Grafting of commercial tomato varieties and hybrids on the tomato ecotype Manduria resulted in high levels of tolerance to the infection of Sw5 resistance-breaking strains of tomato spotted wilt ...virus and of severe cucumber mosaic virus strains supporting hypervirulent satellite RNAs that co-determine stunting and necrotic phenotypes in tomato. To decipher the basis of such tolerance, here we used a RNAseq analysis to study the transcriptome profiles of the Manduria ecotype and of the susceptible variety UC82, and of their graft combinations, exposed or not to infection of the potato virus Y recombinant strain PVY
-to. The analysis identified graft- and virus-responsive mRNAs differentially expressed in UC82 and Manduria, which led to an overall suitable level of tolerance to viral infection confirmed by the appearance of a recovery phenotype in Manduria and in all graft combinations. The transcriptome analysis suggested that graft wounding and viral infection had diverging effects on tomato transcriptome and that the Manduria ecotype was less responsive than the UC82 to both graft wounding and potyviral infection. We propose that the differential response to the two types of stress could account for the tolerance to viral infection observed in the Manduria ecotype as well as in the susceptible tomato variety UC82 self-grafted or grafted on the Manduria ecotype.
RNA interference (RNAi) is a sequence identity-dependent RNA degradation mechanism conserved in eukaryotic organisms. One of the roles of RNAi is as a defense system against viral infections, which ...has been demonstrated in filamentous fungi but not in oomycetes. We investigated the virus-RNAi interplay in the oomycete Phytophthora infestans using a crucifer-infecting strain of the plant virus tobacco mosaic virus (TMVcr) and its derivative TMVcr-Δ122 that is mutated in the sequence of the p122 replicase subunit and thus inhibited in RNA suppression activity. In this study we provide evidence that replication of TMVcr-Δ122 but not of TMVcr was impaired in P. infestans as well as in tobacco plants used as positive control. The interference was associated with induction of high transcription of dicer-like genes Pidcl2 and NtDCL2 and of RNA-dependent-RNA-polymerase Pirdr1 and NtRDR1 in P. infestans and tobacco, respectively. These high transcription levels suggest an RNAi-based response that TMVcr-Δ122 mutant was not able to suppress. Taken altogether, results of this study demonstrated that an antiviral silencing activity operates also in P. infestans and that a plant virus could be a simple and feasible tool for functional studies also in oomycetes.
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 1α, 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.
Most of the 26 viruses that infect globe artichoke are detrimental to the crop’s production, quality, and nutritional value, hamper the development of a nursery activity in the respect of current EU ...legislation and pose risks for the new plantings. Worldwide movement of virus-controlled plant propagative material require fast, accurate and feasible methods to guarantee reliable results and detection of multiple viruses in a single assay to reduce time and costs in routine tests. Here we describe the use of a digoxigenin-labelled polyprobe assembled from synthetic 50 nt single-stranded DNA sequences derived from genomes of the most commonly occurring viruses in globe artichoke. These included artichoke Italian latent virus (AILV), artichoke latent virus (ArLV), artichoke mottled crinkle virus (AMCV), turnip mosaic virus (TuMV), tomato infectious chlorosis virus (TICV), bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), pelargonium zonate spot virus (PZSV), tomato spotted wilt virus (TSWV) and tobacco mosaic virus (TMV). The polyprobe described herein represent the first cheap and reliable example of a technology for broad-spectrum surveys in globe artichoke.
RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted ...through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.
Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging begomovirus (
family) listed in the EPPO Alert List 2, present in the Mediterranean area and in Italy, where it was reported in 2015 in ...Sicilian courgette. The virus is widespread in cucurbits where it causes up to 100% production losses. In 2018, ToLCNDV was isolated in Apulia (southern Italy) in commercial fields of zucchini squash and since then its recurrent outbreaks generated justified concern among growers. Thus, a sustainable and environmentally friendly approach must be adopted. Genetic resistances have been identified in
and
but, compared to genetic resistance, grafting could provide a faster and more flexible solution because the graft wounding induces tolerance rather than resistance against airborne virus infection. Compared to tolerance, the up-regulation of resistance genes requires energy resources mobilized at the expense of primary metabolism, plant growth, and development. Results of screening among twenty-one local cucurbit cvs. ecotypes and accessions to evaluate tolerance levels against rub-inoculation of ToLCNDV led to the identification of potential rootstocks to attain suitable levels of tolerance against the virus in commercial cucurbit varieties. Cucurbit plants were challenged by a ToLCNDV isolated in Apulia denoted ToLCNDV-Le and evaluated for disease symptoms development and viral DNA accumulation up to 28 days after inoculation. On the basis of disease symptoms developed, plants were classified as tolerant, moderately tolerant, moderately susceptible, and susceptible.
cv. Barattiere did not show any detectable disease symptoms and very low levels of viral DNA accumulation was recorded; thus, it was used as rootstock for some of the remaining cucurbit genotypes that were used as scions. The tolerance trait was transmitted to the otherwise susceptible and moderately susceptible cucurbit genotypes grafted onto the cv. Barattiere. The results of this study suggest practical implications of the approach described.
A headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME/GC-MS) method was used to study the volatile organic compounds (VOCs) associated with the differential immune ...response of tomato plants infected with the recombinant strain of potato virus Y (PVYC-to), necrogenic to tomato. Analysis was carried out in UC82 (UC), a virus susceptible tomato variety, comparing the same UC plants grafted or not onto a virus tolerant tomato ecotype, Manduria (Ma); the three types of samples used for the GC-MS analysis were mock-inoculated UC/Ma plants, UC/Ma+PVYC-to and UC+PVYC-to plants; the VOCs obtained were 111. Results from symptomatic PVYC-to-infected UC plants showed a VOCs composition enriched in alcohols, fatty acid derivates, benzenoids, and salicylic acid derivatives, while in mock-(UC/Ma)-inoculated plants VOCs were mainly characterized by methyl ester compounds. The VOC profile was in line with RNAseq data analyses, denoting that PVYC-to viral RNA accumulation and disease symptoms induce the specific transcriptional activation of genes involved in VOCs biosynthesis. Furthermore, principal component analysis highlighted that VOCs of PVYC-to-infected and mock-inoculated grafted plants were much closer each other than that of symptomatic PVYC-to-infected non-grafted UC plants. These results suggest that VOCs profiles of tomato plants are related to the viral RNA accumulation, disease intensity and graft-derived tolerance to PVYC-to infection.
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•Metagenomic and NGS analyses reveal census of complex viral populations in plants.•Viruses, mutants, virus-like agents and sequences embedded in host genome create complex ...viromes.•Complemented, enhanced or mutually excluded viral traits lead to synergism and antagonism in plants.•Similar or different tissue localization influence virus interactions.•Mixed infections alter relations with host plant and components of its microbiome.
Metagenomic surveys and data from next generation sequencing revealed that mixed infections among plant viruses are probably a rule rather than an exception in natural pathosystems. The documented cases may range from synergism to antagonism, which may depend from the spatiotemporal order of arrival of the viruses on the host and upon the host itself. In synergistic interactions, the measurable differences in replication, phenotypic and cytopathological changes, cellular tropism, within host movement, and transmission rate of one of the two viruses or both are increased. Conversely, a decrease in replication, or inhibition of one or more of the above functions by one virus against the other, leads to an antagonistic interaction. Viruses may interact directly and by transcomplementation of defective functions or indirectly, through responses mediated by the host like the defense mechanism based on RNA silencing. Outcomes of these interactions can be applied to the risk assessment of transgenic crops expressing viral proteins, or cross-protected crops for the identification of potential hazards. Prior to experimental evidence, mathematical models may help in forecasting challenges deriving from the great variety of pathways of synergistic and antagonistic interactions. Actually, it seems that such predictions do not receive sufficient credit in the framework of agriculture.
Grafting is routinely implemented in modern agriculture to manage soilborne pathogens such as fungi, oomycetes, bacteria, and viruses of solanaceous crops in a sustainable and environmentally ...friendly approach. Some rootstock/scion combinations use specific genetic resistance mechanisms to impact also some foliar and airborne pathogens, including arthropod or contact-transmitted viruses. These approaches resulted in poor efficiency in the management of plant viruses with superior virulence such as the strains of tomato spotted wilt virus breaking the Sw5 resistance, strains of cucumber mosaic virus carrying necrogenic satellite RNAs, and necrogenic strains of potato virus Y. Three different studies from our lab documented that suitable levels of resistance/tolerance can be obtained by grafting commercial tomato varieties onto the tomato ecotype Manduria (Ma) rescued in the framework of an Apulian (southern Italy) regional program on biodiversity. Here we review the main approaches, methods, and results of the three case studies and propose some mechanisms leading to the tolerance/resistance observed in susceptible tomato varieties grafted onto Ma as well as in self-grafted plants. The proposed mechanisms include virus movement in plants, RNA interference, genes involved in graft wound response, resilience, and tolerance to virus infection.
RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin ...RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.
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