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Evidence for different, host-dependent functioning of Rx against both wild-type and recombinant Pepino mosaic virus.

ABSTRACT: The potato Rx gene provides resistance against Pepino mosaic virus (PepMV) in tomato; however, recent work has suggested that the resistance conferred may not be durable. Resistance breaking can probably be attributed to multiple mutations observed to accumulate in the capsid protein (CP) region of resistance-breaking isolates, but this has not been confirmed through directed manipulation of an infectious PepMV clone. The present work describes the introduction of two specific mutations, A-T78 and A-T114, into the coat protein minimal elicitor region of an Rx-controlled PepMV isolate of the EU genotype. Enzyme-linked immunosorbent assay (ELISA) and phenotypic evaluation were conducted in three Rx-expressing and wild-type solanaceous hosts: Nicotiana benthamiana, Nicotiana tabacum and Solanum lycopersicum. Mutation A-T78 alone was sufficient to confer Rx-breaking activity in N.?benthamiana and S.?lycopersicum, whereas mutation A-T114 was found to be associated, in most cases, with a secondary A-D100 mutation to break Rx-mediated resistance in S.?lycopersicum. These results suggest that the need for a second, fitness-restoring mutation may be dependent on the PepMV mutant under consideration. Both mutations conferred Rx breaking in S.?lycopersicum, whereas neither conferred Rx breaking in N.?tabacum and only A-T78 allowed Rx breaking in N.?benthamiana, suggesting that Rx may function in a different manner depending on the genetic background in which it is present.

SUBMITTER: Duff-Farrier CR

PROVIDER: S-EPMC6638469 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Evidence for different, host-dependent functioning of Rx against both wild-type and recombinant Pepino mosaic virus.

Duff-Farrier Celia R A CR Candresse Thierry T Bailey Andy M AM Boonham Neil N Foster Gary D GD

Molecular plant pathology 20150512 1

The potato Rx gene provides resistance against Pepino mosaic virus (PepMV) in tomato; however, recent work has suggested that the resistance conferred may not be durable. Resistance breaking can probably be attributed to multiple mutations observed to accumulate in the capsid protein (CP) region of resistance-breaking isolates, but this has not been confirmed through directed manipulation of an infectious PepMV clone. The present work describes the introduction of two specific mutations, A-T78 a ...[more]

PMID: 25787776

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Project description:BackgroundPepino mosaic virus (PepMV) is an emerging plant pathogen that infects tomatoes worldwide. Understanding the factors that influence its evolutionary success is essential for developing new control strategies that may be more robust against the evolution of new viral strains. One of these evolutionary factors is the distribution of mutational fitness effect (DMFE), that is, the fraction of mutations that are lethal, deleterious, neutral, and beneficial on a given viral strain and host species. The goal of this study was to characterize the DMFE of introduced nonsynonymous mutations on a mild isolate of PepMV from the Chilean 2 strain (PepMV-P22). Additionally, we also explored whether the fitness effect of a given mutation depends on the gene where it appears or on epistatic interactions with the genetic background. To address this latter possibility, a subset of mutations were also introduced in a mild isolate of the European strain (PepMV-P11) and the fitness of the resulting clones measured.ResultsA collection of 25 PepMV clones each containing a single nucleotide nonsynonymous substitution was created by site-directed mutagenesis and the fitness of each mutant was determined. PepMV-P22 genome showed a high degree of robustness against point mutations, with 80% of mutations being either neutral or even beneficial and only 20% being deleterious or lethal. We found that the effect of mutations strongly depended on the gene in which they were introduced. Mutations with the largest average beneficial effects were those affecting the RdRp gene, in contrast to mutations affecting TGB1 and CP genes, for which the average effects were deleterious. Moreover, significant epistatic interactions were observed between nonsynonymous mutations and the genetic background, meaning that the effect of a given nucleotide substitution on a particular genomic context cannot be predicted by knowing its effect in a different one.ConclusionsOur results indicated that PepMV genome has a surprisingly high robustness against mutations. We also found that fitness consequences of a given mutation differ between the two strains analyzed. This discovery suggests that the strength of selection, and thus the rates of evolution, vary among PepMV strains.

Project description:Virus emergence is a complex phenomenon, which generally involves spread to a new host from a wild host, followed by adaptation to the new host. Although viruses account for the largest fraction of emerging crop pathogens, knowledge about their emergence is incomplete. We address here the question of whether Pepino Mosaic Virus (PepMV) emergence as a major tomato pathogen worldwide could have involved spread from wild to cultivated plant species and host adaptation. For this, we surveyed natural populations of wild tomatoes in southern Peru for PepMV infection. PepMV incidence, genetic variation, population structure, and accumulation in various hosts were analyzed. PepMV incidence in wild tomatoes was high, and a strain not yet reported in domestic tomato was characterized. This strain had a wide host range within the Solanaceae, multiplying efficiently in most assayed Solanum species and being adapted to wild tomato hosts. Conversely, PepMV isolates from tomato crops showed evidence of adaptation to domestic tomato, possibly traded against adaptation to wild tomatoes. Phylogenetic reconstructions indicated that the most probable ancestral sequence came from a wild Solanum species. A high incidence of PepMV in wild tomato relatives would favor virus spread to crops and its efficient multiplication in different Solanum species, including tomato, allowing its establishment as an epidemic pathogen. Later, adaptation to tomato, traded off against adaptation to other Solanum species, would isolate tomato populations from those in other hosts.Virus emergence is a complex phenomenon involving multiple ecological and genetic factors and is considered to involve three phases: virus encounter with the new host, virus adaptation to the new host, and changes in the epidemiological dynamics. We analyze here if this was the case in the recent emergence of Pepino Mosaic Virus (PepMV) in tomato crops worldwide. We characterized a new strain of PepMV infecting wild tomato populations in Peru. Comparison of this strain with PepMV isolates from tomato crops, plus phylogenetic reconstructions, supports a scenario in which PepMV would have spread to crops from wild tomato relatives, followed by adaptation to the new host and eventually leading to population isolation. Our data, which derive from the analysis of field isolates rather than from experimental evolution approaches, significantly contribute to understanding of plant virus emergence, which is necessary for its anticipation and prevention.

Project description:Plant and virus materials, inoculation and symptom evaluation Tomato seedlings, cultivar Tricia (De Ruiter seeds, Bergschenhoek, the Netherlands) were grown in stonewool in climate chamber conditions (22 and 20°C during day and night periods of 10 and 14 hours, respectively, at 75% relative humidity). At 29 days after planting, plants were inoculated with a mild (1906; GenBank accession number FJ457096) and an aggressive (PCH 06/104; GenBank accession number FJ457097) PepMV isolate of the CH2 genotype. Here, a PepMV isolate is defined as the viral inoculum derived from PepMV infected plants from one specific tomato production site. After inoculation, the genotype of both isolates was determined using a previously described RT-PCR-RFLP method (Hanssen et al., 2008). Inoculation was performed on the second fully developed leaf as previously described (Hanssen et al., 2008). The phenotypic response of tomato seedlings upon inoculation was evaluated by recording the development of typical nettlehead-like PepMV symptoms at 4, 8 and 12 days post inoculation (DPI) on 20 plants per treatment. PepMV induced nettlehead-like symptoms are characterized by a reduced leaf surface, leaf bubbling and leaf deformation (Hanssen et al., 2008). Symptoms were scored from 0 (no symptoms) to 3 (severe symptoms) (Figure 1b). Significant (p<0.05) differences in symptom scores were identified by analysis of variance (one-way ANOVA) and post-hoc Bonferroni tests using SPSS software (v. 10.0; SPSS Inc., Chicago, IL, USA). Microarray sample preparation and determination of viral titers Tomato genes that were differentially regulated (more than twofold change with P value < 0,001) upon inoculation with the aggressive and mild PepMV isolates were identified at 4, 8 and 12 DPI using mock-inoculated control plants as a reference. At each time point, the youngest fully developed leaves from CH2 mild, CH2 aggressive and mock-inoculated plants were sampled for tomato gene chip hybridizations. Each plant was sampled only once. Three biological replicates, each consisting of pooled RNA extracts obtained from the youngest fully developed leaves of two seedlings, were analyzed per treatment. Total RNA was extracted using the RiboPure RNA extraction kit (Ambion) and reverse transcribed with labeled oligo-dT primers for hybridization onto custom-designed Affymetrix tomato GeneChip arrays (Syngenta Biotechnology, Inc., Research Triangle Park, North Carolina, US) that contains probe sets to interrogate 22,721 tomato transcripts (Van Esse et al., 2007). Viral accumulation was measured using a PepMV-specific RT-qPCR assay with forward primer Pep5 (5' ATGAAGCATTCATACCAAAT 3') and reverse primer Pep4 (5' AATTCCGTGCACAACTAT 3'; Mumford & Metcalfe, 2001) respectively. PCR amplification was carried out using a Cepheid® Smart Cycler II thermocycler and analyzed using Smart Cycler software. The PCR program consisted of an initial denaturation step at 95°C for 15 min, 45 cycles of 15s at 94 ºC, 30 s at 50 °C and 30 s at 72 °C, followed by a final incubation step of 2 min at 72°C. Standard curves based on cDNA dilution series were generated to determine the relative concentrations of amplified viral RNA. Based on 4 replicates, run in two different analyses, a reaction efficiency of around 90% was obtained. Ct values obtained from the PepMV specific assay were standardized by subtraction from an internal control assay (efficiency 99%) amplifying a partial sequence of the ribulose 1.5-biphosphate carboxylase chloroplast gene (Sánchez-Navarro et al. 2005).

Project description:Recently, Pepino mosaic virus (PepMV) infections causing severe yellowing symptoms in tomato plants have been reported in glasshouse tomato crops. When studying this phenomenon in commercial glasshouses, two different types of yellowing symptoms, occurring in adjacent plants, were distinguished: interveinal leaf yellowing and yellow mosaics. After several weeks, the interveinal leaf yellowing symptoms gradually disappeared and the plant heads became green again, with yellow mosaic patterns on the leaves as an intermediate stage. The sequencing of multiple isolates causing interveinal leaf yellowing identified two point mutations, occurring in positions 155 and 166 of the coat protein (CP), as unique to the yellowing pathotype. Site-directed mutagenesis of infectious clones confirmed that both CP mutations are determinants of the interveinal leaf yellowing symptoms. Sequencing of CP clones from plants or plant parts with the yellow mosaic symptoms resulted in a mixture of wild-type and mutated sequences, whereas sequencing of CP clones from the green heads of recovered plants resulted in only wild-type sequences. Yellow mosaic symptoms could be reproduced by inoculation of an artificial 1:1 mixture of RNA transcripts from the wild-type and mutated infectious clones. These results show that the ratio of mutated versus wild-type sequences can determine the nature and severity of symptom development. The gradual recovery of the plants, which coincides with the disappearance of the yellowing mutations, suggests that selection pressure acts to the advantage of the wild-type virus. Experiments with wild-type and mutated infectious clones showed that reverse mutation events from mutant to wild-type occur and that the wild-type virus does not have a replicative advantage over the mutant. These results suggest that reverse mutation events occur, with subsequent selection pressure acting in favour of the wild-type virus in the growing plant parts, possibly related to a lower long-distance movement efficiency of the mutant.

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Evidence for different, host-dependent functioning of Rx against both wild-type and recombinant Pepino mosaic virus.

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Evidence for different, host-dependent functioning of Rx against both wild-type and recombinant Pepino mosaic virus.

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