Tomato (Solanum lycopersicum) is a crucial vegetable globally, pivotal in securing the world's food supply. However, viral diseases pose a significant threat to tomato cultivation, resulting in ...considerable yield losses. Employing sensitive, rapid, and cost‐effective detection methods is imperative for mitigating such losses in agricultural production. In response, we developed a Taqman® probe‐based real‐time multiplex PCR method capable of concurrently detecting three major plant viruses: Tomato brown rugose fruit virus (ToBRFV), tomato spotted wilt orthotospovirus (TSWV), and pepino mosaic virus (PepMV), all of which are prominent viral pathogens affecting tomato production. Utilizing envelope protein encoding sequences of these pathogens, we designed primers and probes, assigning the FAM reporter for ToBRFV, the HEX reporter for TSWV, and the Cy5 reporter for PepMV detection. The multiplex RT‐qPCR experiment yielded successful results, with Ct values of 29.34, 25.93, and 27.47 for ToBRFV (at 10−6 dilution), TSWV (at 10−2 dilution), and PepMV (at 10−3 dilution), respectively. Developed primers facilitate the early detection of destructive pathogens ToBRFV, TSWV, and PepMV using both RT‐PCR and RT‐qPCR, offering a user‐friendly and cost‐effective approach.
•The pandemic pepino mosaic virus (PepMV) causes huge losses in tomato crops.•Strain mixed infections are frequent and result in asymmetric antagonism.•Tagged PepMV strains colonized the host sharing ...approximately 16% of infected cells.•Viral replication complexes-like structures were shared by two tagged PepMV strains.
Mixed infections of pepino mosaic virus (PepMV) isolates from the EU and CH2 strains are frequent in tomato crops. An asymmetric antagonistic relationship has been described between these strains, making their in planta interaction worthy of study. The aim of this work was to verify if PepMV isolates labelled with fluorescent proteins recapitulate the interactions described for wild type isolates and, if so, to determine the proportion of cells infected with each isolate in single and mixed infected plants. Infectious clones were prepared for PepMV-CH2-GFP and -EU-TagRFP, and also for their reciprocal combination, PepMV-CH2-TagRFP and -EU-GFP, and used to inoculate Nicotiana benthamiana plants. The accumulation of viral RNA followed trends that differed from wild type viruses, with the PepMV-EU-GFP and -CH2-TagRFP pair reproducing more closely the wild type interaction. Protoplasts were isolated from leaves that were systemically infected with PepMV-EU-GFP, -CH2-TagRFP, or both, and flow cytometry was used to determine the proportion of cells infected with each tagged isolate. A significant proportion (16.6%) of cells were found to be infected with both, without strong evidence of virus exclusion in coinfections, as could have been expected for related viruses; in fact, cellular structures reminiscent of viral replication complexes were found to be labelled with both fluorescent reporters.
While recent pepino mosaic virus (PepMV; species
, genus
, family
) epidemics seem to be predominantly caused by isolates of the CH2 strain, PepMV epidemics in intensive tomato crops in Spain are ...caused by both CH2 and EU isolates that co-circulate, representing a challenge in terms of control, including cross-protection. In this work, we hypothesized that mixed infections with two mild isolates of the EU and CH2 strains (PepMV-Sp13 and -PS5, respectively) may be useful in PepMV cross-protection in Spanish epidemics, providing protection against a broad range of aggressive isolates. Thus, we performed a range of field trials and an experimental evolution assay to determine the phenotypic and genetic stability of PepMV-Sp13 and -PS5 mixed infections, as well as their cross-protective efficiency. Our results showed that: (i) the phenotype of PepMV-Sp13 and -PS5 mixed infections was mild and did not change significantly when infecting different tomato cultivars or under different environmental conditions in Spain, (ii) PepMV-Sp13 and -PS5 mixed infections provided more efficient protection against two aggressive EU and CH2 isolates than single infections, and (iii) PepMV-Sp13 and -PS5, either in single or in mixed infections, were less variable than other two PepMV isolates occurring naturally in PepMV epidemics in Spain.
Summary
Pepino mosaic virus (PepMV) is pandemic in tomato crops, causing important economic losses world‐wide. No PepMV‐resistant varieties have been developed yet. Identification of host factors ...interacting with PepMV proteins is a promising source of genetic targets to develop PepMV‐resistant varieties.
The interaction between the PepMV coat protein (CP) and the tomato glutathione S‐transferase (GST) SlGSTU38 was identified in a yeast two‐hybrid (Y2H) screening and validated by directed Y2H and co‐immunoprecipitation assays. SlGSTU38‐knocked‐out Micro‐Tom plants (gstu38) generated by the CRISPR/Cas9 technology together with live‐cell imaging were used to understand the role of SlGSTU38 during infection. The transcriptomes of healthy and PepMV‐infected wild‐type (WT) and gstu38 plants were profiled by RNA‐seq analysis.
SlGSTU38 functions as a PepMV‐specific susceptibility factor in a cell‐autonomous manner and relocalizes to the virus replication complexes during infection. Besides, knocking out SlGSTU38 triggers reactive oxygen species accumulation in leaves and the deregulation of stress‐responsive genes.
SlGSTU38 may play a dual role: On the one hand, SlGSTU38 may exert a proviral function depending on its specific interaction with the PepMV CP; and on the other hand, SlGSTU38 may delay PepMV‐infection sensing by participating in the redox intracellular homeostasis in a nonspecific manner.
Mixed viral infections are common in plants, and the evolutionary dynamics of viral populations may differ depending on whether the infection is caused by single or multiple viral strains. However, ...comparative studies of single and mixed infections using viral populations in comparable agricultural and geographical locations are lacking. Here, we monitored the occurrence of pepino mosaic virus (PepMV) in tomato crops in two major tomato-producing areas in Murcia (southeastern Spain), supporting evidence showing that PepMV disease-affected plants had single infections of the Chilean 2 (CH2) strain in one area and the other area exhibited long-term (13 years) coexistence of the CH2 and European (EU) strains. We hypothesized that circulating strains of PepMV might be modulating the differentiation between them and shaping the evolutionary dynamics of PepMV populations. Our phylogenetic analysis of 106 CH2 isolates randomly selected from both areas showed a remarkable divergence between the CH2 isolates, with increased nucleotide variability in the geographical area where both strains cocirculate. Furthermore, the potential virus-virus interaction was studied further by constructing six full-length infectious CH2 clones from both areas, and assessing their viral fitness in the presence and absence of an EU-type isolate. All CH2 clones showed decreased fitness in mixed infections and although complete genome sequencing indicated a nucleotide divergence of those CH2 clones by area, the magnitude of the fitness response was irrespective of the CH2 origin. Overall, these results suggest that although agroecological cropping practices may be particularly important for explaining the evolutionary dynamics of PepMV in tomato crops, the cocirculation of both strains may have implications on the genetic variability of PepMV populations.
The hyperosmolality-gated calcium permeable channel 4.1 (OSCA4.1) belongs to an evolutionarily conserved small family of mechano-sensitive channels. OSCA members may represent key players in plant ...resistance to drought and to pathogen infection but are scarcely studied. After screening for resistance to pepino mosaic virus (PepMV) a collection of 1000 mutagenized tomato families, we identified a mutant showing no symptoms and reduced virus accumulation. Resistance was mapped to chromosome 2 between positions 46 309 531 to 47 044 163, where a missense mutation caused the putative truncation of the OSCA4.1 protein. A CRISPR/Cas9 slosca4.1 mutant was resistant to PepMV, but not to tobacco mosaic virus or potato virus X. Inoculation of mutant and wild type tomato protoplasts showed that resistance was expressed in single cells, suggesting a role for SlOSCA4.1 in early viral function(s); congruently, SlOSCA4.1 re-localized to structures reminiscent of viral replication complexes. We propose that SlOSCA4.1 contributes to the correct regulation of the Ca
homeostasis necessary for optimal PepMV infection. PepMV is a pandemic virus that causes significant losses in tomato crops worldwide. In spite of its importance, no tomato-resistant varieties have been deployed yet; the mutant identified here has great potential to breed tomato varieties resistant to PepMV.
Pepino mosaic virus (PepMV) is a highly infectious potexvirus that causes a severe disease in tomato (Solanum lycopersicum) crops worldwide. In Sicily, the first outbreak was detected in a single ...greenhouse in 2005 and it was promptly eradicated. However, in 2008, a large number of greenhouses were simultaneously affected, and it was impossible to eradicate or control the virus. This study addressed the dispersion and the genetic diversity of PepMV isolates obtained from the outbreak in Sicily, in comparison with worldwide PepMV isolates, to gain insight into the factors determining the evolution and epidemiology of the virus. A total of 1800 samples from plants with and without symptoms were collected in the Sicilian provinces of Agrigento, Caltanissetta, Palermo, Ragusa, Siracusa and Trapani. Three isolates collected at different times were biologically characterized. The incidence of the virus increased rapidly from 13% in 2011 to 63% in 2013, and phylogenetic analysis showed that all Sicilian isolates of PepMV belonged to the CH2 strain, one of the six strains previously described. Nucleotide diversity of the Sicilian isolates was low, thus suggesting rapid spread and genetic stability.
Plants have evolved well‐tuned surveillance systems, including complex defence mechanisms, to constrain pathogens. TFs are master regulators of host molecular responses against plant pathogens. While ...PepMV constitutes a major threat to the global tomato production, there is still a lack of information on the key TFs that regulate host responses to this virus.
A combinatorial research approach was applied relying on tomato transcriptome analysis, RT‐qPCR validation, phylogenetic classification, comparative analysis of structural features, cis‐regulatory element mining and in silico co‐expression analysis to identify a set of 11 highly responsive TFs involved in the regulation of host responses to PepMV.
An endemic PepMV isolate, generating typical mosaic symptoms, modified expression of ca. 3.3% of tomato genes, resulting in 1,120 DEGs. Functional classification of 502 upregulated DEGs revealed that photosynthesis, carbon fixation and gene silencing were widely affected, whereas 618 downregulated genes had an impact mainly on plant defence and carotenoid biosynthesis. Strikingly, all 11 highly responsive TFs carried abiotic stress response cis‐regulatory elements, whereas five of them were better aligned with rice than with Arabidopsis gene homologues, suggesting that plant responses against viruses may predate divergence into monocots and dicots. Interestingly, tomato C2H2 family TFs, ZAT1‐like and ZF2, may have distinct roles in plant defence due to opposite response patterns, similar to their Arabidopsis ZAT10 and ZAT12 homologues.
These highly responsive TFs provide a basis to study in‐depth molecular responses of the tomato–PepMV pathosystem, providing a perspective to better comprehend viral infections.
A combinatorial research approach was applied primarily relying on transcriptome analysis to identify highly responsive tomato TFs and understand the molecular defensive mechanisms of the tomato‐PepMV pathosystem.
Pepino mosaic virus (PepMV) causes significant economic losses in tomato crops worldwide. Since its first detection infecting tomato in 1999, aggressive PepMV variants have emerged. This study aimed ...to characterize two aggressive PepMV isolates, PepMV-H30 and PepMV-KLP2. Both isolates were identified in South-Eastern Spain infecting tomato plants, which showed severe symptoms, including bright yellow mosaics. Full-length infectious clones were generated, and phylogenetic relationships were inferred using their nucleotide sequences and another 35 full-length sequences from isolates representing the five known PepMV strains. Our analysis revealed that PepMV-H30 and PepMV-KLP2 belong to the EU and CH2 strains, respectively. Amino acid sequence comparisons between these and mild isolates identified 8 and 15 amino acid substitutions for PepMV-H30 and PepMV-KLP2, respectively, potentially involved in severe symptom induction. None of the substitutions identified in PepMV-H30 have previously been described as symptom determinants. The E236K substitution, originally present in the PepMV-H30 CP, was introduced into a mild PepMV-EU isolate, resulting in a virus that causes symptoms similar to those induced by the parental PepMV-H30 in
plants. In silico analyses revealed that this residue is located at the C-terminus of the CP and is solvent-accessible, suggesting its potential involvement in CP-host protein interactions. We also examined the subcellular localization of PepGFPm2
in comparison to that of PepGFPm2, focusing on chloroplast affection, but no differences were observed in the GFP subcellular distribution between the two viruses in epidermal cells of
plants. Due to the easily visible symptoms that PepMV-H30 and PepMV-KLP2 induce, these isolates represent valuable tools in programs designed to breed resistance to PepMV in tomato.
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