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[Taxonomy]: Hop stunt viroid (HSVd) is the type species of the genus Hostuviroid (family Pospiviroidae). The other species of this genus is Dahlia latent viroid, which presents an identical central conserved region (CCR) but lacks other structural hallmarks present in Hop stunt viroid. HSVd replication occurs in the nucleus through an asymmetric rolling-circle model as in the other members of the family Pospiviroidae, which also includes the genera Pospiviroid, Cocadviroid, Apscaviroid, and Coleoviroid., [Physical properties]: Hop stunt viroid consists of a single-stranded, circular RNA of 295–303 nucleotides depending on isolates and sequence variants. The most stable secondary structure is a rod-like or quasi-rod-like conformation with two characteristic domains: a CCR and a terminal conserved hairpin similar to that of cocadviroids. HSVd lacks a terminal conserved region., [Hosts and symptoms]: HSVd infects a very broad range of natural hosts and has been reported to be the causal agent of five different diseases (citrus cachexia, cucumber pale fruit, peach and plum apple apricot distortion, and hop stunt). It is distributed worldwide., [Transmission]: HSVd is transmitted mechanically and by seed., This work was funded by grants BIO2017-88321-R, AGL2016-79825-R, and PID2019-104126RB-I00 from the Spanish Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). J.M.-M. is the recipient of a predoctoral contract from the ACIF programme (ACIF-2017-114) of the Conselleria d'Educació, Investigació, Cultura i Esport Generalitat Valenciana.

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.

The coat protein (CP) of Melon necrotic spot virus (MNSV) is structurally composed of three major domains. The middle S-domain builds a robust protein shell around the viral genome, whereas the C-terminal protruding domain, or P-domain, is involved in the attachment of virions to the transmission vector. Here, we have shown that the N-terminal domain, or R-domain, and the arm region, which connects the R-domain and S-domain, are involved in different key steps of the viral cycle, such as cell-to-cell movement and the suppression of RNA silencing and pathogenesis through their RNA-binding capabilities. Deletion mutants revealed that the CP RNA-binding ability was abolished only after complete, but not partial, deletion of the R-domain and the arm region. However, a comparison of the apparent dissociation constants for the CP RNA-binding reaction of several partial deletion mutants showed that the arm region played a more relevant role than the R-domain in in vitro RNA binding. Similar results were obtained in in vivo assays, although, in this case, full-length CPs were required to encapsidate full-length genomes. We also found that the R-domain carboxyl portion and the arm region were essential for efficient cell-to-cell movement, for enhancement of Potato virus X pathogenicity, for suppression of systemic RNA silencing and for binding of small RNAs. Therefore, unlike other carmovirus CPs, the R-domain and the arm region of MNSV CP have acquired, in addition to other essential functions such as genome binding and encapsidation functions, the ability to suppress RNA silencing by preventing systemic small RNA transport.

F.A. was the recipient of a contract Ramon y Cajal (RYC-2010-06169) program of the Ministerio de Educacion, Cultura y Deporte of Spain. We thank L. Corachan for excellent technical assistance. This work was supported by Grants BIO2014-54862-R from the Spanish grant agency Direccion General de Investigacion Cientifica y Tecnica (DGICT) the Prometeo Program GV2015/010 from the Generalitat Valenciana and PAID-06-10-1496 from the Universitat Politecnica de Valencia (Spain).

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.

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.

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.

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.

*† Summary Phloem proteins (P-proteins) are an enigmatic group of proteins present in most angiosperm species. The best characterized P-proteins (PP1 and PP2) are synthesized in companion cells, transported into sieve elements via pore plasmodesmata and translocated through the plant. Characteristics such as long-distance translocation, RNA-binding activity and capacity of increasing plasmodesmata exclusion size suggest that certain phloem proteins could be involved in RNA transport within the plant, forming translocatable ribonucleoprotein complexes with endogenous or pathogenic RNAs. Long-distance movement of RNA through the phloem is a process known to occur, but both the mechanisms involved and the components constituting this potential information network remain unclear. Here, we demonstrate that several melon phloem proteins have a wide RNA-binding activity. Serological assays strongly suggest that one of these proteins is the melon phloem protein 2 (CmmPP2). Mass spectrometry analysis undoubtedly identifies another one as the recently characterized melon phloem lectin (CmmLec17). Grafting experiments demonstrate that the CmmLec17 is a translocatable phloem protein, able to move through intergeneric grafts from melon to pumpkin. Translocatability and RNA-binding activity was also demonstrated for an uncharacterized protein of approximately 14 kDa. In light of these results the possible involvement of these phloem proteins in the long-distance transport of melon RNAs is discussed.

[EN] Viral movement proteins exploit host endomembranes and the cytoskeleton to move within the cell via routes that, in some cases, are dependent on the secretory pathway. For example, melon necrotic spot virus p7B, a type II transmembrane protein, leaves the endoplasmic reticulum (ER) through the COPII-dependent Golgi pathway to reach the plasmodesmata. Here we investigated the sequence requirements and putative mechanisms governing p7B transport through the early secretory pathway. Deletion of either the cytoplasmic N–terminal region (CR) or the luminal C–terminal region (LR) led to ER retention, suggesting that they are both essential for ER export. Through alanine-scanning mutagenesis, we identified residues in the CR and LR that are critical for both ER export and for viral cell-to-cell movement. Within the CR, alanine substitution of aspartic and proline residues in the DSSP b–turn motif (D7AP10A) led to movement of discrete structures along the cortical ER in an actin-dependent manner. In contrast, alanine substitution of a lysine residue in the LR (K49A) resulted in a homogenous ER distribution of the movement protein and inhibition of ER–Golgi traffic. Moreover, the ability of p7B to recruit Sar1 to the ER membrane is lost in the D7AP10A mutant, but enhanced in the K49A mutant. In addition, fluorescence recovery after photobleaching revealed that K49A but not D7AP10A dramatically diminished protein lateral mobility. From these data, we propose a model whereby the LR directs actin-dependent mobility toward the cortical ER, where the cytoplasmic DSSP b–turn favors assembly of COPII vesicles for export of p7B from the ER., We are grateful to C. Hawes (Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Headington, Oxford, UK) and V. V. Dolja (Department of Botany and Plant Pathology and Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR, USA) for providing the Sar1-YFP construct and some fluorescent-tagged organelle markers, respectively. We would like to thank F. Aniento (Departmento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad de Valencia, Burjassot, Valencia, Spain) and E. Rojo (Departament of Plant Molecular Genetics, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, 28049 Madrid, Spain) for critical reading and valuable comments on this manuscript. This work was funded by grant BIO2011-25018 from the Spanish Departmento Governmental de Investigaciones Cientificas y Tecnologicas and Prometeo Program GV2011/003 from the Generalitat Valenciana. J.A.N. and M. S. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Educacion y Ciencia of Spain. We thank L. Corachan for her technical assistance.

Recombinant DNA technology was used to raise a polyclonal antiserum against the coat protein (CP) of Parietaria mottle virus (PMoV). The CP gene was expressed in Escherichia coli as a fusion to a 6xHis tag and purified by affinity chromatography. Recombinant purified protein was used as antigen to raise a polyclonal antiserum. This polyclonal antiserum consistently detected PMoV specifically infected tomato plants from different commercial tomato crops by indirect enzyme-linked immunosorbent assay (I-ELISA) and direct tissue-printing immunoassay (DTBIA).

Three methods were compared for the detection of prunus necrotic ringspot virus in herbaceous and woody plants: DAS-ELISA, nonisotopic dot-blot hybridization and reverse transcriptional polymerase chain reaction (RT-PCR). When purified virus preparations were used, the detection limit of the RT-PCR technique was 1·28 pg mL π1 whereas nonisotopic molecular hybridization and DAS-ELISA allowed detection of 0·8 ng mL π1 and 4 ng mL π1 , respectively. Several sample processing procedures were evaluated for virus detection by the nonisotopic molecular hybridization technique. When a very short and simple sample processing method was used, the detection limit of the nonisotopic molecular hybridization technique was 25 times higher than that of DAS-ELISA and 625 times lower than that of RTPCR. A comparison of the level of virus accumulation in mature fruits and in leaf tissue showed that, on average, 125 times more virus was found in the fruits.

The authors would like to thank J. A. Daros and S. F. Elena for the critical reading of the manuscript. This work was supported by grant BIO2011-25018 from the Spanish granting agency Dirección General de Investigación Científica and from the Prometeo program 2011/003 from the Generalitat Valenciana. The authors thank the anonymous reviewers for their valuable comments and suggestions.

A non-radioactive molecular hybridization method was developed to detect carnation mottle virus (CarMV) in carnation plants. This method was compared to the standard ELISA test and biological assays. For crude plant extracts, the molecular hybridization technique proved to be at least 125 times more sensitive than double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The method was adapted to avoid the necessity to clarify the samples by centrifugation and to use pipettes to load them. By introducing this modification, the hybridization assay was found to be a suitable method for routine virus detection. The relative benefits of using this procedure for large-scale indexing are discussed.