Genetic diversity and population structure of Pepino mosaic virus in tomato crops of Spain and Morocco

Pepino mosaic virus (PepMV, genus Potexvirus) is an emergent and highly infectious pathogen responsible for economically important diseases in tomato crops. An extensive survey of tomato plants showing PepMV‐like symptoms was carried out in 2017 to study the PepMV genetic diversity and populations structure in different tomato‐producing areas of Spain and Morocco. Molecular dot‐blot hybridization analysis showed that virus populations from Spain and Morocco were mainly composed of isolates belonging to the Chilean 2 (CH2) strain, although isolates of the European (EU) strain were detected in significant proportions in Spanish populations, mainly in mixed infections. A few isolates of the American (US1) strain were also detected in Tenerife (Canary Islands, Spain) crops. Eighty‐five isolates were randomly selected and sequenced in the genomic region that encodes the triple gene block and capsid protein genes. Our phylogenetic and population genetics analyses confirmed the presence of the CH2, EU and US1 PepMV strains. Despite the high genetic similarity observed within populations, variants were maintained at low frequency under purifying selection, and differentiation among more geographically distant locations was identified, with potential gene flow contributing to the shaping of the PepMV populations structure.     

Mixed Infections of Pepino Mosaic Virus Strains Modulate the Evolutionary Dynamics of this Emergent Virus

Pepino mosaic virus (PepMV) is an emerging pathogen that causes severe economic losses in tomato crops (Solanum lycopersicum L.) in the Northern hemisphere, despite persistent attempts of control. In fact, it is considered one of the most significant viral diseases for tomato production worldwide, and it may constitute a good model for the analysis of virus emergence in crops. We have combined a population genetics approach with an analysis of in planta properties of virus strains to explain an observed epidemiological pattern. Hybridization analysis showed that PepMV populations are composed of isolates of two types (PepMV-CH2 and PepMV-EU) that cocirculate. The CH2 type isolates are predominant; however, EU isolates have not been displaced but persist mainly in mixed infections. Two molecularly cloned isolates belonging to each type have been used to examine the dynamics of in planta single infections and coinfection, revealing that the CH2 type has a higher fitness than the EU type. Coinfections expand the range of susceptible hosts, and coinfected plants remain symptomless several weeks after infection, so a potentially important problem for disease prevention and management. These results provide an explanation of the observed epidemiological pattern in terms of genetic and ecological interactions among the different viral strains. Thus, mixed infections appear to be contributing to shaping the genetic structure and dynamics of PepMV populations.Pepino mosaic virus (PepMV; genus Potexvirus, family Flexiviridae) was identified in 1974 as the agent responsible for a viral disease of pepino crops (Solanum muricatum) in Peru (30). PepMV in tomato (Solanum lycopersicum) was first reported in The Netherlands in 1999 (74) but has since spread rapidly in Europe (3, 11, 38, 48, 51, 57) and beyond (20, 35, 36, 42, 68), causing epidemics and severe economic losses (27, 29, 36, 51, 67, 69). The PepMV host range is limited mainly to the Solanaceae (59), and the virus is easily transmitted from plant to plant by contact (30), vectored by bumblebees (65), or seedborne-transmitted (37). PepMV infections in tomato are associated with a wide range of leaf symptoms: mild and severe mosaics, bubbling, laminal distortions, and stunting (26, 27, 51). Fruit symptoms occur with or without leaf symptoms, and the main impact of PepMV is on fruit quality (irregular lycopene distribution [26]) but not on yield (69). Therefore, PepMV is currently considered a dangerous pathogen and is included in the European Plant Protection Organization alert list (15) as one of the most important tomato viruses worldwide (27, 51, 57, 68, 69).The PepMV genome consists of a single, positive-sense, ∼6,400-nucleotide (nt) RNA strand containing five open reading frames (ORFs). ORF1 encodes the putative viral polymerase (RdRp) (3). ORFs 2, 3, and 4 encode the triple gene block (TGB) proteins TGBp1, TGBp2, and TGBp3, which are essential for virus movement (46, 75, 78). Potato virus X TGBp1 is a multifunctional protein that induces plasmodesmal gating, moves from cell to cell, has ATPase and RNA helicase activities, binds viral RNAs, and acts as suppressor of RNA silencing (39, 76-78). ORF5 encodes the coat protein (CP) which, in addition to its structural role, is required for cell-to-cell and long-distance movement (12). Finally, two short untranslated sequences flank the coding regions, and there is a poly(A) tail at the 3′ end of the genomic RNA (3, 11, 48).Previous studies have shown that Spanish PepMV populations sampled between 2000 and 2004 were genetically very homogeneous (∼99% nucleotide identity), most comprising isolates highly similar to the so-called European tomato strain (PepMV-EU). However, a few isolates sampled in 2004 in the Murcia region (Southeastern Spain) were distinct and highly similar to the US2 strain reported in the United States (51). U.S. isolates (US1 and US2) and a Chilean isolate from infected tomato seeds (CH2) share only 79 to 86% nucleotide identity with European (EU) isolates (36, 42). The CH2 type has been reported recently in greenhouses for tomato production in Poland (29) and Belgium (27). In this last study, CH2 was predominant in single infections and also frequent in mixed infections with isolates of the EU type (27). However, all PepMV types (EU, US1, US2, and CH2) have been found in United States, where the PepMV-EU type has been the most prevalent, and mixed infections were found in samples collected from Arizona, Colorado, and Texas (35).Several studies of plant virus populations have reported a reduced genetic diversity of populations separated in time or space (19, 40, 56) with high virus genetic stability (23). Nevertheless, how genetic and ecological factors modulate the evolutionary dynamics of viruses and determine epidemiological patterns is still poorly understood (25, 47).We have characterized the population genetic structure of PepMV in infected samples of commercial tomato crops in the Murcia region (southeastern Spain) between 2005 and 2008. Phylogenetic analysis was performed, and genetic diversity values among PepMV isolates were estimated to determine the structure of the population and the strength and direction of selection. In addition, the biological properties (host range, fitness, and virulence) of two cloned isolates of the CH2 and EU types were studied to understand the evolutionary dynamics of natural PepMV populations.     

Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection

In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro.     

Temperature-dependent survival of Turnip crinkle virus-infected arabidopsis plants relies on an RNA silencing-based defense that requires dcl2, AGO2, and HEN1

While RNA silencing is a potent antiviral defense in plants, well-adapted plant viruses are known to encode suppressors of RNA silencing (VSR) that can neutralize the effectiveness of RNA silencing. As a result, most plant genes involved in antiviral silencing were identified by using debilitated viruses lacking silencing suppression capabilities. Therefore, it remains to be resolved whether RNA silencing plays a significant part in defending plants against wild-type viruses. We report here that, at a higher plant growth temperature (26°C) that permits rigorous replication of Turnip crinkle virus (TCV) in Arabidopsis, plants containing loss-of-function mutations within the Dicer-like 2 (DCL2), Argonaute 2 (AGO2), and HEN1 RNA methyltransferase genes died of TCV infection, whereas the wild-type Col-0 plants survived to produce viable seeds. To account for the critical role of DCL2 in ensuring the survival of wild-type plants, we established that higher temperature upregulates the activity of DCL2 to produce viral 22-nucleotide (nt) small interfering RNAs (vsRNAs). We further demonstrated that DCL2-produced 22-nt vsRNAs were fully capable of silencing target genes, but that this activity was suppressed by the TCV VSR. Finally, we provide additional evidence supporting the notion that TCV VSR suppresses RNA silencing through directly interacting with AGO2. Together, these results have revealed a specialized RNA silencing pathway involving DCL2, AGO2, and HEN1 that provides the host plants with a competitive edge against adapted viruses under environmental conditions that facilitates robust virus reproduction.     

Pepino mosaic virus: a successful pathogen that rapidly evolved from emerging to endemic in tomato crops

Taxonomy: Pepino mosaic virus (PepMV) belongs to the Potexvirus genus of the Flexiviridae family. Physical properties: PepMV virions are nonenveloped flexuous rods that contain a monopartite, positive‐sense, single‐stranded RNA genome of 6.4 kb with a 3′ poly‐A tail. The genome contains five major open reading frames (ORFs) encoding a 164‐kDa RNA‐dependent RNA polymerase (RdRp), three triple gene block proteins of 26, 14 and 9 kDa, and a 25‐kDa coat protein. Genome diversity: Four PepMV genotypes, with an intergenotype RNA sequence identity ranging from 78% to 95%, can be distinguished: the original Peruvian genotype (LP); the European (tomato) genotype (EU); the American genotype US1; and the Chilean genotype CH2. Transmission: PepMV is very efficiently transmitted mechanically, and a low seed transmission rate has been demonstrated. In addition, bumblebees have been associated with viral transmission. Host range: Similar to other Potexviruses, PepMV has a rather narrow host range that is thought to be largely restricted to species of the Solanaceae family. After originally being isolated from pepino (Solanum muricatum), PepMV has been identified in natural infections of the wild tomato species S. chilense, S. chmielewskii, S. parviflorum and S. peruvianum. PepMV is causing significant problems in the cultivation of the glasshouse tomato Solanum lycopersicum, and has been identified in weeds belonging to various plant families in the vicinity of tomato glasshouses. Symptomatology: PepMV symptoms can be very diverse. Fruit marbling is the most typical and economically devastating symptom. In addition, fruit discoloration, open fruit, nettle‐heads, leaf blistering or bubbling, leaf chlorosis and yellow angular leaf spots, leaf mosaic and leaf or stem necrosis have been associated with PepMV. The severity of PepMV symptoms is thought to be dependent on environmental conditions, as well as on the properties of the viral isolate. Minor nucleotide sequence differences between isolates from the same genotype have been shown to lead to enhanced aggressiveness and symptomatology. Control: Prevention of infection through strict hygiene measures is currently the major strategy for the control of PepMV in tomato production. Cross‐protection can be effective, but only under well‐defined and well‐controlled conditions, and the effectiveness depends strongly on the PepMV genotype.     

Transmission of Pepino mosaic virus by the Fungal Vector Olpidium virulentus

Transmission of Pepino mosaic virus (PepMV) by the fungal vector Olpidium virulentus was studied in two experiments. Two characterized cultures of the fungus were used as stock cultures for the assay: culture A was from lettuce roots collected in Castellón (Spain), and culture B was from tomato roots collected in Murcia (Spain). These fungal cultures were maintained in their original host and irrigated with sterile water. The drainage water collected from irrigating these stock cultures was used for watering PepMV‐infected and non‐infected tomato plants to constitute the acquisition–source plants of the assay, which were divided into six different plots: plants containing fungal culture A (non‐infected and PepMV‐infected); plants containing fungal culture B (non‐infected and PepMV‐infected); PepMV‐infected plants without the fungus; and plants non‐infected either with PepMV and the fungus. Thirty‐six healthy plants grouped into six plots, which constituted the virus acquisition–transmission plants of the assay, were irrigated with different drainage waters obtained by watering the different plots of the acquisition–source plants. PepMV was only transmitted to plants irrigated with the drainage water collected from PepMV‐infected plants whose roots contained the fungal culture B from tomato with a transmission rate of 8%. No infection was detected in plants irrigated with the drainage water collected from plots with only a fungus or virus infection. Both the virus and fungus were detected in water samples collected from the drainage water of the acquisition–source plants of the assay. These transmission assays demonstrated the possibility of PepMV transmission by O. virulentus collected from tomato crops.     

Arabidopsis RNA-Dependent RNA Polymerases and Dicer-Like Proteins in Antiviral Defense and Small Interfering RNA Biogenesis during Turnip Mosaic Virus Infection

Plants respond to virus infections by activation of RNA-based silencing, which limits infection at both the single-cell and system levels. Viruses encode RNA silencing suppressor proteins that interfere with this response. Wild-type Arabidopsis thaliana is immune to silencing suppressor (HC-Pro)-deficient Turnip mosaic virus, but immunity was lost in the absence of DICER-LIKE proteins DCL4 and DCL2. Systematic analysis of susceptibility and small RNA formation in Arabidopsis mutants lacking combinations of RNA-dependent RNA polymerase (RDR) and DCL proteins revealed that the vast majority of virus-derived small interfering RNAs (siRNAs) were dependent on DCL4 and RDR1, although full antiviral defense also required DCL2 and RDR6. Among the DCLs, DCL4 was sufficient for antiviral silencing in inoculated leaves, but DCL2 and DCL4 were both involved in silencing in systemic tissues (inflorescences). Basal levels of antiviral RNA silencing and siRNA biogenesis were detected in mutants lacking RDR1, RDR2, and RDR6, indicating an alternate route to form double-stranded RNA that does not depend on the three previously characterized RDR proteins.     

An antiviral defense role of AGO2 in plants

Background

Argonaute (AGO) proteins bind to small-interfering (si)RNAs and micro (mi)RNAs to target RNA silencing against viruses, transgenes and in regulation of mRNAs. Plants encode multiple AGO proteins but, in Arabidopsis, only AGO1 is known to have an antiviral role.

Methodology/Principal Findings

To uncover the roles of specific AGOs in limiting virus accumulation we inoculated turnip crinkle virus (TCV) to Arabidopsis plants that were mutant for each of the ten AGO genes. The viral symptoms on most of the plants were the same as on wild type plants although the ago2 mutants were markedly hyper-susceptible to this virus. ago2 plants were also hyper-susceptible to cucumber mosaic virus (CMV), confirming that the antiviral role of AGO2 is not specific to a single virus. For both viruses, this phenotype was associated with transient increase in virus accumulation. In wild type plants the AGO2 protein was induced by TCV and CMV infection.

Conclusions/Significance

Based on these results we propose that there are multiple layers to RNA-mediated defense and counter-defense in the interactions between plants and their viruses. AGO1 represents a first layer. With some viruses, including TCV and CMV, this layer is overcome by viral suppressors of silencing that can target AGO1 and a second layer involving AGO2 limits virus accumulation. The second layer is activated when the first layer is suppressed because AGO2 is repressed by AGO1 via miR403. The activation of the second layer is therefore a direct consequence of the loss of the first layer of defense.     

Knockout of SlTOM1 and SlTOM3 results in differential resistance to tobamovirus in tomato

During tobamovirus–host coevolution, tobamoviruses developed numerous interactions with host susceptibility factors and exploited these interactions for replication and movement. The plant‐encoded TOBAMOVIRUS MULTIPLICATION (TOM) susceptibility proteins interact with the tobamovirus replicase proteins and allow the formation of the viral replication complex. Here CRISPR/Cas9‐mediated mutagenesis allowed the exploration of the roles of SlTOM1a, SlTOM1b, and SlTOM3 in systemic tobamovirus infection of tomato. Knockouts of both SlTOM1a and SlTOM3 in sltom1a/sltom3 plants resulted in an asymptomatic response to the infection with recently emerged tomato brown rugose fruit virus (ToBRFV). In addition, an accumulation of ToBRFV RNA and coat protein (CP) in sltom1a/sltom3 mutant plants was 516‐ and 25‐fold lower, respectively, than in wild‐type (WT) plants at 12 days postinoculation. In marked contrast, sltom1a/sltom3 plants were susceptible to previously known tomato viruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), indicating that SlTOM1a and SlTOM3 are not essential for systemic infection of TMV and ToMV in tomato plants. Knockout of SlTOM1b alone did not contribute to ToBRFV and ToMV resistance. However, in triple mutants sltom1a/sltom3/sltom1b, ToMV accumulation was three‐fold lower than in WT plants, with no reduction in symptoms. These results indicate that SlTOM1a and SlTOM3 are essential for the replication of ToBRFV, but not for ToMV and TMV, which are associated with additional susceptibility proteins. Additionally, we showed that SlTOM1a and SlTOM3 positively regulate the tobamovirus susceptibility gene SlARL8a3. Moreover, we found that the SlTOM family is involved in the regulation of plant development.     

Pseudomonas syringae pv. tomato infection of tomato plants is mediated by GABA and l‐Pro chemoperception

Foliar bacterial pathogens have to penetrate the plant tissue and access the interior of the apoplast in order to initiate the pathogenic phase. The entry process is driven by chemotaxis towards plant‐derived compounds in order to locate plant openings. However, information on plant signals recognized by bacterial chemoreceptors is scarce. Here, we show that the perception of GABA and l‐Pro, two abundant components of the tomato apoplast, through the PsPto‐PscC chemoreceptor drives the entry of Pseudomonas syringae pv. tomato into the tomato apoplast. The recognition of both compounds by PsPto‐PscC caused chemoattraction to both amino acids and participated in the regulation of GABA catabolism. Mutation of the PsPto‐PscC chemoreceptor caused a reduced chemotactic response towards these compounds which in turn impaired entry and reduced virulence in tomato plants. Interestingly, GABA and l‐Pro levels significantly increase in tomato plants upon pathogen infection and are involved in the regulation of the plant defence response. This is an example illustrating how bacteria respond to plant signals produced during the interaction as cues to access the plant apoplast and to ensure efficient infection.     

Epidemics of Tomato torrado virus,Pepino mosaic virus and Tomato chlorosis virus in tomato crops: do mixed infections contribute to torrado disease epidemiology?

Torrado disease was first observed in protected tomato crops in the Murcia province of Spain in spring 2001, causing serious concern to regional tomato producers. The disease-causing agent was initially identified as a picorna-like bipartite plant RNA virus, now known as Tomato torrado virus (ToTV), but several additional torradoviruses inducing similar disease symptoms have been described more recently. We studied the incidence of torradoviruses between 2005 and 2008 in two parts of Murcia (Spain) where tomato crops are grown commercially. We also analysed the potential association among ToTV, Pepino mosaic virus (PepMV) and Tomato chlorosis virus (ToCV) in samples showing torrado symptoms of varying severity. ToTV was the only torradovirus found in the samples (predominantly as single infections), but double and triple infections comprising ToTV, PepMV and/or ToCV were also detected. There was no evidence of a specific association among the viruses as the frequencies of mixed infections did not deviate from those expected to occur by chance. Statistical analysis of the potential association between torrado symptoms and the type of infection (single or multiple) was inconclusive. To determine whether co-infections with ToTV and PepMV have any marked influence on the torrado disease, we analysed torrado symptom severity and virus accumulation in tomato plants experimentally infected with ToTV-CE, PepMV-Sp13 and PepMV-PS5 in single and mixed infections. The severity of the torrado symptoms was not affected by the presence of PepMV. In single infections, the ToTV titre remained very low, reaching its maximum in the early stages of infection and declining rapidly thereafter, whereas the disease symptoms became more severe over the same timescale. In mixed infections, the accumulation of both ToTV and PepMV was altered with respect to single infections, and the magnitude of this alteration appeared to be virus and strain specific. Therefore, ToTV and PepMV mixed infections may modulate the epidemiology of both viruses in a complex way by altering virus fitness. The impact of our studies on efforts to track and prevent the spread of torrado disease is discussed.     

Immune recognition of the secreted serine protease ChpG restricts the host range of Clavibacter michiganensis from eggplant varieties

Bacterial wilt and canker caused by Clavibacter michiganensis (Cm) inflict considerable damage in tomato‐growing regions around the world. Cm has a narrow host range and can cause disease in tomato but not in many eggplant varieties. The pathogenicity of Cm is dependent on secreted serine proteases, encoded by the chp/tomA pathogenicity island (PI), and the pCM2 plasmid. Screening combinations of PI deletion mutants and plasmid‐cured strains found that Cm‐mediated hypersensitive response (HR) in the Cm‐resistant eggplant variety Black Queen is dependent on the chp/tomA PI. Singular reintroduction of PI‐encoded serine proteases into Cm^∆PI identified that the HR is elicited by the protease ChpG. Eggplant leaves infiltrated with a chpG marker exchange mutant (CmΩchpG) did not display an HR, and infiltration of purified ChpG protein elicited immune responses in eggplant but not in Cm‐susceptible tomato. Virulence assays found that while wild‐type Cm and the CmΩchpG complemented strain were nonpathogenic on eggplant, CmΩchpG caused wilt and canker symptoms. Additionally, bacterial populations in CmΩchpG‐inoculated eggplant stem tissues were c.1000‐fold higher than wild‐type and CmΩchpG‐complemented Cm strains. Pathogenicity tests conducted in multiple Cm‐resistance eggplant varieties demonstrated that immunity to Cm is dependent on ChpG in all tested varieties, indicating that ChpG‐recognition is conserved in eggplant. ChpG‐mediated avirulence interactions were disabled by alanine substitution of serine231 of the serine protease catalytic triad, suggesting that protease activity is required for immune recognition of ChpG. Our study identified ChpG as a novel avirulence protein that is recognized in resistant eggplant varieties and restricts the host range of Cm.