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50 Pág., In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratifcation vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV, This work was supported in part through Laulima Govern ment Solutions, LLC prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272201800013C. J.H.K. performed this work as an employee of Tunnell Government Services (TGS), a subcontractor of Laulima Gov ernment Solutions, LLC under Contract No. HHSN272201800013C. This work was also funded in part by Contract No. HSHQDC 15-C-00064 awarded by DHS S&T for the management and operation of The National Biodefense Analysis and Countermeasures Center, a federally funded research and development center operated by the Battelle National Biodefense Institute (V.W.); and NIH contract HHSN272201000040I/HHSN27200004/D04 and grant R24AI120942 (N.V., R.B.T.). S.S. acknowledges support from the Mississippi Agri cultural and Forestry Experiment Station (MAFES), USDA-ARS pro ject 58-6066-9-033 and the National Institute of Food and Agriculture, US Department of Agriculture, Hatch Project, under Accession Num ber 1021494.

Although the coat protein (CP) has a relevant role in the long-distance movement of alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), its precise function is not fully understood. Previous results showed that a specific interaction between the C termini of the movement protein (MP) and the cognate CP is required for systemic transport. Thus, we have performed a compensatory evolution experiment using an AMV RNA3 derivative defective in long-distance transport that carries a BMV MP lacking the C-terminal 48 residues and unable to interact with the AMV CP. After several passages, five independent evolution lineages were able to move long distance. The analysis of the viral RNA of these lineages showed the presence of three different modifications located exclusively at the 5' untranslated region (5' UTR). The three evolved 5' UTR variants accumulated comparable levels of viral RNA and CP but reduced the accumulation of virus particles and the affinity between the 5' UTR and the AMV CP. In addition, the evolved 5' UTR increased cell-to-cell transport for both the AMV RNA3 carrying the BMV MP and that carrying the AMV MP. Finally, the evolved 5' UTRs allowed the systemic transport of an AMV RNA3 carrying a CP mutant defective in virus particles and increased the systemic transport of several AMV RNA3 derivatives carrying different viral MPs associated with the 30K superfamily. Altogether, our findings indicate that virus particles are not required for the systemic transport of AMV but also that BMV MP is competent for the short- and long-distance transport without the interaction with the CP. IMPORTANCE The results obtained in the present work could challenge the view of the role of the virus particle in the systemic transport of plant viruses. In this sense, we show that two different MPs are competent to systemically transport the AMV genome without the requirement of the virus particles, as reported for viruses lacking a CP (e.g., Umbravirus). The incapability of the viral MP to interact with the CP triggered virus variants that evolved to reduce the formation of virus particles, probably to increase the accessibility of the MP to the viral progeny. Our results point to the idea that virus particles would not be necessary for the viral systemic transport but would be necessary for vector virus transmission. This idea is reinforced by the observation that heterologous MPs also increased the systemic transport of the AMV constructs that have reduced encapsidation capabilities.

To counteract RNA interference-mediated antiviral defence, virus genomes evolved to express proteins that inhibit this plant defence mechanism. Using six independent biological approaches, we show that orchid fleck dichorhavirus citrus strain (OFV-citrus) movement protein (MP) may act as a viral suppressor of RNA silencing (VSR). By using the alfalfa mosaic virus (AMV) RNA 3 expression vector, it was observed that the MP triggered necrosis response in transgenic tobacco leaves and increased the viral RNA (vRNA) accumulation. The use of the potato virus X (PVX) expression system revealed that the cis expression of MP increased both the severity of the PVX infection and the accumulation of PVX RNAs, further supporting that MP could act as an RNA silencing suppressor (RSS). From the analysis of the RSS-defective turnip crinkle virus (TCV), we do not find local RSS activity for MP, suggesting a link between MP suppressor activity and the prevention of systemic silencing. In the analysis of local suppressive activity using the GFP-based agroinfiltration assay in Nicotiana benthamiana (16 c line), we do not identify local RSS activity for the five OFV RNA1-encoded proteins. However, when evaluating the small interfering RNA (siRNA) accumulation, we find that the expression of MP significantly reduces the accumulation of GFP-derived siRNA. Finally, we examine whether the MP can prevent systemic silencing in 16c plants. Our findings show that MP inhibits the long-distance spread of RNA silencing, but does not affect the short-distance spread. Together, our findings indicate that MP is part of OFV's counter-defence mechanism, acting mainly in the prevention of systemic long-distance silencing. This work presents the first report of a VSR for a member of the genus Dichorhavirus.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV., La lista completa de autores que integran el documento puede consultarse en el archivo., Facultad de Ciencias Agrarias y Forestales, Centro de Investigaciones en Fitopatología, Facultad de Ciencias Exactas, Instituto de Biotecnologia y Biologia Molecular

[EN] Brevipalpus-transmitted viruses (BTVs) belong to the genera Dichorhavirus and Cilevirus and are the main causal agents of the citrus leprosis (CL) disease. In this report, we explored aspects related to the movement mechanism mediated by dichorhaviruses movement proteins (MPs) and the homologous and heterologous interactions among viral proteins related to the movement of citrus leprosis-associated viruses. The membrane-spanning property and topology analysis of the nucleocapsid (N) and MP proteins from two dichorhaviruses revealed that the MPs are proteins tightly associated with the cell membrane, exposing their N- and C-termini to the cytoplasm and the inner part of the nucleus, whereas the N proteins are not membrane-associated. Subcellular localization analysis revealed the presence of dichorhavirus MPs at the cell surface and in the nucleus, while the phosphoproteins (P) were located exclusively in the nucleus and the N proteins in both the cytoplasm and the nucleus. Co-expression analysis with the MP, P, and N proteins showed an interaction network formed between them. We highlight the MP capability to partially redistribute the previously reported N-P core complex, redirecting a portion of the N from the nucleus to the plasmodesmata at the cell periphery, which indicates not only that the MP might guide the intracellular trafficking of the viral infective complex but also that the N protein may be associated with the cell-to-cell movement mechanism of dichorhaviruses. The movement functionality of these MPs was analyzed by using three movement-defective infectious systems. Also, the MP capacity to generate tubular structures on the protoplast surface by ectopic expression was analyzed. Finally, we evaluated the in vivo protein-protein interaction networks between the dichorhavirus MP and/or N proteins with the heterologous cilevirus movement components, which suggest a broad spectrum of interactions, highlighting those among capsid proteins (CP), MPs, and Ns from citrus leprosis-associated viruses. These data may aid in understanding the mixed infection process naturally observed in the field caused by distinct BTVs., This work was supported by the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), proc. 2014/08459, 2015/10249-1, 2017/50222-0, and 2017/19898-8. This work was also supported by grant BIO2017-88321-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT) and the Prometeo Program GV2015/010 from the Generalitat Valenciana.

[EN] Although citrus leprosis disease has been known for more than a hundred years, one of its causal agents, citrus leprosis virus C2 (CiLV-C2), is poorly characterized. This study described the association of CiLV-C2 movement protein (MP) and capsid protein (p29) with biological membranes. Our findings obtained by computer predictions, chemical treatments after membrane fractionation, and biomolecular fluorescence complementation assays revealed that p29 is peripherally associated, while the MP is integrally bound to the cell membranes. Topological analyses revealed that both the p29 and MP expose their N- and C-termini to the cell cytoplasmic compartment. The implications of these results in the intracellular movement of the virus were discussed., This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), proc. 2014/0845-9, 2017/50222-0, 2015/10249-1 and 2017/19898-8. This work was also supported by grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), and the Prometeo Program GV2015/010 from the Generalitat Valenciana.

[EN] Previous results using a movement defective alfalfa mosaic virus (AMV) vector revealed that citrus leprosis virus C (CiLV-C) movement protein (MP) generates a more efficient local movement, but not more systemic transport, than citrus leprosis virus C2 (CiLV-C2) MP, MPs belonging to two important viruses for the citrus industry. Here, competition experiment assays in transgenic tobacco plants (P12) between transcripts of AMV constructs expressing the cilevirus MPs, followed by several biological passages, showed the prevalence of the AMV construct carrying the CiLV-C2 MP. The analysis of AMV RNA 3 progeny recovered from P12 plant at the second viral passage revealed the presence of a mix of progeny encompassing the CiLV-C2 MP wild type (MPWT) and two variants carrying serines instead phenylalanines at positions 72 (MPS72F) or 259 (MPS259F), respectively. We evaluated the effects of each modified residue in virus replication, and cell-to-cell and long-distance movements. Results indicated that phenylalanine at position 259 favors viral cell-to-cell transport with an improvement in viral fitness, but has no effect on viral replication, whereas mutation at position 72 (MPS72F) has a penalty in the viral fitness. Our findings indicate that the prevalence of a viral population may be correlated with its greater efficiency in cell-to-cell and systemic movements., This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant numbers 2014/0845-9, 2017/50222-0, 2015/10249-1, 2017/19898-8 and by the Spanish Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), grant number PID2020-115571RB-100.

Unfortunately, the inclusion of original names (in non-Latin script) of the following authors caused problems with author name indexing in PubMed. Therefore, these original names were removed from XML data to correct the PubMed record. Mengji Cao, Yuya Chiaki, Hideki Ebihara, Jingjing Fu, George Fú Gāo, Tong Han, Jiang Hong, Ni Hong, Seiji Hongo, Masayuki Horie, Dàohóng Jiāng, Fujio Kadono, Hideki Kondō, Kenji Kubota, Shaorong Li, Longhui Li, Jiànróng Lǐ, Huazhen Liu, Tomohide Natsuaki, Sergey V. Netesov, Anna Papa, Sofia Paraskevopoulou, Liying Qi, Takahide Sasaya, Mang Shi, Xiǎohóng Shí, Zhènglì Shí, Yoshifumi Shimomoto, Jin‑Won Song, Ayato Takada, Shigeharu Takeuchi, Yasuhiro Tomitaka, Keizō Tomonaga, Shinya Tsuda, Changchun Tu, Tomio Usugi, Nikos Vasilakis, Jiro Wada, Lin‑Fa Wang, Guoping Wang, Yanxiang Wang, Yaqin Wang, Tàiyún Wèi, Shaohua Wen, Jiangxiang Wu, Lei Xu, Hironobu Yanagisawa, Caixia Yang, Zuokun Yang, Lifeng Zhai, Yong‑Zhen Zhang, Song Zhang, Jinguo Zhang, Zhe Zhang, Xueping Zhou. In addition, the publication call-out in the supplementary material was updated from issue 11 to issue 12. The original article has been corrected.

[EN] Members of the family Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA-RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/pospiviroidae., Production of this summary, the online chapter, and associated resources was funded by a grant from the Wellcome Trust (WT108418AIA).

[EN] Citrus leprosis virus C (CiLV-C) belongs to the genusCilevirus, familyKitaviridae, and is considered the most devastating virus infecting citrus in Brazil, being the main viral pathogen responsible for citrus leprosis (CL), a severe disease that affects citrus orchards in Latin America. Here, proteins encoded by CiLV-C genomic RNA 1 and 2 were screened for potential RNA silencing suppressor (RSS) activity by five methods. Using the GFP-based reporter agroinfiltration assay, we have not found potential local suppressor activity for the five CiLV-C encoded proteins. However, when RSS activity was evaluated using the alfalfa mosaic virus (AMV) system, we found that the p29, p15, and p61 CiLV-C proteins triggered necrosis response and increased the AMV RNA 3 accumulation, suggesting a suppressive functionality. From the analysis of small interfering RNAs (siRNAs) accumulation, we observed that the ectopic expression of the p29, p15, and p61 reduced significantly the accumulation of GFP derived siRNAs. The use of the RSS defective turnip crinkle virus (TCV) system revealed that only thetrans-expression of the p15 protein restored the cell-to-cell viral movement. Finally, the potato virus X (PVX) system revealed that the expression of p29, p15, and p61 increased the PVX RNA accumulation; in addition, the p29 and p15 enhanced the pathogenicity of PVX resulting in the death of tobacco plants. Furthermore, PVX-p61 infection resulted in a hypersensitive response (HR), suggesting that p61 could also activate a plant defense response mechanism. This is the first report describing the RSS activity for CiLV-C proteins and, moreover, for a member of the familyKitaviridae., This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), proc. 2014/0845-9, 2017/50222-0, 2015/10249-1, and 2017/19898-8. This work was also supported by Instituto para la Formacion y Aprovechamiento de Recursos Humanos, Becas IFARHU-SENACYT, contrato 270-2018-361, grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI), Fondo Europeo de Desarrollo Regional (FEDER), and the Prometeo Program GV2015/010 from the Generalitat Valenciana.

Cucurbit chlorotic yellows virus (CCYV) is a new member of the genus Crinivirus (family Closteroviridae) with a bi-partite genome. CCYV RNA 1-encoded p22 has recently been reported to be a weak local suppressor of RNA silencing for which an interaction with cucumber SKP1LB1 through an F-box-like motif was demonstrated to be essential. Using a bacterially expressed maltose-binding protein (MBP) fusion of CCYV p22 in electrophoretic mobility shift assays (EMSA), we have examined in vitro its ability to bind different RNA templates. Our experiments showed that CCYV p22 is able to bind to ss and ds long RNAs, in addition to ss and ds small interfering (si) RNA molecules. CCYV p22 deletion mutants (MBP_CCYV DEL1-4) were produced that covered the entire protein, with MBP_CCYV DEL2 corresponding to the F-box motif and its flanking sequences. None of these deletions abolished the capacity of CCYV p22 to bind ss- and dsRNA molecules. However, deletions affecting the C-terminal half of the protein resulted in decreased binding efficiency for either ss- or dsRNA molecules indicating that essential elements for these interactions are located in this region. Taken together, our data add to current knowledge of the mode of action of suppressors of RNA silencing encoded by genes sited at the 3′-terminus of crinivirus genomic RNA 1, and shed light on the involvement of CCYV p22 in the suppression of RNA silencing and/or in another role in the virus life cycle via RNA binding.

[EN] In this work, we describe the complete sequence and genome organization of a novel tobamovirus detected in a prickly pear plant (Opuntia sp.) by high-throughput sequencing, tentatively named "opuntia virus 2". The full genome of opuntia virus 2 is 6,453 nucleotides in length and contains four open reading frames (ORFs) coding for the two subunits of the RNA polymerase, the movement protein, and the coat protein, respectively. Phylogenetic analysis using the complete nucleotide sequence revealed that the virus belongs to the genus Tobamovirus (family Virgaviridae), showing the highest nucleotide sequence identity (49.8%) with cactus mild mottle virus (CMMoV), being indicating that it belongs in the Cactaceae subgroup of tobamoviruses., This study was funded by project no. IN216317 of UNAM-Programa de Apoyo a Proyectos de Investigacion e Innovacion Tecnologica (PAPIIT) and by Grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER).

The stone fruit genus Prunus, within the family Rosaceae, comprises more than 230 species, some of which have great importance or value as ornamental or fruit crops. Prunus are affected by numerous viruses and viroids linked to the vegetative propagation practices in many of the cultivated species. To date, 44 viruses and three viroids have been described in the 9 main cultivated Prunus species. Seven of these viruses and one viroid have been identified in Prunus hosts within the last 5 years. This work addresses recent advances and prospects in the study of viruses and viroids affecting Prunus species, mostly concerning the detection and characterisation of the agents involved, pathogenesis analysis and the search for new control tools. New sequencing technologies are quickly reshaping the way we can identify and characterise new plant viruses and isolates. Specific efforts aimed at virus identification or data mining of high-throughput sequencing data generated for plant genomics-oriented purposes can efficiently reveal the presence of known or novel viruses. These technologies have also been used to gain a deeper knowledge of the pathogenesis mechanisms at the gene and miRNA expression level that underlie the interactions between Prunus spp. and their main viruses and viroids. New biotechnological control tools include the transfer of resistance by grafting, the use of new sources of resistance and the development of gene silencing strategies using genetic transformation. In addition, the application of next generation sequencing and genome editing techniques will contribute to improving our knowledge of virus–host interactions and the mechanisms of resistance. This should be of great interest in the search to obtain new Prunus cultivars capable of dealing both with known viruses and viroids and with those that are yet to be discovered in the uncertain scenario of climate change.

[EN] The lack of infectious tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NSM) of four tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NSM proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NSM of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NSM, but not that of the CSNV, TCSV and TSWV NSM proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NSM mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NSM was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the `30K family¿. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent tospovirus species that have a distinct biological behavior., We thank L. Corachan for her excellent technical assistance. This work was supported by grant BIO2014-54862-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT), the Prometeo Program GV2014/010 from the Generalitat Valenciana, CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), Capes (Conselho de Aperfeicoamento de Pessoal de Nivel Superior) and FAP-DF (Fundacao de Apoio a Pesquisa do Distrito Federal)

[EN] The complete genome sequence of a trichovirus was obtained from peach samples collected from Mexico and found to be 7985 nucleotides long, excluding the poly(A) tail. Phylogenetic analysis using the complete nucleotide sequence revealed that the virus is a member of the genus Trichovirus and is closely related to peach mosaic virus (PcMV) and cherry mottle leaf virus (CMLV). The highest nucleotide sequence identity was 70% to both PcMV and CMLV, indicating that this virus, which we have tentatively named "peach virus M" (PeVM) should be considered a member of a new trichovirus species. We determined, for the first time, the initiation sites of the subgenomic RNAs (sgRNA) of a trichovirus. The sgRNAs for the movement and coat proteins started with the sequence 'GAA', while the smallest one, coding for the nucleotide-binding protein, started with the nucleotides 'GU'. In all cases, the sgRNAs leader ranged between 113 and 121 nt in length., This work was supported by Grant BIO2017-88321-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT) and the Prometeo Program GV2015/010 from the Generalitat Valenciana.

[EN] Non-radioactive molecular hybridization represents an attractive approach for the detection of multiple plant virus and/or viroids and a good alternative to the more extended serological and PCR-based detection methods. The use of polyprobes or riboprobes carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the detection of up to 10 different pathogens or the development of genus-specific probes. In the present article, the polyprobe technology has been adapted for the detection of the main viruses and viroids affecting tomato crops. To do this, three polyprobes have been developed covering four viroids (Poly4), twelve viruses (Poly12) or the four viroids plus the twelve viruses (poly16). The detection limit of the three polyprobes was comparable to the individual probes allowing the detection of up to 0,2 pg/mu l of viral or viroidal RNA. A survey of 50 field samples revealed that all positive samples detected with the individual probes were also detected with the corresponding poly12 (98%) or poly16 (100%) probes. The analysis of tomato seeds revealed that both, single and polyprobes, were able to detect an infected seed in a pool of 250 healthy seeds. Finally, a ring-test analysis among six laboratories revealed a high reproducibility of the non-radioactive molecular hybridization procedure using the three polyprobes. The use of this technology in the routine analysis of tomato samples is discussed., This work was supported by grant BIO2017-88321-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT) and the Prometeo Program GV2015/010 from the Generalitat Valenciana.

[EN] Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA-RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at /http:/www.ictv.global/report/avsunvirodae., Production of this summary, the online chapter and associated resources was funded by a grant from the Wellcome Trust (WT108418AIA).

The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3–12.5 kb divided into 3–4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae.

Clavibacter michiganensis subsp. michiganensis, Pepino mosaic virus, and Mexican papita viroid are three economically important pathogens that infect tomato crops. In this work, a polyprobe (poly-3) was developed and evaluated for the simultaneous detection of these pathogens in tomato plants by non-isotopic molecular hybridization. The endpoint detection limit of the poly-3 with C. michiganensis subsp. michiganensis cell cultures was 10(6) CFU/ml. No differences were found in terms of the sensitivity and specificity when the individual riboprobes were compared to the poly-3 for the detection of the three pathogens. The analysis of 80 tomato field samples by the poly-3 and RT-PCR techniques rendered the same number of positive samples for each pathogen. As far as we know this is the first time that three pathogens with very different life cycle styles (bacteria, virus and viroid) are simultaneously detected in a single assay. The possibility of using this poly-3 technology for the routine diagnosis of field tomato samples is discussed., E.J M-Z. was recipient of a Pre-doctoral fellowship from the Consejo Nacional de Ciencia y Tecnologia of Mexico. F.A. was recipient of a contract Ramon y Cajal (RYC-2010-06169) Program of the Ministerio de Educacion y Ciencia of Spain. We thank L. Corachan for her excellent technical assistance. This work was supported by Grants BIO2014-54862-R from the Spanish Granting Agency DGICYT, the Prometeo Program GV2011/003 from the Generalitat Valenciana and PAID-06-10-1496 from the Universitat Politecnica de Valencia (Spain).

Plant viruses must usurp host factors/routes for their survival. Disturbances derived from this extreme dependence for host resources, together with physiological alterations associated with defensive responses, can cause, in some virus-host combinations, acute or chronic plant diseases. As the coding capacity of these biotrophic pathogens is limited, viral-encoded proteins must essentially be multifunctional proteins involved in very different steps of their life cycle, and are usually elicitors of defensive responses. Thus, most, if not all, viral-encoded proteins can act as pathogenicity determinants. Indeed, the viral proteins involved in the essential processes of their life cycle, such as replication, movement, encapsidation and transmission can be critical players of the pathogenesis process through direct or indirect interactions. This review updates our knowledge on how viral factors affect plant physiology and contribute to the development of symptomatology., The authors would like to apologize to those colleagues whose relevant publications could not be cited because of space constraints. This work was supported by the Spanish Ministerio de Economia y Competitividad grants BIO2011-25018 to VP and BIO2013-49053 and Plant KBBE PCIN2013-056 to JAG.

[EN] The cell-to-cell movement protein (NSM) in members of the tospovirus species Tomato spotted wilt virus (TSWV) has been recently identified as the effector of the single dominant Sw-5b resistance gene from tomato (Solanum lycopersicum L.). Although most TSWV isolates shows a resistance-inducing (RI) phenotype, regular reports have appeared on the emergence of resistance-breaking (RB) isolates in tomato fields, and suggested a strong association with two point mutations (C118Y and T120N) in the NSM protein. In this study the Sw-5b gene has been demonstrated to confer not only resistance against TSWV but to members of five additional, phylogeneticallyrelated tospovirus species classified within the so-called ¿American¿ evolutionary clade, i.e. Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV) and Tomato chlorotic spot virus (TCSV). Remarkably, a member of the species Bean necrotic mosaic virus (BeNMV), a recently discovered tospovirus classified in a distinct American subclade and circulating on the American continent, did not trigger a Sw-5b-mediated hypersensitive (HR) response. Introduction of point mutations C118Y and T120N into the NSM protein of TSWV, TCSV and CSNV abrogated the ability to trigger Sw-5b-mediated HR in both transgenic-N. benthamiana and tomato isolines harboring the Sw-5b gene whereas it had no effect on BeNMV NSM. Truncated versions of TSWV NSM lacking motifs associated with tubule formation, cell-to-cell or systemic viral movement were made and tested for triggering of resistance. HR was still observed with truncated NSM proteins lacking 50 amino acids (out of 301) from either the amino- or carboxy-terminal end. These data altogether indicate the importance of amino acid residues C118 and T120 in Sw-5b-mediated HR only for the NSM proteins from one cluster of tospoviruses within the American clade, and that the ability to support viral cell-to-cell movement is not required for effector functionality, We thank Leonardo Boiteux (Embrapa Vegetable Crops - CNPH, Brasilia, DF, Brazil) for providing the isogenic line "CNPH LAM 147" essential to elaboration of this work. This research was financially supported by CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), CAPES (Conselho de Aperfeicoamento de Pessoal de Nivel Superior), BIO2014-54862-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT), the Prometeo Program GV2014/010 from the Generalitat Valenciana and Wageningen University

[EN] Pokeweed antiviral protein (PAP) of Phytolacca americana L. (pokeweed) is a single-chain ribosome-inactivating protein (RIP) characterized by its ability to depurinate plant ribosomes. Here, we isolated, cloned, and expressed the ribosome inactivating protein (RIP) gene, designated as pokeweed antiviral protein type 1 (PAP I), from the summer leaves of pokeweed collected from the Black Sea region (Turkey). Our findings presented here provide direct evidence that exogenous application of PAP I causes concentration-dependent inhibition of Zucchini yellow mosaic virus (ZYMV) infection on squash plants. Squash plants were exposed to PAP I protein with and without DMSO for four consecutive days. Regular spraying of approximately 30 kDa recombinant PAP I at 2 µg mL¿1 concentration prevented treated plants from mechanical virus infection. PAP I showed antiviral activity in 9 plants out of 15 inoculated plants. Remarkably, simultaneous application of PAP, DMSO, and ZYMV did not prevent virus infection, suggesting that PAP did not have any effect on viral RNA. In the absence of ZYMV the purified peptide was not cytotoxic for squash plants, although a reduction of plant size, possibly caused by host ribosome depurination, was observed., Financial support for this study was provided by the Scientific and Technological Research Council of Turkey (TUBITAK Project No: 110 O 180).

This is the accepted version of the following article: Peiro Morell, A.; Izquierdo Garcia, AC.; Sanchez Navarro, JA.; Pallas Benet, V.; Mulet Salort, JM.; Aparicio Herrero, F. (2014). Patellins 3 and 6, two members of the Plant Patellin family, interact with the movement protein of Alfalfa mosaic virus and interfere with viral movement. Molecular Plant Pathology. 15(9):881-891. doi:10.1111/mpp.12146., which has been published in final form at http://dx.doi.org/10.1111/mpp.12146.

Prunus necrotic ringspot virus (PNRSV) is distributed worldwide, but no molecular data have been previously reported from South American isolates. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of 23 isolates of PNRSV from Chile, Brazil, and Uruguay, and from different Prunus species, have been obtained. Phylogenetic analysis performed with full-length MP and CP sequences from all the PNRSV isolates confirmed the clustering of the isolates into the previously reported PV32-I, PV96-II and PE5-III phylogroups. No association was found between specific sequences and host, geographic origin or symptomatology. Comparative analysis showed that both MP and CP have phylogroup-specific amino acids and all of the motifs previously characterized for both proteins. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that most amino acid sites are under the effect of negative purifying selection.

[EN] Plant viruses express RNA silencing suppressor (RSS) proteins to counteract plant defence mechanisms. Here, we describe a method to assess the RSS activity based on an alfalfa mosaic virus (AMV) RNA 3 expression vector and transgenic Nicotiana tabacum plants that express the P1 and P2 subunits of the AMV replicase (P12 plants). Inoculation of P12 plants with different AMV RNA 3 constructs expressing different HC-Pro mutants that differ in their RSS capabilities, revealed a perfect correlation between necrotic lesions on inoculated leaves and RSS activity. Protoplast analysis showed that the RSS activity correlated with the accumulation of the AMV RNAs. A direct comparison between three RSS activity assays and the AMV-P12 system revealed that the coat protein of carnation mottle virus displays RSS activity with the four assays and reduced the accumulation of the siRNAs., We thank L. Corachan for her excellent technical assistance. We thank Dr. S. Elena, I2SysBio (Valencia, Spain), for supplying us the different HC-Pro mutants. This work was supported by grant BIO2017-88321-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT) and the Prometeo Program GV2015/010 from the Generalitat Valenciana.

The use of riboprobes carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the simultaneous detection of up to six different pathogens using a non-radioactive molecular hybridization procedure. In the present report, we describe the development of a unique polyprobe (poly10) with the capacity to detect viruses and viroids commonly found infecting fruit trees. The poly10 covers eight viruses: Apple mosaic virus (ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern (APLPV), Plum pox virus (PPV), Apple chlorotic leaf spot virus (ACLSV), Apricot latent virus (ApLV), Plum bark necrosis and stem pitting-associated virus (PBNSPaV) and two viroids: Hop stunt viroid (HSVd) and Peach latent mosaic viroid (PLMVd). Poly10 is comparable to the individual riboprobes in terms of end-point dilution limit and specificity, allowing the detection of up to 2.5 picograms of viral or viroidal RNA. However, the polyprobe requires a hybridization temperature of 60 degrees C instead of the standard 68 degrees C. The validation of the new simultaneous detection strategy was confirmed by the analysis of 60 field samples, which came from seven different hosts. The use of the polyprobe as an alternative to other routinely used detection methods is discussed., A. P. M. is the recipient of a JAE-Pre contract from the Consejo Superior de Investigaciones Cientificas (CSIC). We thank L. Corachan for her excellent technical assistance. This work was supported by grant BIO2008-03528 from the Spanish granting agency DGICYT and by grant ACOMP/2010/214 from the Generalitat Valenciana.

[EN] Plant viruses are biotrophic pathogens that need living tissue for their multiplication and thus, in the infection-defence equilibrium, they do not normally cause plant death. In some instances virus infection may have no apparent pathological effect or may even provide a selective advantage to the host, but in many cases it causes the symptomatic phenotypes of disease. These pathological phenotypes are the result of interference and/or competition for a substantial amount of host resources, which can disrupt host physiology to cause disease. This interference/competition affects a number of genes, which seems to be greater the more severe the symptoms that they cause. Induced or repressed genes belong to a broad range of cellular processes, such as hormonal regulation, cell cycle control and endogenous transport of macromolecules, among others. In addition, recent evidence indicates the existence of interplay between plant development and antiviral defence processes, and that interference among the common points of their signalling pathways can trigger pathological manifestations. This review provides an update on the latest advances in understanding how viruses affect substantial cellular processes, and how plant antiviral defences contribute to pathological phenotypes. © 2011 SGM., We would like to apologize to those people whose relevant publications could not be cited because of space constraints. Work in the laboratories of Juan Antonio Garcı´a and Vicente Pallas were supported by grants from the Spanish MICINN (BIO2010-18541) and the EU (KBBE-204429), and from the Spanish MICINN (BIO08- 03528), respectively

Small RNA (sRNA)-guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self-replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of viroid-derived sRNAs (vd-sRNAs) is usually associated with viroid infection. However, the pathways involved in the biogenesis of vd-sRNAs are largely unknown. Here, we analyse, by high-throughput pyrosequencing, the profiling of the Hop stunt viroid (HSVd) vd-sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd-sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd-sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd-sRNAs. Our sequence data suggest that viroid-derived double-stranded RNA functions as one of the main precursors of vd-sRNAs. Remarkably, phloem vd-sRNAs accumulated preferentially as 22-nucleotide species with a consensus sequence over-represented. This bias in size and sequence in the HSVd vd-sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd-sRNAs to the phloem tissue of infected cucumber plants.

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for viral transport. Previous analysis with MPs of other members of the family Bromoviridae has shown that the C-terminal part of these MPs plays a critical role in the interaction with the cognate coat protein (CP) and in cell-to-cell transport. Bimolecular fluorescence complementation and overlay analysis confirm an interaction between the C-terminal 38 aa of PNRSV MP and its cognate CP. Mutational analysis of the C-terminal region of the PNRSV MP revealed that its C-terminal 38 aa are dispensable for virus transport, however, the 4 aa preceding the dispensable C terminus are necessary to target the MP to the plasmodesmata and for the functionality of the protein. The capacity of the PNRSV MP to use either a CP-dependent or a CP-independent cell-to-cell transport is discussed.

The aim of this work was to follow Prunus necrotic ringspot virus (PNRSV) infection in apricot reproductive tissues and transmission of the virus to the next generation. For this, an analysis of viral distribution in apricot reproductive organs was carried out at different developmental stages. PNRSV was detected in reproductive tissues during gametogenesis. The virus was always present in the nucellus and, in some cases, in the embryo sac. Studies within infected seeds at the embryo globular stage revealed that PNRSV infects all parts of the seed, including embryo, endosperm and testa. In the torpedo and bent cotyledon developmental stages, high concentrations of the virus were detected in the testa and endosperm. At seed maturity, PNRSV accumulated slightly more in the embryo than in the cotyledons. In situ hybridization showed the presence of PNRSV RNA in embryos obtained following hand-pollination of virus-free pistils with infected pollen. Interestingly, tissue-printing from fruits obtained from these pistils showed viral RNA in the periphery of the fruits, whereas crosses between infected pistils and infected pollen resulted in a total invasion of the fruits. Taken together, these results shed light on the vertical transmission of PNRSV from gametes to seedlings.

A RT-PCR was developed for the simultaneous detection and identification of three groups of Pepino mosaic virus (PepMV): European/Peruvian, Chilean 1/US1 and Chilean 2/US2 groups, followed by a restriction analysis that allowed the separation of the European, Peruvian, Chilean 2 and US2 isolates (patent pending). The multiplex RT-PCR reaction was performed by a mix of six primers that amplified a part of the RNA-dependent RNA polymerase gene of PepMV plus an internal control. Amplifications resulted in a 980 bp, 703 bp or 549 bp PCR product for European/Peruvian, Chilean 1/US1 or Chilean 2/US2 groups, respectively. For the identification of the isolates present within the European/Peruvian and Chilean 2/US2 groups, the amplified PCR fragments were directly digested with SacI enzyme. The multiplex RT-PCR method presented higher sensitivity to detect CH1/US1 isolates in field samples than the RFLP-PCR method described by Hanssen et al. (European Journal of Plant Pathology 121:131–146, 2008). The detection limit observed with the multiplex RT-PCR was equal to or 3,125 times higher when compared to single RT-PCR or ELISA-DAS and molecular hybridisation methods, respectively. The use of the multiplex RT-PCR method in routine analysis of field tomato samples allowed the detection of 36.2 and 33.4% more positives when compared to the serological and molecular hybridisation methods, respectively, and the identification of plants infected with one, two or three isolates of PepMV.

In this paper, we report a large-scale survey for the incidence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) in stone fruit collections and commercial orchards in the Czech Republic. From the 645 samples analysed, PLMVd was detected in 80 (26.6%) of peaches and the HSVd in 3 (1.3%) of apricot and 1 (0.33%) of peach trees. Sixty-seven accession of peach (44.6%) from the Czech Clonal GeneBank were infected by PLMVd. In addition, we used naturally infected trees to standardise the simultaneous detection of PLMVd and HSVd plus host mRNA as the control by means of one-step multiplex RTC-PCR. Eleven PLMVd and two HSVd isolates were sequenced and analysed. All the PLMVd variants were highly homologous (97–100%) to previously reported PLMVd variants from Tunisian peach and almond trees, and clustered together in the previously reported phylogenetic group III. The HSVd variants obtained from apricot and peach trees were included in the previously proposed recombinant group PH/cit3.

A polyprobe for the simultaneous detection by non-isotopic molecular hybridisation has been developed to detect any of the following six viruses causing important economic losses in tomato crops: Tomato spotted wilt virus, Tomato mosaic virus, Pepino mosaic virus, Cucumber mosaic virus, Potato Y virus and Parietaria mottle virus. The polyprobe detected all six viruses with similar sensitivity to that obtained using individual riboprobes. In addition, we evaluated the possible use of the tissue-printing as a sample preparation technique applied to routine diagnosis of tomato plants with the polyprobe.

Viroids are small self-replicating RNAs that infect plants. How these noncoding pathogenic RNAs interact with hosts to induce disease symptoms is a long-standing unanswered question. Recent experimental data have led to the suggestive proposal of a pathogenic model based on the RNA silencing mechanism. However, evidence of a direct relation between key components of the RNA silencing pathway and symptom expression in infected plants remains elusive. To address this issue, we used a symptomatic transgenic line of Nicotiana benthamiana that expresses and processes dimeric forms of Hop stunt viroid (HSVd). These plants were analyzed under different growing temperature conditions and were used as stocks in grafting assays with the rdr6i-Nb line, in which the RNA-dependent RNA polymerase 6 (RDR6) is constitutively silenced. Here, we show that the symptom expression in N. benthamiana plants is independent of HSVd accumulation levels but dependent on an active state of the viroid-specific RNA silencing pathway. The scion of rdr6i-Nb plants remained asymptomatic when grafted onto symptomatic plants, despite an accumulation of a high level of mature forms of HSVd, indicating the requirement of RDR6 for viroid-induced symptom production. In addition, the RDR6 requirement for symptom expression was also observed in wild-type N. benthamiana plants mechanically infected with HSVd. These results provide biological evidence of the involvement of the viroid-specific RNA silencing pathway in the symptom expression associated with viroid pathogenesis.

Until very recently isolates of American plum line pattern virus (APLPV) had not been reported from outside North America. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of eight APLPV isolates from five Mediterranean countries were determined. Sequence analysis showed that both MP and CP genes are highly conserved irrespective of geographic origin. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that these proteins are under the effect of negative purifying selection. The MP and CP of APLPV possess most of the functional motifs described for other members of the genus Ilarvirus.

Changes in gene expression of Mexican lime plants in response to infection with a severe (T305) or a mild (T385) isolate of Citrus tristeza virus (CTV) were analyzed using a cDNA microarray containing 12,672 probes to 6875 different citrus genes. Statistically significant (P

Cell-to-cell movement of the Melon Necrotic Spot Virus (MNSV) is controlled by two small proteins working in trans, an RNA-binding protein (p7A) and an integral membrane protein (p7B) separated by an amber stop codon. p7B contains a single hydrophobic region. Membrane integration of this region was observed when inserted into model proteins in the presence of microsomal membranes. Furthermore, we explored the topology and targeting mechanisms of full-length p7B. Here we present evidence that p7B integrates in vitro into the ER membrane cotranslationally and with an Nt-cytoplasmic/Ct-luminal orientation. The observed topology was monitored in vivo by fusing GFP to the Ct of p7B, enabling the overexpression in Escherichia coli cultures. Finally, the topology of a putative p14 movement protein was established by replacing the amber stop codon located between p7A and p7B.

The genomic regions encoding the putative movement protein (MP), coat protein (CP) and intergenic region (IGR) of seven Spanish isolates of the Parietaria mottle virus which infects tomato plants (PMoV-T) were sequenced. Values for the genetic diversity of the PMoV-T isolates were 0.056, 0.047 and 0.013 for the CP, MP genes and IGR, respectively. Nucleotide and amino acid sequence comparison of the seven PMoV-T isolates with those of PMoV revealed significant differences. All of them had a cytosine deletion at position 1366, also confirmed in an Italian tomato isolate, which involves a start codon for the CP gene different from that for the PMoV sequence, resulting in a CP 16 amino acids shorter than the PMoV CP. The certainty of a cytosine deletion only associated to the tomato isolates or the possibility of a mistake in the PMoV published sequence are the two hypotheses that could explain this difference. Structural motifs highly conserved in Ilarviruses were identified in PMoV-T MP and CP. A stable hairpin structure is proposed for IGR, by the initiation site for subgenomic RNA 4 synthesis. Phylogenetic analysis of CP and MP amino acid sequences showed that Spanish PMoV-T isolates form a separate group from PMoV and other members of the Ilarvirus genus. Comparative analysis with different PMoV isolates including tomato isolates from other regions and isolates from different hosts are necessary to confirm this differentiation.

Viroids, small non-coding pathogenic RNAs, are able to induce RNA silencing, a phenomenon that has been associated with the pathogenesis and evolution of these small RNAs. It has been recently suggested that viroids may resist this plant defense mechanism. However, the simultaneous degradation of non-replicating full-length viroid RNA, and the resistance of mature forms of viroids to RNA silencing, have not been experimentally demonstrated. Transgenic Nicotiana benthamiana plants expressing a dimeric form of Hop stunt viroid (HSVd) that have the capability to cleave and circularize this viroid RNA were used to address this question. A reporter construct, consisting of a full-length HSVd RNA fused to GFP-mRNA, was agroinfiltrated in these plants and its expression was suppressed. Interestingly, both circular and linear HSVd molecules were stable and able to traffic through grafts in these restrictive conditions, indicating that the mature forms of HSVd are able, in some way, to resist the RNA-silencing mechanism. The observation that a full-length HSVd RNA fused to GFP-mRNA, but not circular and/or linear viroid forms, was fully susceptible to RNA degradation strongly suggests that structures adopted by the free mature monomer protect the pathogenesis-associated forms of the viroid from RNA silencing.

We previously showed that the movement protein (MP) gene of Alfalfa mosaic virus (AMV) is functionally exchangeable for the cell-to-cell transport of the corresponding genes of Tobacco mosaic virus (TMV), Brome mosaic virus, Prunus necrotic ringspot virus, Cucumber mosaic virus and Cowpea mosaic virus. We have analysed the capacity of the heterologous MPs to systemically transport the corresponding chimeric AMV genome. All MPs were competent in systemic transport but required the fusion at their C terminus of the coat protein-interacting C-terminal 44 aa (A44) of the AMV MP. Except for the TMV MP, the presence of the hybrid virus in upper leaves correlated with the capacity to move locally. These results suggest that all the MPs assigned to the 30K superfamily should be exchangeable not only for local virus movement but also for systemic transport when the A44 fragment is present., We thank L. Corachan for her excellent technical assistance. This work was supported by the Spanish granting agency DGICYT via grant BIO2011-25018 and by the Generalitat Valenciana via grant PROMETEO 2011-003.

Conclusions: We compare for the first time the sRNA profile of four different tissues, including source, sink and conductive (phloem) tissues, in two plant-virus pathosystems. Our results indicate that antiviral silencing machinery in melon and cucumber acts mainly through DCL4. Upon infection, the total sRNA pattern in phloem remains unchanged in contrast to the rest of the analyzed tissues indicating a certain tissue-tropism to this polulation. Independently of the accumulation level of the vsRNAs both viruses were able to modulate the host sRNA pattern., We thank Dr A. Niehl for critical reading and helpful comments on the manuscript. This work was funded by a supporting program for the research from the Universidad Politecnica de Valencia (PAID-05-10), a grant BIO2011-25018 from the Spanish granting agency Direccion General de Investigacion Cientifica and the PROMETEO program 2011/003 from the Generalitat Valenciana. MCH is the recipient of a contract from JAE-DOC program of the CSIC, JAN is the recipient of a postdoctoral contract from the Ministerio de Educacion y Ciencia of Spain.

[EN] Tospovirus is the only genus containing virus species which infect plants in the Bunyaviridae family. The aims of this study were to understand the in vivo membrane association of the movement protein (NSm) of the tospovirus species Bean necrotic mosaic virus, Chrysanthemum stem necrosis virus, Tomato chlorotic spot virus and Tomato spotted wilt virus and the homologous and heterologous interactions among NSm and nucleocapsid protein (N). The results obtained by bimolecular fluorescence complementation (BiFC) assay and chemical treatments after membrane fractionation revealed that the four NSm proteins are associated with the biological membranes with the N- and C-termini oriented to the cytoplasm. BiFC analysis for protein protein interactions showed: i) dimer formation for all NSm and N proteins; ii) interaction between NSm and the cognate N and iii) heterologous interactions between the NSm and N proteins. The implications of these interactions in the life cycle of tospoviruses are discussed. (C) 2015 Elsevier Inc. All rights reserved., We thank L. Corachan for her excellent technical assistance. This work was supported by Grant BIO2011-25018 from the Spanish granting agency DGICYT, joint Grant 564633/2010-0 CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) and CAPES (Conselho de Aperfeicoamento de Pessoal de Nivel Superior) and Grant 2009/00102-2 from FAP-DF (Fundacao de Apoio a Pesquisa do Distrito Federal).

Carnation mottle virus (CarMV), Carnation etched ring virus (CERV), Carnation vein mottle virus (CVMV), Carnation ringspot virus (CRSV), Carnation Italian ringspot virus (CIRV) and Carnation latent virus (CLV) are the most important viruses affecting carnation crops. All except CERV are RNA viruses. Viral RNA or DNA accumulation on root, stem, leaf, sepal, petal, stamen, pistil and ovary tissues of infected carnation or Saponaria vaccaria plants was analysed by non-isotopic molecular hybridisation. High-titres of CarMV, CRSV, CIRV, and CLV accumulated in all plant tissues whereas CERV and CVMV were irregularly distributed over the plant. High-titres of all viruses accumulated in leaf, petal, stamen, pistil, and ovary tissues, so leaves or petals are a good tissue for routine diagnosis. Six chemicals were evaluated for inactivation of all carnation viruses in infected extracts. Commercial bleach at 7% (v/v) or NaOH at 0.5% (w/v) was found to inactivate all viruses after 60 s treatment in a systemic S. vaccaria bioassay.

SUMMARY Viroids are small, single-stranded, circular, pathogenic RNAs that do not code for proteins and thus depend on host factors for their replication and movement. They induce diseases in plants by direct interaction with host factors through a mechanism as yet unidentified. Hop stunt viroid (HSVd) has been found in a wide range of herbaceous and woody hosts. Nicotiana benthamiana is one of the most frequently used experimental systems in the study of plant-virus interactions. However, this plant is a non-host for HSVd. To go further in the study of the requirements of a plant to be a host for viroids N. benthamiana was transformed with cDNA expressing dimeric (+) HSVd. Correct processing to the circular (+) monomers was always observed, demonstrating that N. benthamiana has the appropriate machinery to cleave and circularize (+) HSVd molecules. Additionally, N. benthamiana plants agroinoculated with the dimeric (-) HSVd accumulated the circular and linear (+) monomers, indicating that (-) HSVd transcripts can be used as template for the RNA-RNA transcription. Grafting assays showed that HSVd is able to move to distal plant parts. In addition, alterations in the normal flower development were observed. Taken together, these results indicate that deficiencies in the interaction with host factors related either to the early steps of the replication process or to cell to cell movement appear to be the factors that limit infectivity of HSVd in N. benthamiana. These transgenic plants can be an useful tool to study the HSVd-host factor interactions.

Function of the melon necrotic spot virus (MNSV) genome-encoded proteins (p29, p89, p7A, p7B and p42) has been studied. Protein-expression mutants of an infectious, full-length cDNA clone of a Spanish MNSV-Al isolate and a recombinant green fluorescent protein (GFP)-expressing virus were used in infection bioassays on melon plants. Results revealed that p29 and p89 are both essential for virus replication, whereas small proteins p7A and p7B are sufficient to support viral movement between adjacent cells operating in trans. It is also demonstrated that, in addition to its structural role as coat protein, p42 is an important factor controlling symptoms and is required for systemic transport. Moreover, both p42 and p7B, among all of the MNSV-encoded proteins, were able to delay RNA silencing in transient-expression assays on GFP-transgenic Nicotiana benthamiana plants. Finally, the presence of p42 also produced an enhancing effect on local spread similar to that of potyviral helper component proteinase (HC-Pro), probably due to its RNA silencing-suppression ability.

RNA 3 of Alfalfa mosaic virus (AMV) encodes the movement protein (MP) and coat protein (CP). Chimeric RNA 3 with the AMV MP gene replaced by the corresponding MP gene of Prunus necrotic ringspot virus, Brome mosaic virus, Cucumber mosaic virus or Cowpea mosaic virus efficiently moved from cell-to-cell only when the expressed MP was extended at its C-terminus with the C-terminal 44 amino acids of AMV MP. MP of Tobacco mosaic virus supported the movement of the chimeric RNA 3 whether or not the MP was extended with the C-terminal AMV MP sequence. The replacement of the CP gene in RNA 3 by a mutant gene encoding a CP defective in virion formation did not affect cell-to-cell transport of the chimera's with a functional MP. A GST pull-down technique was used to demonstrate for the first time that the C-terminal 44 amino acids of the MP of a virus belonging to the family Bromoviridae interact specifically with AMV virus particles. Together, these results demonstrate that AMV RNA 3 can be transported from cell-to-cell by both tubule-forming and non-tubule-forming MPs if a specific MP–CP interaction occurs.

A sensitive and reliable one step RT-PCR reaction with an internal control has been developed to detect and differentiate eight important viruses that affect stone fruit tress: Apple mosaic virus (ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern virus (APLPV), Plum pox virus (PPV), Apple chlorotic leaf spot virus (ACLSV), Apricot latent virus (ApLV) and Plum bark necrosis stem pitting associated virus (PBNSPaV). In addition, we investigated the detection limit and the efficiency of three different nucleic acid extraction methods that avoid the use of organic solvents, for both multiplex RT-PCR and dot-blot hybridisation assays. The primer cocktail was used to analyse 38 stone fruits originating from nine different countries and six species. A large number of virus combinations was detected and up to three different viruses were observed in five samples. A decrease in sensitivity was observed when the primer cocktail contained more than five different pair primers. However, comparative analyses showed that the multiplex RT-PCR containing the eight virus pair primers was even more sensitive than the ELISA or molecular hybridisation assays. The use of the multiplex RT-PCR technology in routine diagnosis of stone fruit tree viruses is discussed.

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is involved in intercellular virus transport. In this study, putative RNA-binding properties of the PNRSV MP were studied. The PNRSV MP was produced in Escherichia coli using an expression vector. Electrophoretic mobility shift assays (EMSAs) using DIG-labelled riboprobes demonstrated that PNRSV MP bound ssRNA cooperatively without sequence specificity. Two different ribonucleoprotein complexes were found to be formed depending on the molar MP : PNRSV RNA ratio. The different responses of the complexes to urea treatment strongly suggested that they have different structural properties. Deletion mutagenesis followed by Northwestern analysis allowed location of a nucleic acid binding domain to aa 56-88. This 33 aa RNA-binding motif is the smallest region delineated among members of the family Bromoviridae for which RNA-binding properties have been demonstrated. This domain is highly conserved within all phylogenetic subgroups previously described for PNRSV isolates. Interestingly, the RNA-binding domain described here and the one described for Alfamovirus are located at the N terminus of their corresponding MPs, whereas similar domains previously characterized in members of the genera Bromovirus and Cucumovirus are present at the C terminus, strongly reflecting their corresponding phylogenetic relationships. The evolutionary implications of this observation are discussed.

The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRSV) varying in the symptomatology they cause in six different Prunus spp. were determined. Analysis of the molecular variability has allowed, in addition to study the phylogenetic relationships among them, to evaluate the minimal requirements for the synthesis of the subgenomic RNA in Ilarvirus genus and their comparison to other members of the Bromoviridae family. Computer assisted comparisons led recently to Jaspars (Virus Genes 17, 233-242, 1998) to propose that a hairpin structure in viral minus strand RNA is required for subgenomic promoter activity of viruses from at least two, and possibly all five, genera in the family of Bromoviridae. For PNRSV and Apple mosaic virus two stable hairpins were proposed whereas for the rest of Ilarviruses and the other four genera of the Bromoviridae family only one stable hairpin was predicted. Comparative analysis of this region among the fifteen PNRSV isolates characterized in this study revealed that two of them showed a 12-nt deletion that led to the disappearance of the most proximal hairpin to the initiation site. Interestingly, the only hairpin found in these two isolates is very similar in primary and secondary structure to the one previously shown in Brome mosaic virus to be required for the synthesis of the subgenomic RNA. In this hairpin, the molecular diversity was concentrated mostly at the loop whereas compensatory mutations were observed at the base of the stem strongly suggesting its functional relevance. The evolutionary implications of these observations are discussed.