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 structur

Characterization of begomoviruses sampled during severe epidemics in tomato cultivars carrying the Ty-1 gene

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) is a major species that causes a tomato disease for which resistant tomato hybrids (mainly carriers of the Ty-1/Ty-3 gene) are being used widely. We have characterized begomoviruses severely affecting resistant tomato crops in Southeast Spain. Circular DNA was prepared from samples by rolling circle amplification, and sequenced by massive sequencing (2015) or cloning and Sanger sequencing (2016). Thus, 23 complete sequences were determined, all belonging to the TYLCV Israel strain (TYLCV-IL). Massive sequencing also revealed the absence of other geminiviral and beta-satellite sequences. A phylogenetic analysis showed that the Spanish isolates belonged to two groups, one related to early TYLCV-IL isolates in the area (Group 1), and another (Group 2) closely related to El Jadida (Morocco) isolates, suggesting a recent introduction. The most parsimonious evolutionary scenario suggested that the TYLCV isolates of Group 2 are back recombinant isolates derived from TYLCV-IS76, a recombinant virus currently predominating in Moroccan epidemics. Thus, an infectious Group 2 clone (TYLCV-Mu15) was constructed and used in in planta competition assays against TYLCV-IS76. TYLCV-Mu15 predominated in single infections, whereas TYLCV-IS76 did so in mixed infections, providing credibility to a scenario of co-occurrence of both types of isolates

Stable and Broad Spectrum Cross-Protection Against Pepino Mosaic Virus Attained by Mixed Infection

While recent pepino mosaic virus (PepMV; species Pepino mosaic virus, genus Potexvirus, family Alphaflexiviridae) 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

Interacción MNSV-planta de melón : una aproximación transcriptómica y celular

El Virus de las manchas necróticas del melón (Melon necrotic spot virus, MNSV; género Carmovirus; Familia Tombusviridae) es un virus de ARN de sentido positivo endémico en cultivos de melón. Este virus ha sido empleado como modelo de estudio de aspectos relacionados con la superación de resistencias recesivas, la traducción de ARNs virales carentes de estructuras cap o el movimiento intercelular. Sin embargo, se sabe poco acerca de las respuestas que la infección por este virus induce en las plantas o los genes alterados como consecuencia del desarrollo de su ciclo viral en su huésped natural, el melón. Así mismo, se desconocen los lugares, dentro de la célula vegetal, donde este virus lleva a cabo su replicación viral. Todos estos procesos podrían aportar conocimiento sobre los genes necesarios en la interacción planta-virus que permiten el establecimiento de una infección satisfactoria por parte del virus, o los factores necesarios para la puesta en marcha de una respuesta de defensa eficaz para contrarrestar la infección y que podrían ser utilizados en estrategias antivirales. Con el objetivo de ahondar en el conocimiento de la interacción MNSV-melón, esta tesis ha sido estructurada en dos capítulos. En el primer capítulo, utilizando los microarrays como herramienta principal de análisis, se ha llevado a cabo un estudio de las alteraciones transcriptómicas inducidas por MNSV en melón, con especial énfasis en aquellos cambios asociados con: (i) una región no codificante del genoma del virus, (ii) diferentes cultivares de melón (iii) diferentes tejidos de la planta, (iv) y con el desarrollo de infecciones de tipo local o sistémico. Los resultados obtenidos han permitido identificar la desregulación específica de dos grupos de genes asociados con una región no traducible del genoma viral, así como genes que responden de forma específica de cultivar. La comparación entre tejidos ha mostrado una respuesta cuantitativa diferente entre hoja y cotiledón, pero cualitativamente muy similar, aportando validez a los resultados obtenidos en cotiledón. Así mismo, una aproximación tradicional al estudio de la posible activación de una respuesta sistémica adquirida por parte de la planta, ha descartado la activación de la misma. Finalmente, la comparación entre estos resultados con los obtenidos en trabajos previos para otros virus, ha permitido identificar un sólo gen compartido por los tres virus analizados, que podría ser importante en las infecciones virales al menos en melón. Por otro lado, estos análisis han puesto de manifiesto la importancia de utilizar diferentes tiempos de muestreo a la hora de realizar comparaciones entre los cambios transcriptómicos inducidos por diferentes virus. En el segundo capítulo, se ha llevado a cabo el análisis tisular de las lesiones inducidas por MNSV para delimitar las regiones de interés donde realizar los análisis ultraestructurales. Este análisis identificó una región idónea para tales efectos. El estudio ultraestructural mediante microscopía electrónica de transmisión de la región identificada como Z2, ha puesto de manifiesto la existencia de grandes orgánulos originados como consecuencia de la infección por MNSV. Estos orgánulos han sido identificados como mitocondrias modificadas estructuralmente y representan los lugares donde el virus lleva a cabo su replicación ya que, a través de experimentos de hibridación in situ e inmunocitoquímica, se han localizado los ARNs virales, la proteína de la cápsida viral (CP) así como el dsRNA (intermediario de replicación) en estos orgánulos. La reconstrucción tridimensional de las mitocondrias modificadas mostró la presencia de grandes dilataciones internas, interconectadas entre ellas y con el citoplasma circundante a través de poros y/o estructuras complejas así como su conexión con cuerpos lipídicos. Así mismo, se llevó a cabo el estudio de la implicación de la proteína p29 de MNSV en la localización y generación de los sitios de replicación de MNSV. Para ello se generaron construcciones de la proteína p29 fusionada a GFP que fueron localizadas mediante microscopía laser confocal en mitocondrias. El análisis de la ultraestructura de las células que expresaron la proteína de fusión p29-GFP y su localización por inmunocitoquímica identificó la presencia de esta proteína en mitocondrias, así como la inducción de modificaciones estructurales en estos orgánulos muy semejantes a las inducidas por el virus. SUMMARY Melon necrotic spot virus (MNSV) (genus Carmovirus, family Tombusviridae) is a single-stranded, positive-sense RNA virus endemic of cucurbit crops worldwide. This virus has become an experimental model for the analysis of cell-to-cell virus movement and translation of uncapped viral RNAs, whereas little is known about its replication or the transcriptome changes induced in melon plant by the virus during the viral cycle and in response to the infection. So far it is unknown the cellular compartment in which this virus carries out its viral replication. Addressing all these processes could provide information about genes involved in the plant-virus interaction that lead to a successful viral infection and the cellular factors involved in the defense response to counteract the infection. This knowledge may be used in future antiviral strategies. To clarify all these questions about MNSV-melon interaction this thesis has been structured into two chapters. The first chapter is focused on transcriptomic alterations induced by MNSV in melon plants, with special emphasis on those changes associated with: (i) a non-coding region of the genome virus, (ii) different melon cultivars, (iii) different plant tissues, and, (iv) development of local or systemic infections. Microarray analyses have led to identify a specific deregulation of two groups of genes associated with an untranslated region of the viral genome, and also several sets of genes that respond specifically in each cultivar. The comparison between tissues has shown a different quantitative response between leaf and cotyledon, but qualitatively similar, providing validity to the results obtained in cotyledon. Also, a traditional approach to the study of the putative activation of a systemic acquired response from the plant has dismissed the activation of such response. Finally, the comparison of these results with those obtained in previous work for other viruses, has identified one gene shared by all the three viruses tested. This gene could play a key role in melon viralinfection. On the other hand, these results have shown the need of using different sampling times when comparing transcriptome changes induced by different viruses. In the second chapter, an histological analysis of the MNSV infected tissues was performed in order to define the most adequate region where to focuses the ultrastructural analysis. This analysis identified an ideal region for this purpose. The ultrastructural study by transmission electron microscopy of the region identified as Z2, has revealed the existence of large organelles originated as a result of MNSV infection. Immunolocalization of the glycine decarboxylase complex (GDC) P protein in these organelles confirmed their mitochondrial origin. In situ hybridization and immunolocalization experiments showed the specific localization of positive-sense viral RNA, capsid protein (CP), and double-stranded (ds)RNA (a replication intermediate) in these organelles meaning that replication of the virus takes place in association with them. The three-dimensional reconstructions of the altered mitochondria showed the presence of large, interconnected, internal dilations which appeared to be linked to the outside cytoplasmic environment through pores and/or complex structures, and with lipid bodies. Transient expression of MNSV p29 revealed that its specific target is mitochondria. Our data document the extensive reorganization of host mitochondria induced by MNSV, which provides a protected environment to viral replication, and show that the MNSV p29 protein is the primary determinant of this effect in the host

In situ hybridization for the localization of two pepino mosaic virus isolates in mixed infections

In situ hybridization (ISH) is an informative and relatively accessible technique for the localization of viral genomes in plant tissue and cells. However, simultaneous visualization of related plant viruses in mixed infections may be limited by the nucleotide similarity in the genomes and the single chromogenic detection over the same sample preparation. To address this issue, we used two Pepino mosaic virus isolates and performed ISH over consecutive serial cross-sections of paraffin-embedded leaf samples of single and mixed infected Nicotiana benthamiana plants. Moreover, the probe design was optimized to reduce cross-hybridisation, and co-localization was based on the overlapping of consecutive cross-sections from mixed infected leaves; thus, our results showed that both Pepino mosaic virus isolates co-localized in the same leaf tissue. In turn, both isolates were localized in the cytoplasm of the same cells. These results provide valuable information for studying mixed infections in plants by using a simple ISH procedure that is accessible to any pathology laboratory.P.G. and M.A.A. acknowledge funding from MINECO (grants AGL2014-59556-R and PCIN-2017-055, the latter within the ERANET-ARIMNet2 (ref. 302) program), and C.A. was supported by funding from the Ministry of Industry, Economy and Competitiveness (MINECO, Spain) within the PhD programme grant (FPU16/02569).Peer reviewe

Transcriptomic profiling of Melon necrotic spot virus-infected melon plants revealed virus strain and plant cultivar-specific alterations

- Background: Viruses are among the most destructive and difficult to control plant pathogens. Melon (Cucumis melo L.) has become the model species for the agriculturally important Cucurbitaceae family. Approaches that take advantage of recently developed genomic tools in melon have been extremely useful for understanding viral pathogenesis and can contribute to the identification of target genes for breeding new resistant cultivars. In this work, we have used a recently described melon microarray for transcriptome profiling of two melon cultivars infected with two strains of Melon necrotic spot virus (MNSV) that only differ on their 3'-untranslated regions.- Results:Melon plant tissues from the cultivars Tendral or Planters Jumbo were locally infected with either MNSV-Mα5 or MNSV-Mα5/3'264 and analysed in a time-course experiment. Principal component and hierarchical clustering analyses identified treatment (healthy vs. infected) and sampling date (3 vs. 5 dpi) as the primary and secondary variables, respectively. Out of 7566 and 7074 genes deregulated by MNSV-Mα5 and MNSV-Mα5/3'264, 1851 and 1356, respectively, were strain-specific. Likewise, MNSV-Mα5/3'264 specifically deregulated 2925 and 1618 genes in Tendral and Planters Jumbo, respectively. The GO categories that were significantly affected were clearly different for the different virus/host combinations. Grouping genes according to their patterns of expression allowed for the identification of two groups that were specifically deregulated by MNSV-Mα5/3'264 with respect to MNSV-Mα5 in Tendral, and one group that was antagonistically regulated in Planters Jumbo vs. Tendral after MNSV-Mα5/3'264 infection. Genes in these three groups belonged to diverse functional classes, and no obvious regulatory commonalities were identified. When data on MNSV-Mα5/Tendral infections were compared to equivalent data on cucumber mosaic virus or watermelon mosaic virus infections, cytokinin-O-glucosyltransferase2 was identified as the only gene that was deregulated by all three viruses, with infection dynamics correlating with the amplitude of transcriptome remodeling. - Conclusions: Strain-specific changes, as well as cultivar-specific changes, were identified by profiling the transcriptomes of plants from two melon cultivars infected with two MNSV strains. No obvious regulatory features shared among deregulated genes have been identified, pointing toward regulation through differential functional pathways

Imaging Techniques to Study Plant Virus Replication and Vertical Transmission

This article belongs to the Special Issue Application of Advanced Imaging to the Study of Virus-Host Interactions.Plant viruses are obligate parasites that need to usurp plant cell metabolism in order to infect their hosts. Imaging techniques have been used for quite a long time to study plant virus–host interactions, making it possible to have major advances in the knowledge of plant virus infection cycles. The imaging techniques used to study plant–virus interactions have included light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopies. Here, we review the use of these techniques in plant virology, illustrating recent advances in the area with examples from plant virus replication and virus plant-to-plant vertical transmission processes.Research on PepMV in our laboratory is currently funded by grant RTI2018-097099-B-100 (Ministerio de Ciencia e Innovación, Spain).Peer reviewe

Pepino mosaic virus RNA-Dependent RNA Polymerase POL Domain Is a Hypersensitive Response-Like Elicitor Shared by Necrotic and Mild Isolates

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Virus-infected melon plants emit volatiles that induce gene deregulation in neighboring healthy plants

It is well described that viral infections stimulate the emission of plant volatiles able to recruit viral vectors thereby promoting virus spread. In contrast, much less is known on the effects that emitted volatiles may have on the metabolism of healthy neighboring plants, which are potential targets for new infections through vector transmission. Watermelon mosaic virus (WMV) (genus Potyvirus, family Potyviridae) is an aphid-transmitted virus endemic in cucurbit crops worldwide. We have compared gene expression profiles of WMV-infected melon plants with those of healthy or healthy-but-cohabited-with-infected plants. Pathogenesis-related (PR) and small heat shock protein encoding genes were deregulated in cohabited plants, and PR deregulation depended on the distance to the infected plant. The signaling was short distance in the experimental conditions used, and cohabiting had a moderate effect on the plant susceptibility to WMV. Static headspace experiments showed that benzaldehyde and g-butyrolactone were significantly over-emitted by WMV-infected plants. Altogether, our data suggest that perception of a volatile signal encoded by WMV-infected tissues triggers a response to prepare healthy tissues or/and healthy neighboring plants for the incoming infections.Ministry of Science, Innovation and Universities Grant/Award Number: BES-2016-077826 Región de Murcia, Consejería de Empleo Universidades y Empresa; RIS3MUR program Grant/Award Number: 2I16SA000057 Ministry of Economy, Industry and Competitiveness Grant/Award Number: PTQ-15-07646 Grant/Award Number: PTQ-16-0836

Small RNA-Seq to Characterize Viruses Responsible of Lettuce Big Vein Disease in Spain

The emerging lettuce big-vein disease (LBVD) is causing losses in lettuce production ranging from 30 to 70% worldwide. Several studies have associated this disease with Mirafiori lettuce big-vein virus (MiLBVV) alone or in mixed infection with lettuce big-vein associated virus (LBVaV). We used Illumina small RNA sequencing (sRNA-seq) to identify viruses present in symptomatic lettuce plants from commercial fields in Southern Spain. Data analysis using the VirusDetect tool showed the consistent presence of MiLBVV and LBVaV in diseased plants. Populations of MiLBVV and LBVaV viral small RNAs (sRNAs) were characterized, showing features essentially similar to those of other viruses, with the peculiarity of an uneven asymmetric distribution of MiLBVV virus-derived small RNAs (vsRNAs) for the different polarities of genomic RNA4 vs. RNAs1 to 3. Sanger sequencing of coat protein genes was used to study MiLBVV and LBVaV phylogenetic relationships and population genetics. The Spanish MiLBVV population was composed of isolates from three well-differentiated lineages and reflected almost all of the diversity reported for the MiLBVV species, whereas the LBVaV population showed very little genetic differentiation at the regional scale but lineage differentiation at a global geographical scale. Universal primers were used to detect and quantify the accumulation of MiLBVV and LBVaV in field samples; both symptomatic and asymptomatic plants from affected fields carried equal viral loads, with LBVaV accumulating at higher levels than MiLBVV.CG-A was recipient of grant PTQ-15-07646 from the Torres-Quevedo program (Ministry of Economy, Industry and Competitiveness; Spain) and CT of fellowship DI-14-06825 from the Industrial Doctoral program (Ministry of Economy, Industry and Competitiveness, Spain).Peer reviewe