ancora ampiamente dipendente da diversi stress, incluso le malattie causate da batteri, funghi e virus, che hanno un forte impatto sulle produzioni. Nell'ambito del PSR "SaVeGraINPuglia" abbiamo valutato lo stato fitosanitario, per quanto concerne i virus, di landraces di cereali pugliesi campionate in diversi siti e habitat regionali. Determinarne lo stato sanitario è il primo passo per proteggere e migliorare la qualità delle collezioni. Le sintomatologie variano in funzione delle landraces e degli agenti virali responsabili dell'infezione. In alcuni casi le malattie non sono sintomatiche, passando inosservate. Inoltre, i virus mostrano alte frequenze di mutazione, con possibile insorgenza di ceppi differenti. Per superare questo ostacolo abbiamo usato tecniche di diagnosi molecolare che hanno permesso la preparazione di saggi specifici per l'identificazione di specie virali. Un saggio diagnostico virale è stato condotto su 195 campioni di cereali, per valutare la prevalenza di alcuni virus specifici. Sono state saggiate diverse accessioni delle seguenti specie/varietà: Triticum aestivum L., T. aestivum var. lutescens (Alef.) Mansf., T. aestivum var. villosum (Alef.) Mansf., T. aestivum var. aestivum, T. aestivum var. graecum (Korn.) Mansf., Hordeum vulgare L., Avena sativa L., Triticum sp., T. durum (Desf.) Husn., T. durum var. hordeiforme (Host) Korn, T. durum var. melanopus (Alef.) Korn. e T. durum var. affine Korn. I saggi sono stati condotti con RNA dot-blot e trascrizione inversa - reazione a catena della polimerasi (RT-PCR). I seguenti virus sono stati considerati: Barley yellow mosaic virus (BaYMV), Barley mild mosaic virus (BaMMV), Soil-borne cereal mosaic virus (SBCMV), Wheat spindle streak mosaic virus (WSSMV) ed Oat mosaic virus (OMV). Solo tre virus sono stati rinvenuti con RT-PCR o RNA dot-blot, usando sonde specifiche: SBCMV, rinvenuto con RT-PCR in due campioni di H. vulgare ed A. sativa; BaMMV, rinvenuto con RT-PCR nel 9% del materiale saggiato di T. aestivum L. var. lutescens e T. durum var. hordeiforme. Infine, circa l'83% dei campioni è risultato positivo per WSSMV con entrambi i metodi, principalmente nelle varietà saggiate di T. aestivum, T. durum, H. vulgare ed A. sativa. Un sequenziamento high-throughput con piattaforma Illumina per smallRNAs è attualmente in corso, per identificare e caratterizzare ulteriormente virus noti o sconosciuti, da piante infette selezionate.

RNA silencing (RS) is a conserved mechanism in a broad range of eukaryotes. In plants, RSacts as an antiviral system and a successful virus infection requires suppression or evasion of theinduced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected withRNA and DNA viruses and provide specificity to this RNA mediated immune system.High-throughput sequencing has contributed to expanding our knowledge of siRNApopulations better describing their abundance, complexity and diversity in infected tissues. VirusderivedsiRNAs (vsiRNAs, 21-24 nt) from virus-infected plants are extraordinarily abundant anddiverse. However, certain regions of viral genomes ("hot spots") are usually more represented thanothers in sequenced vsiRNA populations.Potato virus Y (PVY) is an important pathogen of solanaceous crops belonging to the largestplant virus family, Potyviridae. The PVY genome is a single-stranded, positive-sense RNA of about10 kb. PVYC-to and PVY-SON41 are two PVY isolates that induce different disease symptoms ontomato (Solanum lycopersicum): while the former provokes severe leaf distortion, the latterproduces in the same host no visible phenotype.This study propose an innovative in silico approach, which consisted in mining genomicregions of PVY isolates and looking at possible PVY vsiRNAs putatively able, by sequencecomplementarity, to targeting and suppressing accumulation of host messenger RNA (mRNA) aspredicted by RS mechanisms, leading to dysfunctional biological processes that could explainisolate-specific disease phenotypes.A computational pipeline was implemented, which allowed the retrieval within the viralgenome of 21-nt vsiRNAs from PVYC-to and PVY-SON41 isolates complementary to tomatopredicted mRNA sequences (database Solgenomics, release ITAG2.3). The pipeline was based onfive bioinformatic algorithms and a relational database (DBMS MySQL) for management of theresults obtained in each steps. The comparative study for identifying the putative tomato targetgenes was performed with NCBI blast+ package 2.2 (option -task 'blastn-short' identity > 94%,max 2 mismatch or gap, alignment length > 19 bp). RandFold was employed for searching in thePVY genomes secondary structures containing putative vsiRNAs identified in previous steps. Withthis tool, five regions in the viral genome showing potential tRNA-like or microRNA-likesecondary structures were identified, that might account for as many "hot spots" of vsiRNAsaccumulation.

Motivations. RNA silencing, or post-transcriptional gene silencing (PTGS), is a conserved mechanism in a broad range of eukaryotes. In plants, PTGS acts as an antiviral system and a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with RNA and DNA viruses and provide specificity to this RNA-mediated immune system. High-throughput sequencing has contributed to expanding our previously knowledge of siRNA populations better describing their abundance, complexity and diversity in infected tissues. It is now known that siRNAs from virus-infected plants are extraordinarily abundant and diverse, and are widespread in near saturation at any region of either positive and negative genomic RNAs. However, certain regions of viral genomes ("hot spots") are usually more represented than others in sequenced siRNA populations. A gene involved in chlorophyll biosynthesis has been shown targeted by a siRNA derived from viral satellite RNA, revealing PTGS mechanism at basis of the symptom. Potato virus Y (PVY) is the type species of Potyvirus, a genus of agricultural importance belonging to the largest plant virus family, Potyviridae. The potyviral genome is a single-stranded, positive-sense RNA of ca. 10 kilobases (kb). PVYc-to and PVY-SON41 are two isolates of PVY that infect solanaceous hosts. While PVYc-to induces severe leaf distortion in different cultivars of tomato (Solanum lycopersicum), PVY-SON41 produces in the same host only a mild mosaic, followed by recovery. In order to elucidate the molecular mechanism underlying the different symptoms induced by PVYc-to and PVY-SON41 infections on tomato, we set up an in silico approach, mining genomic regions of PVY isolates and looking at possible RNA-based mechanisms where siRNA putatively generated from the PVY genome could target and suppress accumulation of host messenger RNA (mRNA), leading to dysfunctional biological processes that could explain different disease phenotypes.Methods. A Perl script was used to extract the complete datasets of 21-mers from isolates PVYc-to and PVY-SON41 complete genomes, leading the scanning of the complete sequence, shifted by one base at the time. MySQL was used for identification of the 21-mers shared and unique between the two viral genomes, highlighting sequence differences that could be at the base of diverse induced symptoms. The data obtained in the previous step were used to build the genomic map of identical regions, by fancyGene. A BLAST analysis was conducted with the identified 21-mer dataset, considering only the dissimilar sequences between the two isolates (blastn-short identity > 94%, max 2 mismatch or gap, alignment length 21 bp). The 21-mer were used as query and the tomato mRNA database (Solgenomics, release ITAG2.3) was used as target. The results were further used as input in a gene ontology analysis, through Blast2GO.Results. Despite the hig

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

MicroRNAs (miRNAs) are small RNAs (21-24 bp) providing an RNA-based system of gene regulation highly conserved in plants and animals. In plants, miRNAs control mRNA degradation or restrain translation, affecting development and responses to stresses. Plant miRNAs show imperfect but extensive complementarity to mRNA targets, making their computational prediction possible, useful when data mining is applied on different species. In this study we used a comparative approach to identify both miRNAs and their targets, in artichoke and safflower.ResultsTwo complete expressed sequence tags (ESTs) datasets from artichoke (3.6·104 entries) and safflower (4.2·104), were analysed with a bioinformatic pipeline and in vitro experiments, identifying 17 potential miRNAs. For each EST, using RNAhybrid program and 953 non redundant miRNA mature sequences, available in mirBase as reference, we searched matching putative targets. 8730 out of 42011 ESTs from safflower and 7145 of 36323 ESTs from artichoke showed at least one predicted miRNA target. BLAST analysis showed that 75% of all ESTs shared at least a common homologous region (E-value < 10-4) and about 50% of these displayed 400 bp or longer aligned sequences as conserved homologous/orthologous (COS) regions. 960 and 890 ESTs of safflower and artichoke organized in COS shared 79 different miRNA targets, considered functionally conserved, and statistically significant when compared with random sequences (signal to noise ratio > 2 and specificity >= 0.85). Four highly significant miRNAs selected from in silico data were experimentally validated in globe artichoke leaves.ConclusionsMature miRNAs and targets were predicted within EST sequences of safflower and artichoke. Most of the miRNA targets appeared highly/moderately conserved, highlighting an important and conserved function. In this study we introduce a stringent parameter for the comparative sequence analysis, represented by the identification of the same target in the COS region. After statistical analysis 79 targets, found on the COS regions and belonging to 60 miRNA families, have a signal to noise ratio > 2, with >= 0.85 specificity. The putative miRNAs identified belong to 55 dicotyledon plants and to 24 families only in monocotyledon.

MotivationThe family Asteraceae represents one of the largest evolutive radiation of flowering plants, including more than 1500 genera and 23000 species, and comprising economically important as well as ornamental crops (Jansen, 1987; Bremer, 2008). Among members of this dicotyledon family two edible species, Carthamus tinctorius L. and Cynara cardunculus var. scolymus L., also have a phytopharmaceutical interest. The former, known as safflower, is the only member of this genus widely cultivated for industrial oil, as a livestock feed or for use in traditional medicine (Han et al., 2009). The second species, the globe artichoke, apart of its importance as a food, is popular for its dietary and therapeutic potentials, especially for hepato-biliary dysfunctions and digestive complaints (Gebhardt, 1998; López-Molina, 2005). The discovery of microRNAs (miRNAs) in different genomes provided a new paradigm in evolution studies, due to their ancient origin and the important role played in development regulation, since the egress of multicellular organisms. This system is highly conserved in plant and animal cells. Plant miRNAs derive from long primary transcripts giving rise to mature 21-24nt RNAs products, fundamental in RNA-based gene regulation (Bartell, 2004). In plants, miRNAs control messengers degradation or restrain translation, affecting somatic development and the response to biotic and abiotic stresses (Jones-Rhoades, 2006). A feature of miRNAs is their imperfect but extensive complementarity to corresponding mRNA targets, thus making their computational prediction possible. This approach is useful when data mining is performed on the basis of miRNA:mRNA targets conservation, among different species, and was herein applied to the study of Carthamus and Cynara spp.MethodsA comparative approach was used to identify both evolutionary and functionally conserved miRNAs as well as their targets, by detection of common expressed sequence tags (ESTs) in Carthamus and Cynara spp. A bioinformatic pipeline was developed at this regard to analyze publicly available ESTs dataset and identify miRNA candidates in these two related species. Complete EST datasets from Carthamus and Cynara (36323 and 42011 sequences, respectively) were screened looking for putative miRNA targets by means of RNAhybrid (Rehmsmeier, et al., 2004) software and using the mirBase mature miRNA sequences from Arabidopsis as a reference set. Afterward the NCBI Blast algorithm (Altschul et al., 1990) embedded in a Perl script was used to identify the homologous region in Carthamus and Cynara EST datasets. The RNAhybrid results and the homologous region calculated, have been downloaded into a relational database (DBMS MySql) ad hoc developed. We generate the "mapping" of the miRNA target on the "EST homologous region" by means SQL queries. Finally, the signal-to-noise ratio and specificity were assessed using two approaches: in the first one, for each Arabidopsis mature miRNA, the

The nematophagous fungus Pochonia chlamydosporia var. chlamydosporia is one of the most studied biological control agentsagainst plant (semi-) endo-parasitic nematodes of the genera Globodera, Heterodera, Meloidogyne, Nacobbus and, more recently,Rotylenchulus. In this paper we present highlights from more than three decades of worldwide research on this biological controlagent. We cover different aspects and key components of the complex plant-fungus-nematode tri-trophic interaction, an interactionthat needs to be addressed to ensure the efficient use of P. chlamydosporia as a biopesticide as part of an integrated pest managementapproach.

Jasmonic acid (JA) is an important regulator of the plant immune system, playing a central role in the modulation of developmental processes and signalling networks. The JA pathway is an indispensable component of plant resistance to nematodes, and is involved in wound response. Another hormone regulating the systemic wound response in tomato is systemin, an 18-amino-acid peptide signal molecule. Systemin and JA constructively interact in the same signalling pathway, coordinating wound-induced systemic expression of defence-related genes. In the present study we evaluated the expression pattern of selected genes involved in the JA pathway and the susceptibility of different tomato genotypes, including plants expressing different levels of Prosystemin, challenged with the root-knot nematode (RKN) Meloidogyne incognita. Six tomato genotypes: wild type, transformed lines expressing different levels of prosystemin and a homozygous Mi gene resistant tomato, were selected for the infection assay. The seedlings were inoculated four weeks after transplanting with freshly hatched juveniles. Genotypes showing significant differences between measurements were used for gene expression analysis. For this purpose, in vivo plant assays were carried out with tissues collected 5 and 6 days after RKN infection, processed for Real-Time PCR analysis (qRT-PCR). Preliminary data showed that transgenic plants overexpressing Prosystemin had significant less galls and showed milder symptoms, suggesting a Prosystemin role in inducing tolerance to RKN. The genotypes were further analysed in an expression assays with five different genes that appeared involved in the JA pathway. Comparison at two different time points showed that, in the early response, Prosystemin and LOX-D are differentially regulated in the JA pathway. Data at 5 dpi suggested that RKNs suppress the JA pathway in wild type plants and that Prosystemin overexpression could therefore be necessary to induce tolerance to RKNs.

Most of the 24 viruses which infect globe artichokeare detrimental to the crop's performance and hamperthe development of a nursery activity in the respect of currentEU legislation. We describe a procedure to sanitize globeartichoke ''Brindisino'' from Artichoke Italian latent virus(AILV) and Artichoke latent virus (ArLV), while preservingits valuable early flowering trait. ArLV was successfullyeliminated by meristem-tip culture,whileAILVwas removedwhen two rounds ofmeristem-tip culture were spaced outwithin vitro thermotherapy. In vivo thermotherapy, followed bymeristem-tip culture, was also successful in producing virusfreematerial but was less efficient in terms of the number ofplants recovered post treatment. Due to the multi-clonalcomposition of the populations at present in cultivation, theselected and sanitised clones were fingerprinted by applyingmicrosatellite and AFLP (amplified fragment length polymorphism)markers.OneAFLP primer combination produced28 informative fragments used to evaluate genetic relatednessamong the clones in study. Our results demonstrates thatAFLP-based molecular fingerprinting enables to verify thetrue to clone correspondence in nurseries, ensure the effectivecorrespondence between the real and the declared identity of aclone, so that to avoid commercial frauds, and might representsa valuable tool for assessing somaclonal variationoccuring during 'in vitro' propagation

A study was carried out for identification and detection of Phytophthora spp. from soil and plant samples, collected fromSolanaceous crops in Egypt and Italy. The samples were screened with specific and universal primers of Phytophthora 18S,ITS1-ITS2 and 28S regions, followed by PCR product sequencing. The Phytophthora spp. detected were P. infestans(Egypt) and P. parasitica (Italy). A molecular beacon probe was also developed based on the avr3a gene of P. infestans todetect a variant associated with virulence traits. The probe was suitable for avr3a allele identification from P. infestans andalso from P. parasitica PCR products.

I virus rappresentano da sempre una delle principali minacce per la produzione dei cereali. Allo scopo di assicurare un'accurata diagnosi dei virus presenti nelle varietà locali di Triticum aestivum L., Hordeum vulgare L., T. durum Desf., Secale cereale L., Avena sativa L., reperite nell'ambito del progetto SaVeGralNPuglia, tali varietà sono state caratterizzate con marcatori molecolari associati a fitopatie ad eziologia virale. Per evitare i falsi negativi, sono stati considerati più marcatori per ciascun virus e regioni diverse del genoma virale. I saggi molecolari hanno previsto la retro­trascrizione dell'RNA estratto, seguita dall'amplificazione mediata dalla polimerasi (RT-PCR). L'incidenza di campioni positivi è risultata pari al 23 % per il virus del mosaico giallo dell'orzo (BaYMV), al 49 % per il virus del mosaico comune del frumento (SBCMV), al 95 % per l'agente del mosaico dell'avena (OMV) , al 55 % per il virus del mosaico striato del frumento (WSMV), e all'84 % per il virus della striatura fusiforme del frumento (WSSMV). I saggi per il virus del mosaico moderato dell'orzo (BaMMV) e quello del nanismo giallo dell'orzo (BYDV) hanno dato esito negativo. Nel caso dell'OMV si è fatto ricorso all'ibridazione molecolare a macchia o "dot-blot" di frammenti amplificati. I risultati ottenuti hanno inoltre consentito di accertare in alcuni campioni I presenza d'infezione multipla da fitovirus in quanto erano presenti contemporaneamente 2, 3 o 4 virus diversi, presenti rispettivamente con percentuali del 23, 8, e 5. Per una diagnosi approfondita di tutta la complessità del "viroma" del campione in esame sono state infine selezionate varietà locali di Triticum durum L., T. aestivum L., e H. vulgare L. più rappresentative, da sottoporre ad analisi di metagenomica con tecnologie NGS.

Nel passato la Puglia era particolarmente ricca di varietà locali di leguminose e cereali dagranella, coltivate in alternanza seguendo tradizionali tecniche di rotazione delle colture chepermettevano di preservare la fertilità del terreno. Queste antiche varietà, ben adattate allecondizioni pedoclimatiche del territorio regionale, sono da tempo minacciate da fenomeni dierosione conseguenti alla progressiva diffusione di nuove varietà maggiormente produttive.Gli agricoltori, la comunità scientifica ed i rappresentanti politici pugliesi, hanno da qualchetempo sottolineato l'importanza e l'urgenza di recuperare, preservare e custodire in Banchedel Germoplasma l'antico e prezioso patrimonio varietale autoctono. Obiettivo prioritario delprogetto PSR-SaVeGraINPuglia è il recupero, la caratterizzazione, la salvaguardia e lavalorizzazione di leguminose e cereali da granella e foraggio di Puglia. Le varietà locali sonoreperite nei diversi areali ed habitat regionali, caratterizzate e salvaguardate applicandoopportuni protocolli di tutela Regionali ed Internazionali, e conservate sia in ex situ che insitu. Le attività progettuali coordinate dall'Istituto di Bioscienze e BioRisorse del ConsiglioNazionale delle Ricerche (IBBR-CNR), sono svolte in collaborazione con 20 partner operantisul territorio regionale ed appartenenti a Enti pubblici di ricerca, Università, Enti Parco,aziende private ed associazioni.

Nel passato la Puglia era particolarmente ricca di varietà locali di leguminose e cereali da granella, coltivate in alternanza seguendo le tradizionali tecniche di rotazione delle colture impiegate da secoli in agricoltura per preservare la fertilità del terreno. La diversità genetica esistente in queste antiche varietà, ben adattate alle condizioni pedoclimatiche del territorio regionale, è da tempo minacciata da fenomeni di erosione conseguenti alla progressiva diffusione di nuove varietà maggiormente produttive. Gli agricoltori, la comunità scientifica ed i politici pugliesi più attenti, hanno da qualche tempo sottolineato l"importanza e l"urgenza di recuperare, preservare e custodire in Banche del Germoplasma questo antico e prezioso patrimonio varietale regionale. Per le varietà locali pugliesi di leguminose e cereali da granella, vi è carenza di informazioni storiche, sulle origini, tradizioni, usi e saperi popolari, sulle motivazioni che hanno contribuito alla loro persistenza in alcuni areali e di dati precisi sulla loro attuale diffusione sul territorio, stato fitosanitario, struttura genetica, caratteristiche agronomiche, nutrizionali e di resistenza a stress biotici ed abiotici. Insufficienti sono inoltre le strutture volte alla promozione, divulgazione e valorizzazione di questo antico patrimonio colturale strettamente connesso alla dieta mediterranea recentemente riconosciuta come patrimonio immateriale dell"umanità. Obiettivo prioritario del progetto PSRMisura 214 SaVeGraINPuglia sarà pertanto il recupero, la caratterizzazione, la salvaguardia e la valorizzazione di leguminose e cereali da granella e foraggio in Puglia. Le varietà locali pugliesi saranno reperite nei diversi areali ed habitat regionali, caratterizzate, e salvaguardate sia mediante conservazione ex situ che in situ applicando protocolli di tutela Regionali ed Internazionali. Le attività progettuali coordinate dall"Istituto di Bioscienze e BioRisorse del Consiglio Nazionale delle Ricerche saranno svolte in collaborazione con Enti pubblici (Consiglio Nazionale delle Ricerche, Università di Bari e del Salento, Consiglio per la Ricerca e la sperimentazione in Agricoltura, Ente Parco Nazionale dell"Alta Murgia), Aziende ed Associazioni (Agrinpro, Azienda Agricola Iannone, Azienda Agricola Lamarcavotta, BioNat Italia, Centro Studi Lino Lana Lenticchia, Centri Regionali per le Tecnologie Agroalimentari, Consorzio Daunia & Bio, CSQA Certificazione, Eco-Spo, Ferventazione, Intini & C.) operanti sul territorio regionale.

Legume, cereal and forage landraces, selected over the time by local farmers and well adaptedto the climatic conditions of the Apulia region, are threatened by genetic erosion resulting from thegradual spread of more productive varieties. In order to collect, preserve and store these landracesin National or Regional gene banks (ex situ conservation) and/or directly in field under the strictlycontrol of local farmers (on farm conservation, a type of in situ conservation) a regional safeguardproject was established within the framework of the European Agricultural Programms for RuralDevelopment. The primary objective of the project, named with the acronym SaVeGraINPuglia, isthe collection, safeguard and preliminary evaluation and valorization of legume, cereal and foragelandraces present in the Apulia region.About 150 landraces were collected in different habitat and areas outside and inside protectedareas where traditional agricultural practices still survive. The collected germplasm was stored andsafeguarded both ex situ and in situ through the application of appropriate, international protocols.A preliminar characterisation is in progress. The project activities, coordinated by the Institute ofBiosciences and Bio-Resources of the National Research Council (IBBR-CNR) was carried out incollaboration with 20 partners operating in the regional territory and belonging to public researchorganizations, universities, parks, private companies and associations.The preliminary results of the project will be presented to enphasize the potential use oflandraces for the promotion of local products and the development of sustainable agricultu

Expression profiles were identified in the fungusPochonia chlamydosporia, a biological control agent ofplant parasitic nematodes, through a cDNA-amplifiedfragment length polymorphism approach. Two isolates withdifferent host ranges, IMI 380407 and IMI 331547, wereassayed in conditions of saprotrophic-to-parasitic transition,through in vitro assays. Gene expression profiles from threedifferent nutritional conditions and four sampling timeswere generated, with eggs of host nematodes Globoderapallida and Meloidogyne incognita. Expression of transcriptschanged in RNA fingerprints obtained underdifferent nutritional stresses (starvation in presence/absenceof eggs, or rich growth media). Transcript derived fragments(TDFs) obtained from the expression profilescorresponded to 6,800 products. A subset was sequencedand their expression profile confirmed through RT PCR. Atotal of 57 TDFs were selected for further analysis, basedon similarities to translated or annotated sequences. Genesexpressed during egg parasitism for both IMI 380407 andIMI 331547 were involved in metabolic functions, cellularsignal regulation, cellular transport, regulation of geneexpression, DNA repair, and other unknown functions.Multivariate analysis of TDF expression showed threegroups for IMI 380407 and one for IMI 331547, eachcharacterized by expression of genes related to eggsparasitism. Common amplification profiles among TDFclusters from both isolates also reflected a pool ofconstitutive genes, not affected by the nutritional conditionsand nematode associations, related to general metabolicfunctions. The differential expression of parasitism relatedgenes suggest a network of induced/repressed products,playing a role in fungal signaling and infection, with partialoverlaps in host infection and parasitism traits.

Potato virus Y (PVY) is one of the most important pathogens of pepper, potato, tobacco,tomato and other solanaceous and non-solanaceous hosts. Two PVY isolates, PVY-Cto and PVYSON41,induce very different disease phenotypes on tomato (Solanum lycopersicum), the formerbeing responsible of leaf distortions and growth reduction and the latter inducing only very mildsymptoms. With the aim of investigating the role of RNA silencing mechanisms in the differentpathogenic behaviour of the two isolates, we performed a systematic analysis of expression profilesof Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) genes thatform the core components of the RNA silencing-based plant immune system. The effects onexpression profiles of some of these genes were significantly higher in tomato plants infected by theaggressive isolate PVY-Cto in comparison with PVY-SON41-infected and healthy plants.We further investigated on whether effects on expression of silencing genes could lead to adifferential accumulation of small RNAs (sRNAs) in plants. Both endogenous microRNA (miRNA)and viral small interfering RNA (vsiRNA) populations were analysed by high-throughputsequencing. SRNAs from tomato plants, healthy or infected by PVY-Cto and PVY-SON41, wereisolated at 21 and 30 days post-inoculation (dpi) and sequenced using the Illumina platform at theNGS Laboratory, IGA (Udine, Italy). Total reads from samples at 21 dpi ranged between 13 to 17million, and at 30 dpi between 23 to 28 million. From these data, vsiRNA and miRNA populationswere analysed separately. VsiRNA from PVY-Cto-infected samples were abundant andcorresponded to 44% and 29% of total reads at 21 and 30 dpi, respectively. Surprisingly, anegligible number of vsiRNA were found in PVY-SON41-infected samples, representing less than1% of total reads at both timepoints.As expected, virus infection affected also the composition of the miRNAome. The totalnumber of sRNAs corresponding to known plant miRNAs was proportional to each library size andvaried in a narrow range from 2.8 % ot total reads in mock-inoculated at 21 dpi to 4,8% in PVY-Ctosamples at the same timepoint. A total of 156 known miRNA was identified in the six libraryfollowing stringent criteria. To compare miRNA abundance in different libraries, the count of eachmiRNA was normalized to transcripts per million (TPM) and subjected to a Z-test. Change inmiRNA read counts between virus- and mock-inoculated plants was scored as fold change andrecorded. On this basis, PVY-Cto infection was associated to the differential accumulation (eitherup- or downregulated) of 71 and 39 miRNA at 21 and 30 dpi, respectively. The mild PVY-SON41 isolate affected the composition of miRNA population at significantly lower extent, and adifferential accumulation was observed only for 25 (21 dpi) and 6 (30 dpi) miRNAs.Our results provide a comparative analysis, via deep sequencing, of changes i

Potato virus Y (PVY) is a pathogen of pepper, potato, tobacco, tomato and other plant hosts. Two PVY isolates, PVY-Cto and PVY-SON41, induce very different disease phenotypes on tomato (Solanum lycopersicum), the former being responsible of leaf distortions and growth reduction and the latter inducing only mild symptoms. With the aim of investigating the role of RNA silencing mechanisms in the different pathogenic behaviour of the two isolates, we performed a systematic analysis of expression profiles of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) genes that form the core components of the RNA silencing-based plant immune system. The effects on expression profiles of some of these genes were higher in tomato plants infected by the aggressive isolate PVY-Cto in comparison with PVY-SON41-infected and healthy plants.We further investigated on whether effects on expression of silencing genes could lead to a differential accumulation of small RNAs (sRNAs) in plants. Both endogenous microRNA (miRNA) and viral small interfering RNA (vsiRNA) populations were analysed by high-throughput sequencing. SRNAs from tomato plants, healthy or infected by PVY-Cto and PVY-SON41, were isolated at 21 and 30 days post-inoculation (dpi) and sequenced using the Illumina platform. Total reads from samples at 21 dpi ranged between 23 to 28 million, and at 30 dpi between 13 to 17 million. From these data, vsiRNA and miRNA populations were analysed separately. VsiRNA from PVY-Cto-infected or PVY-SON41-infected samples were identified using the Bowtie software (Langmead et al., 2009, Genome Biol 10:R25) by mapping reads to the reference genome of the two viral isolates (EU482153.1, AJ439544). As a result, a non-uniform distribution of VsiRNA was observed. PVY-Cto-infected plants accumulated vsiRNA with a peak of 44% at 21 dpi. Surprisingly, a negligible number of vsiRNA were found in PVY-SON41-infected samples, representing less than 1% of total reads. More than 150 known miRNAs were found to be expressed in our six libraries, some of which had relatively high expression abundance and exhibited evolutionary conservation across multiple plant species. MiRNAs read count was proportional to each library size and varied in a narrow range from 2.8 % of total reads in mock-inoculated at 21 dpi to 4,8% in PVY-Cto samples at the same timepoint. Virus infection affected also the profile of miRNAs expression. MiRNA abundance, in different libraries, was normalized to transcripts per million (TPM) and subjected to a Z-test with a significance value of 0.01. Change in miRNA read counts between virus- and mock-inoculated plants was scored as fold change and recorded. On this basis, PVY-Cto infection was associated to the differential accumulation (either up- or downregulated) of 71 and 39 miRNA at 21 and 30 dpi, respectively. The mild PVY-SON41 isolate affected the composition of miRNA population at significantly lower extent, and a differential accu

RNA silencing (RS) is a conserved eukaryotic mechanism acting in plants also as antiviral immune system. A successful virus infection requires suppression or evasion of host RS. Small interfering RNAs (siRNAs) are potent RS effectors, and accumulate in plants infected by RNA and DNA viruses as components of this plant immune system, providing targer specificity for pathogen RNA post-transcriptional degradation. Virus-derived siRNAs (vsiRNAs, 21-24 nt) are abundant and diverse in infected plants. Although any viral genomic regions can potentially be targeted to generating vsiRNAs, certain regions (also referred to as "hot spots") are more represented than others in vsiRNA sequenced libraries.Potato virus Y (PVY, Potyviridae) is an important plant pathogen on solanaceous hosts. The two PVY isolates PVYC-to and PVY-SON41 induce very different disease phenotypes on tomato (Solanum lycopersicum), the former being responsible of leaf distortions and the latter inducing to mild symptoms. In this study, we applied combined in silico and molecular biological approaches to identify PVY vsiRNAs putatively able to suppress host mRNAs by sequence complementarity and deriving RS-based suppression, a mechanisms that would induce dysfunctional processes in infected host plants. As the final aim of the study, we explored differential expression of specific host-targeting vsiRNA by the two isolates as one of the possible sources of diverse disease phenotype expression.A computational pipeline was implemented to retrieve 21nt vsiRNAs from PVY isolates, complementary to tomato predicted mRNAs (Solgenomics, ITAG2.3). The comparative study used NCBI blast+ package 2.2. RandFold searched secondary structures in the PVY RNA genome containing putative vsiRNAs sequences identified in previous steps.Two PVYC-to regions showed potential tRNA-like or microRNA-like secondary structures, not present in the other isolate, possibly accounting for vsiRNAs accumulation "hot spots". Moreover, we obtained two lists of tomato transcripts perfectly or imperfectly (one or two mismatches were allowed, according to typical microRNA recognition sites) complementary to vsiRNA computed for either of the two PVY isolates, and therefore putative target of vsiRNA-driven RS suppression. Putative targets common to the two isolates were discarded, so to identify a list of genes that could represent targets specific to the aggressive isolate PVYC-to only. Some host transcription factors (e.g. NAC, MYB, TCP, HD-ZIP, MADS-box families) active in vegetative development and leaf morphogenesis were selected for further investigation. Quantitative RT-PCR showed differential expression levels of selected host transcripts upon PVYC-to and PVY-SON41 infections, differing also from healthy plants. For some genes, lower isolate-specific mRNA accumulation suggested RS-driven post-transcriptional regulation, thus apparently confirming the starting hypothesis of a correlation between symptoms and RS