Virus-induced gene silencing (VIGS) is a well-established reverse genetics technology for assessment of gene functions in plants. VIGS is a transient loss-of-function assay that involves three steps: engineering viral genomes to include fragments of host genes that are targeted to be silenced, infecting the plant hosts and suppressing the target genes expression by post-transcriptionalgene silencing (PTGS).Suppression of specific mRNA accumulation allows correlation between gene silencing and the deriving phenotype, providing clues on gene functions.However, the efficiency of this technology may be scarce. In these cases, weak and/or non-homogeneous distribution of VIGS through the plant may generate results not fully coherent,. This often limits the extensive application of the technique to more permissive plant species such as Nicotiana benthamiana.Aiming at increasing VIGS efficiency in functional studies,particularly in key crop species, we produced and tested new constructs using a Tobacco rattle virus (TRV)-based vector in tomato (Solanum lycopersicum) and other solanaceous crops. This innovative approach consisted in cloning into the TRV vector a short fragment of a host gene containing at its termini mutations designed forthe expression of small interfering RNAs (siRNAs) that mimicked a microRNA(miRNA) structure.The recently developed artificial microRNAs (amiRNAs) technology modifies an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs targeting transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed to contain mismatches at specific nucleotides with respect to their target sites.We designed a vector where amiRNA-like small RNAs are generated when viral intermediate dsRNA forms are targeted by the host PTGS machinery in the cytoplasm.In the viral vector, we inserted mutant sequences designed to contain at both their 5' and 3' termini one or two mismatches at selected positions. Mismatched sequences were computed by the WMD3 web tool (wmd3.weigelworld.org), an algorithm that generates all possible amiRNAs using full-length target gene sequences as input.Short (110nt) amiRNA-like containing sequences were compared for their VIGS efficiency with wild-type sequences, shorter- or longer-sized inserts and inverted-repeat constructs. Upon inoculation of our constructs , VIGS established earlier and more extensively than its wild-type counterpart in tomato, N. benthamiana and N. tabacum. For instance, suppression of the tomato reporter gene magnesium chelatase (ChlI or SU) with the VIGS-amiRNA-like construct produced the typical yellow phenotype earlier and more extensively than the standard TRV-PDS (phytoene desaturase) VIGS vector. Quantitative RT-PCR confirmed the efficiency of our VIGSamiRNA-like constructs in terms of post-transcriptional suppression of host target mRNAs. Our results are discussed in the light of their beneficial contribution t

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.

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.

Virus-induced gene silencing (VIGS) is a well-established reverse genetics technology for assessment of gene functions in plants. VIGS is a transient loss-of-function assay that involves three steps: engineering viral genomes to include fragments of host genes that are targeted to be silenced, infecting the plant hosts and suppressing the target genes expression by post-transcriptional gene silencing (PTGS), the defense mechanism deployed by plants against virus infections. Suppression of specific mRNA accumulation allows correlation between gene silencing and the deriving phenotype, providing clues on gene functions. However, the efficiency of this technology may be affected by various factors, including virus vector properties and susceptibility of plant host species. In several cases, weak and/or non-homogeneous distribution in the plant (or in the single leaf) of VIGS may generate results not fully coherent, particularly in terms of correlation between phenotype and accumulation levels of the specifically suppressed RNA. This often limits the extensive application of the technique to more permissive plant species such as Nicotiana benthamiana. Aiming at increasing VIGS efficiency in functional studies, particularly in key crop species, we produced and tested new constructs using a Tobacco rattle virus (TRV)-based vector in tomato (Solanum lycopersicum) and other solanaceous crops. This innovative approach consisted in cloning into the TRV vector a short fragment of a host gene containing at its termini mutations designed for the expression of small interfering RNAs (siRNAs) that mimicked a microRNA (miRNA) structure. The recently developed artificial microRNAs (amiRNAs) technology modifies an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs targeting transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed to contain mismatches at specific nucleotides with respect to their target sites. Unlike the conventional amiRNA strategy, where target-specific 21nt small silencing RNAs derive from longer double-stranded RNA (dsRNA) precursors that are processed in the nucleus by DCL1, we designed a vector where amiRNA-like small RNAs are generated when viral intermediate dsRNA forms are targeted by the host PTGS machinery in the cytoplasm. In the viral vector, we inserted mutant sequences designed to contain at both their 5' and 3' termini one or two mismatches at selected positions. Mismatched sequences were computed by the WMD3 web tool (wmd3.weigelworld.org), an algorithm that generates all possible amiRNAs using full-length target gene sequences as input. Short (110nt) amiRNA-like containing sequences were compared for their VIGS efficiency with wild-type sequences, shorter- or longer-sized inserts and inverted-repeat constructs. Upon inoculation of our constructs , VIGS established earlier and more extensively than its wild-type counterpart in tomato, N.

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).

Ozone is widely used as a disinfectant, and ozonated water has been known to confer some protection of plants against several biotic stresses. By applying four foliar spray treatments of ozonated water (10 ppm ozone) on tomato seedlings, i.e. two pre- and two post-inoculation with Tomato spotted wilt virus (TSWV), we observed reduction of disease incidence and severity by 20%, as well as a virus titrrreduction by 80% at 19 days post-inoculation. The same treatments also reduced the number of galls induced by root knot nematode (RKN; Meloidogyne incognita) by 29%. Soil drenching with ozonated water for four consecutive days before inoculation reduced RKN gall formation by 60%, but not TSWV infection. Overall, in mock-inoculated plants, foliar sprays induced PR1b1 expression in leaves, though other salicylate- (PAL and PR-5x) or jasmonate-dependent genes (LoxD, AOS and PinI) were substantially unaffected. Soil drenching promptly enhancedtranscription of PAL and PR1b1 in roots and leaves, down-regulated PR-5x and did not affect expression of LoxD and AOS. PinI was significantly down-regulated only in leaves. The impact of ozonated water applications on the expression of these genes did not correlate with that of benzothiadiazole, a known inducer of systemic acquired resistance. This demonstrates that ozonated water may protect tomato fromtwo very different biotic stresses, especially when applied at the sites of their infection, and modulates salicylate and jasmonate pathways differently from benzothiadiazole. This research was supported by the Fondazione Cassa di Risparmio di Puglia, Italy, within the Project 'Risposte di difesa contro nematodi e virus indotte da trattamenti di ozono in pomodoro'.

Cytosine methylation is a stable and heritable modification of the DNA that imparts epigenetic control throughout the genome, including regulation of coding and noncoding elements. In plants, previous studies have contributed to shape the epigenetic landscape in developmental processes, whereas the potential for these pathways to be dynamically regulated during non-developmental processes, such as stress responses, has not been investigated thoroughly at date. Recently, it has been demonstrated that DNA methylation is involved in controlling the Arabidopsis thaliana defence response against bacterial pathogens, and several lines of evidence suggest that plant immune response to viral infections imply an involvement of DNA methylation. It has been shown that plant viruses have the capability to modify the methylation profile of the host genome, although with scarce loci-specific methylation. However a high-resolution quantitative analysis of DNA methylation changes, a technology now available for genome-wide investigations (also known as BS-Seq) is still missing in the case of plant-virus interactions. With the aim of investigating how epigenetic mechanisms contribute to the onset and progression of diseases, we used BS-Seq to analyse methylation processes on the host genome induced by a RNA virus (Cucumber mosaic virus, CMV) and a DNA virus (Cauliflower mosaic virus, CaMV). First analysis of the BS-seq data demonstrates that infection of each virus modifies the methylation landscape of all 5 chromosomes. Our data strongly indicate that the mechanism induced by RNA- and DNA-virus infection must be different. Interestingly, the RNA-virus infection mainly induces de-methylation whereas CaMV infection triggers a general hyper-methylation. Investigations on the genomic localization of these modifications result in a similar list of regions affected by the different viruses. On the other side, analysis of the differentially methylated sites according to annotated genes reveals significantly more differentially methylated genes in CMV-than in CaMV-infected plants. Furthermore, gene feature analysis shows prevalent promoter regions de-methylation upon infection of both viruses. However, we find many hypermethylated coding regions in CMV infected plants versus hardly differentially methylated coding regions in DNA-virus infected Arabidopsis.Finally, perhaps the most intriguing concluding remark of this preliminary analysis is that despite we find differentially methylated regions within gene features, such as transcripts, transposon and promoter, the large majority of methylation modifications are located in intergenic (not yet annotated) regions and might cover and regulate still unknown non-coding RNA products.

The establishment and maintenance of DNA methylation are relatively well understood whereas little is known about their dynamics and biological relevance in innate immunity [1-2]. In plants, modulation of DNA methylation might be an effective mechanism to regulate gene expression in response to abiotic and biotic stresses. Recent evidences from large-scale epigenomic approaches indicate that dynamic DNA methylation changes are not limited to gene imprinting but can regulate the plant's immune system in response to pathogens.In plants, virus infections trigger the expression of non-coding small RNAs (smRNAs) by also influencing the epigenetic status of the host genome; however, the involvement of DNA methylation in regulation of plant immune system in response to virus infection has not been so far investigated. In this context, we are carrying out a study aiming to elucidate the impact of DNA and RNA virus infections on genomic DNA methylation in plants, and their correlation with also the expression of smallRNA, by integrating the analysis of multiple "omics" datasets obtained by using next-generation sequencing technologies.In this paper we present the results of the analysis on the methylation modifications induced by the viruses infection on the whole genome and on coding and non-coding gene regions.

We analyzed the transcriptome (RNA-Seq) of leaf samples collected from a field crop of tomato cv. Docet (Sw5 resistance gene) in Apulia, southern Italy, with different symptom severity and accumulation levels of a resistance-breaking strain of Tomato spotted wilt virus (TSWV). Four groups of samples were assumed to be different stages of plant tissue colonization by the virus: plants without symptoms anda virus titre (group A) or 1 ×?102 TSWV reads per million (rpm; B), and plants with symptoms and 1 ×?104 rpm (C) or 2 ×?105 rpm (D). Transcriptome sequencing revealed that plant response to TSWV infection is profoundly related to its accumulation level in the tissues. At an early stage of infection (B vs. A comparison), genes related to photosystem I were down-regulated, and oxidoreductase activityincreased. Considerable virus colonization (C vs. B) activated defense-related mechanisms such as cell surface receptor signalling, phenylpropanoid biosynthesis and transcription factor activity. In contrast, photosynthesis, transmembrane transporter activity, and biosynthesis of monosaccharides and peptides were down-regulated. This scenario increased at an advanced stage of colonization (D vs.C), with attenuation of response to stimuli (e.g., surface receptor signaling and protein kinase activity) and an increase of catalytic activities such as ubiquitin- protein transferase and ribonuclease. TSWV infection constantly injured tomato cell metabolism(e.g., photosynthesis, monosaccharide and peptide biosynthesis, ion transporter activity) while plant defense (e.g., cell surface receptor signaling, phenylpropanoid pathway), clearly ineffective in such compatible plant-virus interaction, occurred late and disappeared soon after.

The quantification of messenger RNA expression levels by real-time reverse-transcription polymerase chain reaction requires the availability of reference genes that are stably expressed regardless of the experimental conditions under study. We examined the expression variations of a set of eight candidate reference genes in tomato leaf and root tissues subjected to the infection of five taxonomically and molecularly different plant viruses and a viroid, inducing diverse pathogenic effects on inoculated plants. Parallel analyses by three commonly used dedicated algorithms, geNorm, NormFinder and BestKeeper, showed that different viral infections and tissues of origin influenced, to some extent, the expression levels of these genes. However, all algorithms showed high levels of stability for glyceraldehyde 3-phosphate dehydrogenase and ubiquitin, indicated as the most suitable endogenous transcripts for normalization in both tissue types. Actin and uridylate kinase were also stably expressed throughout the infected tissues, whereas cyclophilin showed tissue-specific expression stability only in root samples. By contrast, two widely employed reference genes, 18S ribosomal RNA and elongation factor 1?, demonstrated highly variable expression levels that should discourage their use for normalization. In addition, expression level analysis of ascorbate peroxidase and superoxide dismutase showed the modulation of the two genes in virus-infected tomato leaves and roots. The relative quantification of the two genes varied according to the reference genes selected, thus highlighting the importance of the choice of the correct normalization method in such experiments.

In October 2013, unusual chlorotic patches were observed on the middle leaves of a few tomato plants cv. Lotty grown in a greenhouse located in the countryside of Fasano (Apulia, Southern Italy). Younger leaves and fruits were symptomless and no aggravation of symptoms was detected as time went by. Electron microscope observations of leaf dips revealed the presence of filamentous virus particles ca. 520 nm in length. Mechanical inoculations with leaf extracts from symptomatic tomatoes elicited a mosaic reaction in Nicotiana benthamiana, but not in tomato plants cv. UC82, which, however, were systemically infected, as shown by RT-PCR. A RT-PCR product of the expected size (ca. 700 bp) was obtained using the degenerate broad-spectrumpotexvirus primers Potex5/Potex1RC (van der Vlugt and Berendsen, 2002). The amplicon was custom sequenced (BMR Genomics, Italy) and the sequence deposited in GenBank under the accession No. KM923762. BLAST alignment showed that the 700 bp amplicon shared 97-99% homology with the RNA-dependent RNA polymerase (RdRp) gene of several isolates of Pepino mosaic virus (PepMV) genotype CH2, 82% homology with the LP and EU genotypes, and 79-80% with the US genotype. A RT-PCR-restriction fragment length polymorphism (RFLP) analysis of the RdRp amplicon with EcoRI and BglII restriction endonucleases confirmed that our isolate, designated PUG1, belongs to the CH2 genotype (Hanssen, 2010). Our observations and assays are consistent with the presence of PepMV in the tomato plants tested, and the fact that PUG1 is a mild isolate of the virus. PepMV has previously been recorded from tomato in three Italian regions, i.e. Sardinia, Sicily and Campania, but this the first report from Apulia.

Virus-induced gene silencing (VIGS) is a transient loss-of-function assay that involves threesteps: engineering the genome of a viral vector to include a fragment of host gene that is targeted tobe silenced, infecting the plant hosts and suppressing the target gene expression by posttranscriptionalgene silencing (PTGS). VIGS is a well-established reverse genetics technology forassessment of gene functions in plants. However, the efficiency of this technology may be low insome plant species, and this often limits the application of the technique to more permissive modelhosts. Aiming at increasing VIGS efficiency in functional studies, particularly in key crop speciessuch as tomato (Solanum lycopersicum), we tested an innovative approach that consisted in: a)enhancement of the target gene cleavage efficiency by exploiting the artificial microRNA(amiRNA) technology; and b) validation of a bioinformatic method for selecting the most suitablegene fragments for induction of gene silencing.The recently developed amiRNA technology modifies an endogenous gene silencingmechanism that processes natural miRNA precursors to small silencing RNAs targeting specifictranscripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designedto contain mismatches at specific nucleotides with respect to their target sites, thus increasingeffectiveness of target gene cleavage as compared to RNA silencing processes guided by otherperfectly matching small RNAs.The WMD3 software (wmd3.weigelworld.org) was used for both identification of putativeamiRNA sequences and selection of suitable gene regions. In WMD3, an algorithm generates insilico all possible amiRNAs putatively able to anneal to full-length target mRNA. We selected andcompared cDNA fragments (110-120 nt) from gene regions with either high or low content ofputative amiRNAs, inserted point mutations to express amiRNA-like small RNAs from the viralvectors, and cloned these cDNAs into tobacco rattle virus (TRV)-based VIGS vectors. The variableVIGS effects of such vectors were analyzed on two tomato reporter genes, phytoene desaturase(PDS) and magnesium chelatase (ChlI or SU), whose VIGS phenotypes consist in leaf bleachingand yellowing, respectively, and therefore could be visually assessed. VIGS efficiency by severaldifferent inserts was compared by evaluating intensity of VIGS phenotype, target mRNA levels andaccumulation of VIGS-target specific small interfering RNAs. Overall, our results clearly indicatedthat: i) VIGS efficiency increased when gene sequences inserted in TRV vectors included amiRNAlikepoint mutations; ii) VIGS efficiency was significantly reduced when cDNA fragments fromgene regions with low amiRNA content were expressed in TRV vectors; and iii) WMD3 wasproved an effective bioinformatic tool to select proper target gene sequences in VIGS experiment.Our results are discussed in the light of their beneficial contri

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.

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60 years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F2 individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection.

Different bacterial groups in irrigation well water are strongly implicated in soil health and plant development. Herein, 48 bacterial strains were isolated from agricultural well water in northern Algeria. Among them, four strains were selected based on their antifungal potential and their ability to express Plant Growth Promoting traits such as Indole Acetic Acid (IAA), hydrolytic enzymes, siderophores etc. The isolates were identified as Pseudomonas sp. (B, D and N strains) and Serratia sp. (C strain) by 16S rRNA gene sequencing. Mycelial growth inhibition against Botrytis cinerea and Aspergillus niger ranged from 60 to 90% for the four strains. Moreover, volatiles compounds emission by the isolates resulted in Plant Growth Inhibition values ranging from 13 to 50%, specifically against B. cinerea. Impressively, the strains' antifungal activity showed high inducibility as it was obtained only by the filtered supernatants from bacterial cultures previously in contact with the fungus. Finally, a greenhouse assay, carried out to determine the strains' efficacy in promoting plant growth and protecting seedlings under Pythium aphanidermatum-infected soil, revealed that the strain N markedly enhanced pea germination (+250%) and fresh weight (+43%) and tomato fresh weigh (+10%). The results constitute an attempt for better use of the bacterial functional diversity from irrigation wells in sustainable agriculture.

The establishment and maintenance of DNA methylation are relatively well understood whereas little is known about their dynamics and biological relevance in innate immunity. In plants, modulation of DNA methylation might be an effective mechanism to regulate gene expression in response to abiotic and biotic stresses. Recent evidence through large-scale epigenomicapproaches indicate that dynamic DNA methylation changes are not limited to gene imprinting but can regulate the plant's immune system in response to pathogens. In plants, virus infections trigger expression and regulation of non-coding smallRNAs, and genomic regions are epigenetically modified through the action of the same molecules; however, the involvement of DNA methylation in regulation of plant immune system in response to virus infection was not investigated before. We have examined for the first time the impact of virus infections on genomic DNA methylation and the correlation with smallRNA regulation and gene expression by integrating together analysis of multiple "omics" datasets based on next-generation sequencing platforms. To investigate the possibility that DNA methylation dynamically responds to virus infection, we performed whole-genome bisulfite sequencing on Arabidopsis leaves systemically infected with either the DNA genome virus Cauliflower mosaic virus (CaMV-Arabidopsis) or the RNA virus Cucumber mosaic virus (CMV-Arabidopsis). Single-base resolution methylome analysis revealed more than 3.7million methyl-cytosines (mCs) for the control plant. Interestingly in CMV Arabidopsis we found 300.000 more mCs (hypermethylated) and in CaMV-Arabidopsis 700.000 mCs less (hypomethylated). Focusing on differentially methylated regions (DMR, 250nt in length) we observed a balanced distribution of hyper- and hypomethylation in CG and CHH context in CMV-Arabidopsis (total DMRs 2700) but in CaMV-Arabidopsis we have predominantly hypomethylated DMRs in CHH context (total DMRs 5600). Gene features including coding, non-coding and promoter sequences were assigned to unique gene identifiers according to the TAIR nomenclature. Among differentially methylated gene features, promoter regions were the vast majority, accounting, in specific mCs contexts, for up to 80% of the total. The whole gene ID dataset was subjected to gene functional enrichment analysis by using the DAVID package tool. Interestingly, definite functional categories such as "plant defense" and "auxin signalling pathway" resulted significantly enriched. The correlation between the DNA methylation status and the transcriptional modulation of those genes is under investigation. A comparison between methylation profiles induced by either CaMV or CMV infections revealed conspicuous qualitative and quantitative differences. Taken together our results indicate that RNA- and DNA-genome virus infection induce different regulation of DNA methylation and, at least in part, different immune response in Arabidopsis.

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.

Systemin is a plant signal peptide hormone involved in the responses to wounding and insect damage in the Solanaceae family. It works in the same signaling pathway of jasmonic acid (JA) and enhances the expression of proteinase inhibitors. With the aim of studying a role for systemin in plant antiviral responses, a tomato (Solanum lycopersicum) transgenic line overexpressing the prosystemin cDNA, i.e. the systemin precursor, was inoculated with Cucumber mosaic virus (CMV) strain Fny supporting either a necrogenic or a non-necrogenic satellite RNA (satRNA) variant. Transgenic plants showed reduced susceptibility to both CMV/satRNA combinations. While symptoms of the non-necrogenic inoculum were completely suppressed, a delayed onset of lethal disease occurred in about half of plants challenged with the necrogenic inoculum. RT-qPCR analysis showed a correlation between the systemin-mediated reduced susceptibility and the JA biosynthetic and signaling pathways (e.g. transcriptional alteration of lipoxygenase D and proteinase inhibitor II). Moreover, transgenically overexpressed systemin modulated the expression of a selected set of receptor- like protein kinase (RLK) genes, including some playing a known role in plant innate immunity. A significant correlation was found between the expression profiles of some RLKs and the systemin-mediated reduced susceptibility to CMV/satRNA. These results show that systemin can increase plant defenses against CMV/satRNA through transcriptional reprogramming of diverse signaling pathways.

In tomato, resistance to Tomato spotted wilt virus (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan(TM) SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.

In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase (RDR) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 52-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 32 half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.

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

L'esistenza del banano nei Paesi tropicali è oggi a rischio: l'obiettivo dei nuovi studi è quindi la ricerca di varietà resistenti e consorzi microbici benefici per un controllo efficace delle diverse malattie.

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

Plant viruses are obligate parasites that exploit host components for replication and spread inside the host. Transport of the viral genome is enabled by movement proteins (MPs) targeting the cell periphery to mediate passage throughout plasmodesmata (PD). Pectin methylesterase (PME) is one of the critical host factors facilitating MPs in PD gating, and a direct interaction of PME with Tobacco mosaic virus (TMV) MP is required for viral movement and in turn for virus viability. PME is a critical enzyme for host development and defence, acting via complex mechanisms involving multigenic and tissue specific isoforms and endogenous inhibitors. This composite activity of PME suggests that level and timing of protein accumulation, with respect to virus inoculation and MP expression, can be critical for the functional outcome of the PME-MP interaction and in turn for the success of a viral infection. Based on this notion, we tested different experimental conditions to evaluate the beneficial effect of the downregulation of PME gene expression on the development of TMV-induced disease and on plant protection. We used virus induced gene silencing technology (VIGS) to downregulate PME gene expression, which resulted in a 30-45 % reduction of TMV symptom severity and, correspondingly, to a 60 % reduction of TMV RNA accumulation in systemic leaves. VIGS proved to be a rapid and effective technology for PME gene silencing in functional assays and for plant defence from viral infection. Our findings indicate that N. benthamiana plants with hindered expression of PME survive a TMV infection, which kills non-silenced plants within a week.

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