The capsid protein of citrus leprosis virus C shows a nuclear distribution and interacts with the nucleolar fibrillarin protein.

Brevipalpus-transmitted viruses (BTVs) have a significant negative economic impact on the citrus industry in Central and South America. Until now, only a few studies have explored the intracellular distribution and interaction of BTVs-encoded proteins with host factors, particularly for cileviruses, the main BTV responsible for the Citrus Leprosis (CL) disease. This study describes the nuclear localization of citrus leprosis virus C (CiLV-C) capsid protein (p29) and its interaction with the fibrillarin (Fib2) within the nucleolar compartment and cell cytoplasm. Our results, obtained by computer predictions and laser scanning confocal microscopy analyses, including colocalization and bimolecular fluorescence complementation (BiFC) approaches, revealed that a fraction of the p29 is localized in the nucleus and colocalizes with the Fib2 in both the nucleolus and cytosol. The nuclear localization of p29 correlated with a smaller nucleus size. Furthermore, co-immunoprecipitation (Co-IP) assays confirmed the interactions between p29 and Fib2. The implications of these findings for the functionalities of the cilevirus capsid protein are discussed.

Rescue of a Cilevirus from infectious cDNA clones.

Reverse genetics systems represent an important tool for studying the molecular and functional processes of viral infection. Citrus leprosis virus C (CiLV-C) (genus Cilevirus, family Kitaviridae) is the main pathogen responsible for the citrus leprosis (CL) disease in Latin America, one of the most economically important diseases of the citrus industry. Molecular studies of this pathosystem are limited due to the lack of infectious clones. Here, we report the construction and validation of a CiLV-C infectious cDNA clone based on an agroinfection system. The two viral RNA segments (RNA1 and RNA2) were assembled into two binary vectors (pJL89 and pLXAS). Agroinfiltrated Nicotiana benthamiana plants showed a response similar to that observed in the natural infection process with the formation of localized lesions restricted to the inoculated leaves. The virus recovered from the plant tissue infected with the infectious clones can be mechanically transmitted between N. benthamiana plants. Detection of CiLV-C subgenomic RNAs (sgRNAs) from agroinfiltrated and mechanically inoculated leaves further confirmed the infectivity of the clones. Finally, partial particle-purification preparations or sections of CiLV-C-infected tissue followed by transmission electron microscopy (TEM) analysis showed the formation of CiLV-C virions rescued by the infectious clone. The CiLV-C reverse genetic system now provides a powerful molecular tool to unravel the peculiarities of the CL pathosystem.

Agave attenuata and Agave amica, new hosts of Tuberose mild mosaic virus in Mexico.

Agave attenuata is a Mexican wild plant originally from highlands in the central and occidental mountains of Mexico. This species, known as "swan´s neck agave", is used only as an ornamental plant in public and private gardens. No virus had previously been reported from A. attenuata before this study. In a survey conducted in a commercial greenhouse in Cuautla, Morelos, in 2018, several plants were observed with symptoms of green mosaic and streaks, consistent with a putative viral infection. Sap inoculation from symptomatic A. attenuata plants to herbaceous indicator plants (Nicotiana benthamiana and N. tabacum) failed to produce symptoms in the mechanically inoculated plants. ELISA specific test to CMV, TEV, AMV, TMV and Potyvirus Group (Agdia, Inc.), was positive only for the last one (Chen and Chang, 1998). To determine the identity of the potyvirus involved, total nucleic acid extracts from 100 mg of symptomatic leaves (Trizol reagent; Gibco BRL Life Technologies, England) were used as template in RT-PCR with genus-specific potyvirus primers POT1-POT2, which targeted the variable 5´ terminal half of the coat protein gene of potyviruses (Colinet et al. 1998). The expected 900 bp amplicon was consistently detected in 10 symptomatic A. attenuata plants whereas no PCR products were obtained from 15 asymptomatic A. attenuata plants collected from the "Agaves de México" section at the 'Botanic Garden' of the Instituto de Biología de la UNAM, México. The amplicons were sequenced by the Sanger´s method and the obtained nucleotide (nt) sequences (Acc. No KY190217.1; OP964597-598) and their derived amino acid (aa) sequences were 94.68% to 95.80% similar to an isolate of Tuberose mild mosaic virus (TuMMV; Potyvirus; (Acc. No ON116187.1) characterized from Agave amica in India (Raj et al. 2009). Interestingly, A. amica (formerly Poliantes tuberose) is also a wild Mexican plant that is geographically distributed in the central and south regions of Mexico and is currently being commercially cultivated as an ornamental plant. Plants of A. amica (n=10) showing yellow mild streak were collected from commercial greenhouse and tested positive for TuMMV by RT-PCR and Sanger sequencing (No Acc. OP964599-601 levels) described above. The derived TuMMV sequences from A. attenuata and A. amica were 99-100% similar to each other at the nt/aa level. To exclude the involvement of additional viral agents in the disease, high-throughput sequencing analysis was performed separately for each species of Agave on total RNA extracts from a composite sample of symptomatic leaf tissues using Illumina´s Next Seq 500 platform. Analysis of the obtained 13,260,700 reads (each 75 nt) by the Trinity software, with a total number of sequences of 22,793, resulted in the identification of a single viral contig of 9500 nt for A. attenuata (Acc. No OP964595). Similarly, for A. amica, 27,262,248 reads were obtained, with a total number of sequences of 23,269, resulting in the identification of a single viral contig of 8500 nt (ACC. No OP964602). These contigs showed an identity percentage of 96%/88% and 98%/96% for nucleotides and amino acids, respectively, compared to an isolate of TuMMV from India (Acc. OM293939). Mexico is a center of origin for numerous species of genus Agave which have high economic, social, and ecological impact. TuMMV could be a threat to these plants and potentially to other unknown susceptible crops. To our knowledge, this is the first report of TuMMV in A. attenuata and A. amica in Mexico. REFERENCE Chen, C. C., and Chang, C. A. 1998. Characterization of a potyvirus causing mild mosaic on tuberose. Plant Dis. 82:45-49. Colinet, D., Nguyen, M., Kummert, J., Lepoivre, P., and Xia, F. Z. 1998. Differentiation among potyviruses infecting sweet potato based on genus- and virus-specific reverse transcription polymerase chain reaction. Plant Dis. 82:223-229. Raj, S.K., Snehi, S.K., Kumar, S., Ram, T. and Goel, A.K. 2009. First report of Tuberose mild mosaic potyvirus from tuberose (Polianthes tuberosa L.) in India. Australasian Plant Dis. Notes 4, 93-95.

Orchid fleck dichorhavirus movement protein shows RNA silencing suppressor activity.

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.

An Evolved 5' Untranslated Region of Alfalfa Mosaic Virus Allows the RNA Transport of Movement-Defective Variants.

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.

Spontaneous Mutation in the Movement Protein of Citrus Leprosis Virus C2, in a Heterologous Virus Infection Context, Increases Cell-to-Cell Transport and Generates Fitness Advantage.

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.

Regulatory circuits involving bud dormancy factor PpeDAM6.

DORMANCY-ASSOCIATED MADS-BOX (DAM) genes have recently emerged as key potential regulators of the dormancy cycle and climate adaptation in perennial species. Particularly, PpeDAM6 has been proposed to act as a major repressor of bud dormancy release and bud break in peach (Prunus persica). PpeDAM6 expression is downregulated concomitantly with the perception of a given genotype-dependent accumulation of winter chilling time, and the coincident enrichment in H3K27me3 chromatin modification at a specific genomic region. We have identified three peach BASIC PENTACYSTEINE PROTEINs (PpeBPCs) interacting with two GA-repeat motifs present in this H3K27me3-enriched region. Moreover, PpeBPC1 represses PpeDAM6 promoter activity by transient expression experiments. On the other hand, the heterologous overexpression of PpeDAM6 in European plum (Prunus domestica) alters plant vegetative growth, resulting in dwarf plants tending toward shoot meristem collapse. These alterations in vegetative growth of transgenic lines associate with impaired hormone homeostasis due to the modulation of genes involved in jasmonic acid, cytokinin, abscisic acid, and gibberellin pathways, and the downregulation of shoot meristem factors, specifically in transgenic leaf and apical tissues. The expression of many of these genes is also modified in flower buds of peach concomitantly with PpeDAM6 downregulation, which suggests a role of hormone homeostasis mechanisms in PpeDAM6-dependent maintenance of floral bud dormancy and growth repression.

The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism.

Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.

Membrane Association and Topology of Citrus Leprosis Virus C2 Movement and Capsid Proteins.

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.

Unravelling the involvement of cilevirus p32 protein in the viral transport.

Citrus leprosis (CL) is a severe disease that affects citrus orchards mainly in Latin America. It is caused by Brevipalpus-transmitted viruses from genera Cilevirus and Dichorhavirus. Currently, no reports have explored the movement machinery for the cilevirus. Here, we have performed a detailed functional study of the p32 movement protein (MP) of two cileviruses. Citrus leprosis-associated viruses are not able to move systemically in neither their natural nor experimental host plants. However, here we show that cilevirus MPs are able to allow the cell-to-cell and long-distance transport of movement-defective alfalfa mosaic virus (AMV). Several features related with the viral transport were explored, including: (i) the ability of cilevirus MPs to facilitate virus movement on a nucleocapsid assembly independent-manner; (ii) the generation of tubular structures from transient expression in protoplast; (iii) the capability of the N- and C- terminus of MP to interact with the cognate capsid protein (p29) and; (iv) the role of the C-terminus of p32 in the cell-to-cell and long-distance transport, tubule formation and the MP-plasmodesmata co-localization. The MP was able to direct the p29 to the plasmodesmata, whereby the C-terminus of MP is independently responsible to recruit the p29 to the cell periphery. Furthermore, we report that MP possess the capacity to enter the nucleolus and to bind to a major nucleolar protein, the fibrillarin. Based on our findings, we provide a model for the role of the p32 in the intra- and intercellular viral spread.

Dichorhaviruses Movement Protein and Nucleoprotein Form a Protein Complex That May Be Required for Virus Spread and Interacts in vivo With Viral Movement-Related Cilevirus Proteins.

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.

Citrus Leprosis Virus C Encodes Three Proteins With Gene Silencing Suppression Activity.

Citrus leprosis virus C (CiLV-C) belongs to the genus Cilevirus, family Kitaviridae, 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 the trans-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 family Kitaviridae.

MBW complexes impinge on anthocyanidin reductase gene regulation for proanthocyanidin biosynthesis in persimmon fruit.

MBW protein complexes containing MYB, bHLH and WD40 repeat factors are known transcriptional regulators of secondary metabolites production such as proanthocyanidins and anthocyanins, and developmental processes such as trichome formation in many plant species. DkMYB2 and DkMYB4 (MYB-type), DkMYC1 (bHLH-type) and DkWDR1 (WD40-type) factors have been proposed by different authors to take part of persimmon MBW complexes for proanthocyanidin accumulation in immature fruit, leading to its characteristic astringent flavour with important agronomical and ecological effects. We have confirmed the nuclear localization of these proteins and their mutual physical interaction by bimolecular fluorescence complementation analysis. In addition, transient expression of DkMYB2, DkMYB4 and DkMYC1 cooperatively increase the expression of a persimmon anthocyanidin reductase gene (ANR), involved in the biosynthesis of cis-flavan-3-ols, the structural units of proanthocyanidin compounds. Collectively, these data support the presence of MBW complexes in persimmon fruit and suggest their coordinated participation in ANR regulation for proanthocyanidin production.

Identification and genomic characterization of a novel tobamovirus from prickly pear cactus.

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.

A sensitive and rapid RNA silencing suppressor activity assay based on alfalfa mosaic virus expression vector.

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.

Key checkpoints in the movement of plant viruses through the host.

Plant viruses cannot exploit any of the membrane fusion-based routes of entry described for animal viruses. In addition, one of the distinctive structures of plant cells, the cell wall, acts as the first barrier against the invasion of pathogens. To overcome the rigidity of the cell wall, plant viruses normally take advantage of the way of life of different biological vectors. Alternatively, the physical damage caused by environmental stresses can facilitate virus entry. Once inside the cell and taking advantage of the characteristic symplastic continuity of plant cells, viruses need to remodel and/or modify the restricted pore size of the plasmodesmata (channels that connect plant cells). In a successful interaction for the virus, it can reach the vascular tissue to systematically invade the plant. The connections between the different cell types in this path are not designed to allow the passage of molecules with the complexity of viruses. During this process, viruses face different cell barriers that must be overcome to reach the distal parts of the plant. In this review, we highlight the current knowledge about how plant RNA viruses enter plant cells, move between them to reach vascular cells and overcome the different physical and cellular barriers that the phloem imposes. Finally, we update the current research on cellular organelles as key regulator checkpoints in the long-distance movement of plant viruses.

Molecular characterization of a new trichovirus from peach in Mexico.

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.

Exploring the role of cellular homologous of the 30K-superfamily of plant virus movement proteins.

Genes orthologous to the 30K-superfamily of movement proteins (MP) from plant viruses have been recently discovered by bioinformatics analyses as integrated elements in the genome of most vascular plants. However, their functional relevance for plants is still unclear. Here, we undertake some preliminary steps into the functional characterization of one of these putative MP genes found in Arabidopsis thaliana. We found that the AtMP gene is expressed at different stages of the plant development, with accumulation being highest in flowers but lowest in mature siliques. We also found down-regulation of the gene may result in a small delay in plant development and in an exacerbation of the negative effect of salinity in germination efficiency. We have also explored whether changes in expression of the endogenous AtMP have any effect on susceptibility to infection with several viruses, and found that the infectivity of tobacco rattle tobravirus was strongly dependent on the expression of the endogenous AtMP. Finally, we have cloned the endogenous MP from four different plant species into an expression vector that allows for specifically assessing their activity as cell-to-cell movement proteins and have shown that though some may still retain the ancestral activity, they do so in a quite inefficient manner, thus suggesting they have acquired a novel function during adaptation to the host genome.

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

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.

Polyvalent Detection of Members of the Genus Potyvirus by Molecular Hybridization Using a Genus-Probe.

The use of a unique riboprobe named polyprobe, carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the polyvalent detection of up to 10 different pathogens by using a nonradioactive molecular hybridization procedure. In the present analysis, we have developed a unique polyprobe with the capacity to detect all members of the genus Potyvirus, which we have named genus-probe. To do this, we have exploited the capacity of the molecular hybridization assay to cross-hybridize with related sequences by reducing the hybridization temperature. We observed that sequences showing a percentage similarity of 68% or higher could be detected with the same probe by hybridizing at 50 to 55°C, with a detection limit of picograms of viral RNA comparable to the specific individual probes. According to this, we developed several polyvalent polyprobes, containing three, five, or seven different 500-nucleotide fragments of a conserved region of the NIb gene. The polyprobe carrying seven different conserved regions was able to detect all the 32 potyviruses assayed in the present work with no signal in the healthy tissue, indicating the potential capacity of the polyprobe to detect all described, and probably uncharacterized, potyviruses being then considered as a genus-probe. The use of this technology in routine diagnosis not only for Potyvirus but also to other viral genera is discussed.