Heterologous RNA silencing suppressors from both plant- and animal-infecting viruses support Plum pox virus infection

[EN] HCPro, the RNA silencing suppressor (RSS) of viruses belonging to the Potyvirus genus in the Potyviridae family, is a multifunctional protein presumably involved in all essential steps of the viral infection cycle. Recent studies have shown that Plum pox potyvirus (PPV) HCPro can be successfully replaced by Cucumber vein yellowing ipomovirus P1b, a sequence unrelated RSS from a virus of the same family. In order to gain insight into the requirement of a particular RSS to establish a successful potyviral infection, we tested the ability of different heterologous RSSs from both plant- and animal-infecting viruses to substitute HCPro. Making use of engineered PPV chimeras, we show that PPV HCPro can be functionally replaced by some, but not all, unrelated RSSs, including the NS1 protein of the mammalian-infecting Influenza A virus. Interestingly, the capacity of a particular RSS to replace HCPro does not strictly correlate with its RNA silencing suppression strength. Altogether, our results suggest that not all suppression strategies are equally suitable for an efficient escape of PPV from the RNA silencing machinery. The approach followed here based on using PPV chimeras in which an under-consideration RSS substitutes for HCPro could further help to study the function of diverse RSSs in a ¿highly-sensitive¿ RNA silencing context, such as that taking place in plant cells during the process of a viral infection.We are especially grateful to those people who sent us plasmids containing the DNA sequence of different viral proteins. We thank Veronique Ziegler-Graff for providing BWYV P0, Ana Giner and Juan Jose Lopez-Moya for providing SPMMV P1, Joel Milner for providing CaMV P6, Maria Rosa Lopez-Huertas and Jose Alcami for providing HIV Tat, Jan Kreuze for providing SPCSV RNase3, and M. Taliansky for providing GRV ORF3. We thank Herman Scholthof and Ariel Rodriguez for providing anti-P19 and anti-NS1 serum, respectively. We are also grateful to David Baulcombe for providing the GFP expression vector and TBSV P19-containing plasmid, and Mark Curtis for providing the pMDC32 destination vector. This work was supported by grants from Spanish MICINN (BIO2010-18541) and the European Union (KBBE-204429). M. C. was the recipient of an 13P fellowship from CSIC-Fondo Social Europeo.Maliogka, VI.; Calvo, M.; Carbonell, A.; Garcia, JA.; Valli, A. (2012). Heterologous RNA silencing suppressors from both plant- and animal-infecting viruses support Plum pox virus infection. Journal of General Virology. 93(7):1601-1611. https://doi.org/10.1099/vir.0.042168-0S16011611937Ala-Poikela, M., Goytia, E., Haikonen, T., Rajamäki, M.-L., & Valkonen, J. P. T. (2011). Helper Component Proteinase of the Genus Potyvirus Is an Interaction Partner of Translation Initiation Factors eIF(iso)4E and eIF4E and Contains a 4E Binding Motif. Journal of Virology, 85(13), 6784-6794. doi:10.1128/jvi.00485-11Ambros, V., & Chen, X. (2007). 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Identification of Ilarviruses in almond and cherry fruit trees using nested PCR assays

In this study nested PCR assays have been developed for the detection of Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV) and Apple mosaic virus (ApMV) modifying a previously reported assay for the generic detection of ilarviruses. In all cases one generic upstream primer was used along with a virus-specific downstream primer in respective nested PCR assays. The application of the same thermocycling profile allowed all amplifications to run in parallel. Ilarvirus isolates from different hosts were used for the evaluation of the detection range of the assays, which were afterwards applied for screening almond and cherry plant material. In almond trees the incidence of PNRSV and PDV was 41% and 21.5%, respectively. In cherry orchards the opposite was observed with PDV (56.6%) being the prevalent virus followed by PNRSV (19.4%). Mixed infections with both viruses were also encountered in approximately 10 and 17% of cherry and almond trees, respectively. ApMV was not detected in any of the samples tested. This is the first extensive survey conducted in Greece in order to monitor the distribution of these viruses using molecular assays. Keywords: Prune dwarf virus, Prunus necrotic ringspot virus, Apple mosaic virus, cherry, almond, nested PC

Virus variants with differences in the P1 protein coexist in a Plum pox virus population and display particular host-dependent pathogenicity features

[EN] Subisolates segregated from an M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the helper component proteinase (HCPro) protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in certain subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings suggested the existence of uncharacterized attenuating factors in these subisolates. In this study, we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host.We wish to thank Elvira Dominguez for technical assistance. This work was supported by grants BIO2010-18541 from the Spanish Ministerio de Educacion y Ciencia (MEC), SAL/0185/2006 from Comunidad de Madrid and KBBE-204429 from the European Union. B. 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Specific Real-Time PCR for the Detection and Absolute Quantitation of Grapevine Roditis Leaf Discoloration-Associated Virus, an EPPO Alert Pathogen

Grapevine Roditis leaf discoloration-associated virus (GRLDaV) is an emerging grapevine pathogen included in the European and Mediterranean Plant Protection Organization (EPPO) alert list due to its ability to damage grapevine crops and cause production losses. This work aimed to develop a specific and reliable diagnostic tool that would contribute to preventing the spread of this pathogen. Therefore, a TaqMan real-time quantitative PCR was developed. The method was validated according to EPPO guidelines showing a high degree of analytical sensitivity, analytical specificity, selectivity, and repeatability and reproducibility. The sensitivity of this method is much higher than the sensitivity reached by previously reported methods even when tested in crude extracts, which could allow rapid testing by avoiding nucleic acid extraction steps. The method was also able to detect GRLDaV isolates from all the geographic origins reported so far, despite their high degree of genetic diversity. In addition, this new technique has been successfully applied for the quantitative detection of GRLDaV in plant material and two mealybug species, Planococcus citri and Pseudococcus viburni. In conclusion, the methodology developed herein represents a significant contribution to the diagnosis and control of this emerging pathogen in grapevine

A Novel and Highly Inclusive Quantitative Real-Time RT-PCR Method for the Broad and Efficient Detection of Grapevine Leafroll Associated Virus 1

Grapevine (Vitis vinifera L.) is one of the most important crops in the world due to its economic and social impact. Like many other crops, grapevine is susceptible to different types of diseases caused by pathogenic microorganisms. Grapevine leafroll-associated virus 1 (GLRaV-1) is a virus associated with grapevine leafroll disease and it is considered at the national and European level as a pathogen that must be absent in propagative plant material. For this reason, the availability of specific, sensitive and reliable detection techniques to ascertain the sanitary status of the plants is of great importance. The objective of this research was the development of a new GLRaV-1 detection method based on a TaqMan quantitative real-time RT-PCR targeted to the coat protein genomic region and including a host internal control in a duplex reaction. To this end, three new GLRaV-1 full genomes were recovered by HTS and aligned with all sequences available in the databases. The method has been validated following EPPO standards and applied for the diagnosis of field plant material and transmission vectors. The new protocol designed has turned out to be highly sensitive as well as much more specific than the current available methods for the detection and absolute quantitation of GLRaV-1 viral titer

Recent Advances on the Multiplex Molecular Detection of Plant Viruses and Viroids

[EN] Plant viruses are still one of the main contributors to economic losses in agriculture. It has been estimated that plant viruses can cause as much as 50 billion euros loss worldwide, per year. This situation may be worsened by recent climate change events and the associated changes in disease epidemiology. Reliable and early detection methods are still one of the main and most effective actions to develop control strategies for plant viral diseases. During the last years, considerable progress has been made to develop tools with high specificity and low detection limits for use in the detection of these plant pathogens. Time and cost reductions have been some of the main objectives pursued during the last few years as these increase their feasibility for routine use. Among other strategies, these objectives can be achieved by the simultaneous detection and (or) identification of several viruses in a single assay. Nucleic acid-based detection techniques are especially suitable for this purpose. Polyvalent detection has allowed the detection of multiple plant viruses at the genus level. Multiplexing RT polymerase chain reaction (PCR) has been optimized for the simultaneous detection of more than 10 plant viruses/viroids. In this short review, we provide an update on the progress made during the last decade on techniques such as multiplex PCR, polyvalent PCR, non-isotopic molecular hybridization techniques, real-time PCR, and array technologies to allow simultaneous detection of multiple plant viruses. Also, the potential and benefits of the powerful new technique of deep sequencing/next-generation sequencing are described.This work was funded by grant BIO2017-88321-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT) and the Prometeo Program GV2014/010 from the Generalitat Valenciana.Pallás Benet, V.; Sanchez Navarro, JÁ.; James, D. (2018). Recent Advances on the Multiplex Molecular Detection of Plant Viruses and Viroids. Frontiers in Microbiology. 9. https://doi.org/10.3389/fmicb.2018.02087S