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It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungus Rosellinia necatrix could replicate in protoplasts of the carrot ( Daucus carota ), Nicotiana benthamiana and Nicotiana tabacum , in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster . Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution., Competing Interests: Competing interests statement:The authors declare no competing interest.

Disease resistance gene (R gene)-encoded nucleotide-binding leucine-rich repeat proteins (NLRs) are critical players in plant host defence mechanisms because of their role as receptors that recognise pathogen effectors and trigger plant effector-triggered immunity (ETI). This study aimed to determine the putative role of a cassava coiled-coil (CC)-NLR (CNL) gene MeRPPL1 ( Manes.12G091600 ) (single allele) located on chromosome 12 in the tolerance or susceptibility to South African cassava mosaic virus (SACMV), one of the causal agents of cassava mosaic disease (CMD). A transient protoplast system was used to knock down the expression of MeRPPL1 by clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9). The MeRPPL1 -targeting CRISPR vectors and/or SACMV DNA A and DNA B infectious clones were used to transfect protoplasts isolated from leaf mesophyll cells from the SACMV-tolerant cassava ( Manihot esculenta ) cultivar TME3. The CRISPR/Cas9 silencing vector significantly reduced MeRPPL1 expression in protoplasts whether with or without SACMV co-infection. Notably, SACMV DNA A replication was higher in protoplasts with lower MeRPPL1 expression levels than in non-silenced protoplasts. Mutagenesis studies revealed that protoplast co-transfection with CRISPR- MeRPPL1 silencing vector + SACMV and transfection with only SACMV induced nucleotide substitution mutations that led to altered amino acids in the highly conserved MHD motif of the MeRPPL1 -translated polypeptide. This may abolish or alter the regulatory role of the MHD motif in controlling R protein activity and could contribute to the increase in SACMV-DNA A accumulation observed in MeRPPL1 -silenced protoplasts. The results herein demonstrate for the first time a role for a CNL gene in tolerance to a geminivirus in TME3.

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.

Background: The study of transient gene expression in cassava plants during virus infection using existing protocols is laborious and may take approximately fifteen weeks due to cassava's recalcitrance to transformation. The combination of a protoplast system with CRISPR-mediated gene editing promises to shorten the turnaround time from plant tissue culture to high-throughput gene expression screening for candidate genes. Here, we detail a protocol for screening genes associated with the response to South African cassava mosaic virus (SACMV) in cassava protoplasts, with reference to the ubiquitin E3 ligase gene, MeE3L., Methods: Cassava protoplasts of model, and SACMV-susceptible and -tolerant genotypes, were transformed with SACMV infectious clones and/or a CRISPR-editing construct targeting the MeE3L using PEG4000-mediated transfection. DNA and RNA were extracted from transformed protoplasts at 24 h post-transfection. Relative SACMV DNA accumulation was determined via qPCR using DpnI-digested total DNA, MeE3L relative expression was determined via reverse transcriptase qPCR, and results were analysed using one-way ANOVA, Tukey's HSD test and the 2 -ΔΔCT statistical method. The MeE3L exonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations using MAFTT and MEGA-X software. Construction of a phylogenetic tree was done using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model., Results: The differential expression of unedited and mutant MeE3L during SACMV infection of model, susceptible and tolerant cassava protoplasts was determined within 7 weeks after commencement of tissue culture. The study also revealed that SACMV DNA accumulation in cassava protoplasts is genotype-dependent and induces multiple mutations in the tolerant landrace MeE3L homolog. Notably, the susceptible cassava landrace encodes a RINGless MeE3Lwhich is silenced by SACMV-induced mutations. SACMV also induces mutations which silence the MeE3L RING domain in protoplasts from and tolerant cassava landraces., Conclusions: This protocol presented here halves the turnaround time for high-throughput screening of genes associated with the host response to SACMV. It provides evidence that a cassava E3 ligase is associated with the response to SACMV and forms a basis for validation of these findings by in planta functional and interaction studies.

The Plasma membrane Cation binding Protein 1 (PCaP1) has been shown to be important for the intra-cellular movement of two members of the Potyvirus genus in arabidopsis and tobacco plants. In this study, the orthologous PCaP1 gene of pepper (Capsicum annuum) was examined for its role in the accumulation of Potato virus Y, type member of the Potyvirus. Downregulation of C. annuum PCaP (CaPCaP) through tobacco rattle virus-induced gene silencing, resulted in lower accumulation of potato virus Y (PVY) in pepper plants. Using an improved pepper protoplast isolation protocol, we showed that knockdown of CaPCaP negatively affected PVY accumulation at the within-cell level in pepper in contrast with the turnip mosaic virus-arabidopsis pathosystem. Conversely, following overexpression of CaPCaP, the accumulation of PVY at the systemic level was increased. The results provide further knowledge on the role of PCaP in the potyvirus infection process and reveal differences of its action among different pathosystems., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ningnanmycin (NNM) belongs to microbial pesticides that display comprehensive antiviral activity against plant viruses. NNM treatment has been shown to efficiently delay or suppress the disease symptoms caused by tobacco mosaic virus (TMV) infection in local-inoculated or systemic-uninoculated tobacco leaves, respectively. However, the underlying molecular mechanism of NNM-mediated antiviral activity remains to be further elucidated. In this study, 414 differentially expressed genes (DEGs), including 383 which were up-regulated and 31 down-regulated, caused by NNM treatment in TMV-infected BY-2 protoplasts, were discovered by RNA-seq. In addition, KEGG analysis indicated significant enrichment of DEGs in the plant-pathogen interaction and MAPK signaling pathway. The up-regulated expression of crucial DEGs, including defense-responsive genes, such as the receptor-like kinase FLS2 , RLK1, and the mitogen-activated protein kinase kinase kinase MAPKKK , calcium signaling genes, such as the calcium-binding protein CML19 , as well as phytohormone responsive genes, such as the WRKY transcription factors WRKY40 and WRKY70 , were confirmed by RT-qPCR. These findings provided valuable insights into the antiviral mechanisms of NNM, which indicated that the agent induces tobacco systemic resistance against TMV via activating multiple plant defense signaling pathways.

Cytosinpeptidemycin (CytPM) is a microbial pesticide that displayed broad-spectrum antiviral activity against various plant viruses. However, the molecular mechanism underlying antiviral activity of CytPM is poorly understood. In this study, the results demonstrated that CytPM could effectively delay the systemic infection of tobacco mosaic virus (TMV) in Nicotiana benthamiana and significantly inhibit the viral accumulation in tobacco BY-2 protoplasts. Results of RNA-seq indicated that 210 and 120 differential expressed genes (DEGs) were significantly up- and down-regulated after CytPM treatment in BY-2 protoplasts, respectively. In addition, KEGG analysis indicated that various DEGs were involved in endoplasmic reticulum (ER) protein processing, suggesting a possible correlation between ER homeostasis and virus resistance. RT-qPCR was performed to validate the gene expression of crucial DEGs related with defense, stress responses, signaling transduction, and phytohormone, which were consistent with results of RNA-seq. Our works provided valuable insights into the antiviral mechanism of CytPM that induced host resistance to viral infection.

Carnation ringspot virus (CRSV) is the prototype virus of the genus Dianthovirus. Full-length cDNAs of CRSV strainsPV-0097 and PV-21 were constructed and the infectivity of in vitro transcripts was analyzed. Infectivity of PV-0097 and PV-21 to several plants was markedly higher than that of 1.30, a previously reported infectious CRSV clone. Overall RNA sequences of these viruses were similar, but PV-0097 and PV-21 contained additional nucleotides at the 5' end of RNA1. Stem-loop structures were predicted in the 5'-terminal region of PV-0097 and PV-21 RNA1 but not in 1.30 RNA1. Mutant CRSV 1.30 RNA1 that contains the terminal 4 nucleotides of PV-0097, predicted to fold a 5'-terminal stem-loop structure, recovered higher level accumulation of viral RNAs in the inoculated protoplasts and leaves of Nicotiana benthamiana. These results suggest that the 5'-terminal stem-loop structure of CRSV RNA1 plays an important role in efficient amplification of the virus., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Plant-viroid interactions represent a valuable model for delineating structure-function relationships of noncoding RNAs. For various functional studies, it is desirable to minimize sample variations by using DNA, RNA, and proteins co-purified from the same samples. Currently, most of the co-purification protocols rely on TRI Reagent (Trizol as a common representative) and require protein precipitation and dissolving steps, which render difficulties in experimental handling and high-throughput analyses. Here, we established a simple and robust method to minimize the precipitation steps and yield ready-to-use RNA and protein in solutions. This method can be applied to samples in small quantities, such as protoplasts. Given the ease and the robustness of this new method, it will have broad applications in virology and other disciplines in molecular biology.

Infectious cDNA clones were developed for Grapevine leafroll-associated virus 3 (GLRaV-3, genus Ampelovirus, family Closteroviridae). In vitro RNA transcripts generated from cDNA clones showed replication via the production of 3'-coterminal subgenomic (sg) mRNAs in Nicotiana benthamiana protoplasts. The detection of sgRNAs and the recovery of progeny recombinant virions from N. benthamiana leaves agroinfiltrated with full-length cDNA clones confirmed RNA replication and virion formation. The 5' non-translated region (5' NTR) of GLRaV-3 was exchangeable between genetic variants and complement the corresponding cognate RNA functions in trans. Mutational analysis of the 5' NTR in minireplicon cDNA clones showed that the conserved 40 nucleotides at the 5'-terminus were indispensable for replication, compared to downstream variable portion of the 5' NTR. Some of the functional mutations in the 5' NTR were tolerated in full-length cDNA clones and produced sgRNAs and virions in N. benthamiana leaves, whereas other mutations affected replication and virion formation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A predicted stem-loop structure of 25 nucleotides, located in the coat protein (CP) gene and 3'-UTR sequences of Tobacco mosaic virus (TMV), was validated previously (Guo et al., 2015). In this study, both disrupted stem-loop and nucleotide deletion mutants of TMV replicated more rapidly in Nicotiana benthamiana protoplasts. The TMV mutant with a complete mirrored stem-loop structure showed similar level of viral RNA accumulation as TMV. Recovering the stem-loop structure also resulted in a similar replication level as TMV. All these mutants induced necrosis in N. benthamiana and assembled into typical rigid rod-shaped virions. TMV mutant without the stem-loop structure induced more local lesions in Chenopodium quinoa. When the putative stem-loop structure in Tomato mosaic virus (ToMV) was disrupted, the mutant also showed an enhanced virus replication. This suggests that the stem-loop structure of TMV is a new cis-acting element with a role in virus replication., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This chapter reviews the discoveries and initial characterizations (1930-1990) of three plant rhabdoviruses, sonchus yellow net virus, potato yellow dwarf virus, and lettuce necrotic yellows virus, that have become model systems for research on this group of enveloped negative-strand RNA plant viruses. We have used our personal perspectives to review the early historical studies of these viruses, the important technologies and tools, such as density gradient centrifugation, that were developed during the research, and to highlight the eminent scientists involved in these discoveries. Early studies on sites of virus replication, virion structure, physicochemical composition, and the use of protoplasts and vector insect cell culture for virus research are discussed, and differences between the nuclear and cytoplasmic lifestyles of plant rhabdoviruses are contrasted. Finally, we briefly summarize the genome organization and more recent developments culminating in the development of a reverse genetics system for plant negative-strand RNA viruses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The transmission of viral infections between plant and fungal hosts has been suspected to occur, based on phylogenetic and other findings, but has not been directly observed in nature. Here, we report the discovery of a natural infection of the phytopathogenic fungus Rhizoctonia solani by a plant virus, cucumber mosaic virus (CMV). The CMV-infected R. solani strain was obtained from a potato plant growing in Inner Mongolia Province of China, and CMV infection was stable when this fungal strain was cultured in the laboratory. CMV was horizontally transmitted through hyphal anastomosis but not vertically through basidiospores. By inoculation via protoplast transfection with virions, a reference isolate of CMV replicated in R. solani and another phytopathogenic fungus, suggesting that some fungi can serve as alternative hosts to CMV. Importantly, in fungal inoculation experiments under laboratory conditions, R. solani could acquire CMV from an infected plant, as well as transmit the virus to an uninfected plant. This study presents evidence of the transfer of a virus between plant and fungus, and it further expands our understanding of plant-fungus interactions and the spread of plant viruses., Competing Interests: The authors declare no conflict of interest.

Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are heterogeneous and highly dynamic nanoscale structures usurped by various viruses. Here, we demonstrate that TBSV p33 and p92 replication proteins can bind to sterol in vitro Mutagenesis analysis of p33 within the CRAC and CARC sequences involved in sterol binding shows the important connection between the abilities of p33 to bind to sterol and to support TBSV replication in yeast and plant cells. Together, the results further strengthen the model that cellular sterols are essential as proviral lipids during viral replication., (Copyright © 2017 American Society for Microbiology.)

Actinobacteria are extremely important to human health, agriculture, and forests. Because of the vast differences of the characteristics of Actinobacteria, a lot of genetic tools have been developed for efficiently manipulating the genetics. Although there are a lot of successful examples of engineering Actinobacteria, they are still more difficult to be genetically manipulated than other model microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis etc. due to the diverse genomics and biochemical machinery. Here, we review the methods to introduce heterologous DNA into Actinobacteria and the available genetic modification tools. The trends and problems existing in engineering Actinobacteria are also covered.

Mal de Río Cuarto virus (MRCV) is a member of the Fijivirus genus, within the Reoviridae family, that replicates and assembles in cytoplasmic inclusion bodies called viroplasms. In this study, we investigated interactions between ten MRCV proteins by yeast two-hybrid (Y2H) assays and identified interactions of non-structural proteins P6/P6, P9-2/P9-2 and P6/P9-1. P9-1 and P6 are the major and minor components of the viroplasms respectively, whereas P9-2 is an N-glycosylated membrane protein of unknown function. Interactions involving P6 and P9-1 were confirmed by bimolecular fluorescence complementation (BiFC) in rice protoplasts. We demonstrated that a region including a predicted coiled-coil domain within the C-terminal moiety of P6 was necessary for P6/P6 and P6/P9-1 interactions. In turn, a short C-terminal arm was necessary for the previously reported P9-1 self-interaction. Transient expression of these proteins by agroinfiltration of Nicotiana benthamiana leaves showed very low accumulation levels and further in silico analyses allowed us to identify conserved PEST degradation sequences [rich in proline (P), glutamic acid (E), serine (S), and threonine (T)] within P6 and P9-1. The removal of these PEST sequences resulted in a significant increase of the accumulation of both proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 (NS M ) 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 NS M 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 NS M 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 NS M , but not that of the CSNV, TCSV and TSWV NS M proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NS M mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NS M 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The terminal ends in the genome of RNA viruses contain features that regulate viral replication and/or translation. We have identified a Y-shaped structure (YSS) in the 3' terminal regions of the bipartite genome of Lettuce chlorosis virus (LCV), a member in the genus Crinivirus (family Closteroviridae). The YSS is the first in this family of viruses to be determined using Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE). Using luciferase constructs/replicons, in vivo and in vitro assays showed that the 5' and YSS-containing 3' terminal regions of LCV RNA1 supported translation activity. In contrast, similar regions from LCV RNA2, including those upstream of the YSS, did not. LCV RNA2 mutants with nucleotide deletions or replacements that affected the YSS were replication deficient. In addition, the YSS of LCV RNA1 and RNA2 were interchangeable without affecting viral RNA synthesis. Translation and significant replication were observed for specific LCV RNA2 replicons only in the presence of LCV RNA1, but both processes were impaired when the YSS and/or its upstream region were incomplete or altered. These results are evidence that the YSS is essential to the viral replication machinery, and contributes to replication enhancement and replication-associated translation activity in the RNA2 replicons.

Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5'- and 3'-UTRs plus the 3'-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants.

Cowpea mosaic virus forms tubules constructed from the movement protein (MP) in plasmodesmata (PD) to achieve cell-to-cell movement of its virions. Similar tubules, delineated by the plasma membrane (PM), are formed protruding from the surface of infected protoplasts. These PM-tubule complexes were isolated from protoplasts by immunoprecipitation and analysed for their protein content by tandem mass spectrometry to identify host proteins with affinity for the movement tubule. Seven host proteins were abundantly present in the PM-tubule complex, including molecular chaperonins and an AAA protein. Members of both protein families have been implicated in establishment of systemic infection. The potential role of these proteins in tubule-guided cell-cell transport is discussed.

The upstream pseudoknots domain (UPD) of Tobacco mosaic virus (TMV) is located at the 3'-untranslated region (UTR). It plays an important role in virus replication and translation. To determine the importance of UPD and 3'-UTR, and the effects of introduced RNA elements in TMV 3'-UTR, a series of TMV mutants with internal poly(A) tract upstream of UPD was constructed for structural analysis by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). TMV(24A+UPD) and TMV(42A+UPD) formed a similar structure as that of TMV 3'-UTR, but TMV(62A+UPD) structures altered by the introduced poly(A) tract. In addition, TMV(24A+UPD) had a higher viral RNAs accumulation than TMV in N. benthamiana protoplasts, and induced lethal symptoms in the infected plants. TMV(62A+UPD) showed a drastically reduced accumulation, its coat protein was undetectable in protoplasts, and the inoculated plants remained symptomless. This study analyzed the structures of 3'-UTR of TMV and found that the longer poly(A) tract introduced upstream of UPD reduced viral RNAs accumulation and induced milder symptoms in N. benthamiana. In conclusion, different lengths of the internal poly(A) tract introduced into the TMV 3'UTR lead to structural variations that affect virus accumulation and symptom expression.

Citrus tristeza virus (CTV), one of the most economically important viruses, produces a unique protein, p33, which is encoded only in the genomes of isolates of CTV. Recently, we demonstrated that membrane association of the p33 protein confers virus ability to extend its host range. In this work we show that p33 shares characteristics of viral movement proteins. Upon expression in a host cell, the protein localizes to plasmodesmata and displays the ability to form extracellular tubules. Furthermore, p33 appears to traffic via the cellular secretory pathway and the actin network to plasmodesmata locations and is likely being recycled through the endocytic pathway. Finally, our study reveals that p33 colocalizes with a putative movement protein of CTV, the p6 protein. These results suggest a potential role of p33 as a noncanonical viral movement protein, which mediates virus translocation in the specific hosts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Plant infection by poleroviruses is restricted to phloem tissues, preventing any classical leaf rub inoculation with viral RNA or virions. Efficient virus inoculation to plants is achieved by viruliferous aphids that acquire the virus by feeding on infected plants. The use of promoter-driven infectious cDNA is an alternative means to infect plants and allows reverse genetic studies to be performed. Using Beet mild yellowing virus isolate 2ITB (BMYV-2ITB), we produced a full-length infectious cDNA clone of the virus (named BMYV-EK) placed under the control of the T7 RNA polymerase and the Cauliflower mosaic virus 35S promoters. Infectivity of the engineered BMYV-EK virus was assayed in different plant species and compared with that of the original virus. We showed that in vitro- or in planta-derived transcripts were infectious in protoplasts and in whole plants. Importantly, the natural aphid vector Myzus persicae efficiently transmitted the viral progeny produced in infected plants. By comparing agroinoculation and aphid infection in a host range assay, we showed that the engineered BMYV-EK virus displayed a similar host range to BMYV-2ITB, except for Nicotiana benthamiana, which proved to be resistant to systemic infection with BMYV-EK. Finally, both the BMYV-EK P0 and the full-length clone were able to strongly interfere with post-transcriptional gene silencing., (© 2013 BSPP AND JOHN WILEY & SONS LTD.)

Cauliflower mosaic virus (CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.

One striking feature of viruses with RNA genomes is the modification of the host membrane structure during early infection. This process requires both virus- and host-encoded proteins; however, the host factors involved and their role in this process remain largely unknown. On infection with Tobacco mosaic virus (TMV), a positive-strand RNA virus, the filamentous and tubular endoplasmic reticulum (ER) converts to aggregations at the early stage and returns to filamentous at the late infectious stage, termed the ER transition. Also, membrane- or vesicle-packaged viral replication complexes (VRCs) are induced early during infection. We used microarray assays to screen the Arabidopsis thaliana gene(s) responding to infection with TMV in the initial infection stage and identified an Arabidopsis gene, PAP85 (annotated as a vicilin-like seed storage protein), with upregulated expression during 0.5 to 6 h of TMV infection. TMV accumulation was reduced in pap85-RNA interference (RNAi) Arabidopsis and restored to wild-type levels when PAP85 was overexpressed in pap85-RNAi Arabidopsis. We did not observe the ER transition in TMV-infected PAP85-knockdown Arabidopsis protoplasts. In addition, TMV accumulation was reduced in PAP85-knockdown protoplasts. VRC accumulation was reduced, but not significantly (P = 0.06), in PAP85-knockdown protoplasts. Coexpression of PAP85 and the TMV main replicase (P126), but not their expression alone in Arabidopsis protoplasts, could induce ER aggregations.

Different regulatory elements function are involved in plant virus gene expression and replication by long-distance RNA-RNA interactions. A cap-independent functional element of the Barley yellow dwarf virus (BYDV) - like translational enhancer (BTE) is present in Tobacco necrosis virus A (TNV-A), a Necrovirus member in the Tombusviridae family. In this paper, an RNA stretch flanking the 5' proximal end of the TNV-A(C) coat protein (CP) gene was shown to be essential for viral replication in Chenopodium amaranticolor plants and tobacco cells. This internal sequence functioned in transient expression of β-glucuronidase (GUS) when present at either the 5' or 3' sides of the GUS open reading frame. Serial deletion analyses revealed that nine nucleotides from nt 2609 to 2617 (-3 to +6 of the CP initiation site) within TNV-A(C) RNA are indispensable for viral replication in whole plants and tobacco cells. Fusion of this RNA element in mRNAs translated in tobacco cells resulted in a remarkable enhancement of luciferase expression from in vitro synthesised chimaeric RNAs or DNA expression vectors. Interestingly, the element also exhibited increased translational activity when fused downstream of the reporter genes, although the efficiency was lower than with upstream fusions. These results provide evidence that an internal RNA element in the genomic (g) RNA of TNV-A(C), ranging approximately from nt 2543 to 2617, plays a bifunctional role in viral replication and translation enhancement during infection, and that this element may use novel strategies differing from those previously reported for other viruses.

The expression of host genes can be altered during the process of viral infection. To investigate the viral infection-induced up-regulated gene expression changes of maize at different time intervals post-inoculation with Sugarcane mosaic virus (SCMV), a suppression subtractive hybridization cDNA library was constructed. A total of 454 cDNA clones were identified to be viral infection-induced up-regulated genes. The influence of Rop1 on the infection of maize by SCMV was investigated. The results showed that transient silencing of the ZmRop1 gene through virus-induced gene silencing enhanced the accumulation and systemic infection of SCMV and another potyvirus (Pennisetum mosaic virus) in maize plants, whereas transient over-expression of ZmRop1 in maize protoplasts reduced SCMV accumulation. Furthermore, it was demonstrated that the heterologous expression of ZmRop1 impaired Potato virus X infection in Nicotiana benthamiana plants. These data suggest that ZmRop1 may play a role in plant defence responses to viral infection., (© 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.)

Satellite RNAs associated with Bamboo mosaic virus (satBaMVs) depend on BaMV for replication and encapsidation. Certain satBaMVs isolated from natural fields significantly interfere with BaMV replication. The 5' apical hairpin stem loop (AHSL) of satBaMV is the major determinant in interference with BaMV replication. In this study, by in vivo competition assay, we revealed that the sequence and structure of AHSL, along with specific nucleotides (C(60) and C(83)) required for interference with BaMV replication, are also involved in replication competition among satBaMV variants. Moreover, all of the 5' ends of natural BaMV isolates contain the similar AHSLs having conserved nucleotides (C(64) and C(86)) with those of interfering satBaMVs, suggesting their co-evolution. Mutational analyses revealed that C(86) was essential for BaMV replication, and that replacement of C(64) with U reduced replication efficiency. The non-interfering satBaMV interfered with BaMV replication with the BaMV-C64U mutant as helper. These findings suggest that two cytosines at the equivalent positions in the AHSLs of BaMV and satBaMV play a crucial role in replication competence. The downregulation level, which is dependent upon the molar ratio of interfering satBaMV to BaMV, implies that there is competition for limited replication machinery.

Potyviruses are a persistent threat to bell pepper (Capsicum annuum L.) production worldwide. Much effort has been expended to study the resistance response of pepper cultivars at whole plant levels but with only limited effort at the cellular level using protoplasts. A pepper protoplast isolation procedure is available but an inoculation procedure is needed that provides consistent and highly efficient infection. An electroporation-based procedure for inoculation of potyviruses was developed using a base procedure developed for Cucumber mosaic virus (CMV). The final parameters identified for efficient potyvirus infection of pepper protoplasts involves two 25ms pulses, 200V each pulse with a 10s interval between pulses. Depending on the method of detection, e.g., ELISA versus RT-PCR, potyvirus RNA inoculum ranged from 10 to 40μg with infection detection occurring with samples of 50,000-100,000 protoplasts., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Packaging of the segmented RNA genome of Brome mosaic virus (BMV) into discrete particles is an essential step in the virus life cycle; however, questions remain regarding the mechanism of RNA packaging and the degree to which the viral coat protein controls the process. In this study, we used a plant-derived glycosidase, Pokeweed antiviral protein, to remove 14 specific bases from BMV RNA3 to examine the effect of depurination on virus assembly. Depurination of A771 within ORF3 and A1006 in the intergenic region inhibited coat protein binding and prevented RNA3 incorporation into particles. The disruption of interaction was not based on sequence identity, as mutation of these two purines to pyrimidines did not decrease coat protein-binding affinity. Rather, we suggest that base removal results in decreased thermodynamic stability of local RNA structures required for packaging, and that this instability is detected by coat protein. These results describe a new level of discrimination by coat protein, whereby it recognizes damage to specific viral RNA elements in the form of base removal and selects against incorporating the RNA into particles.

Many plant host factors are known to interact with viral proteins during pathogenesis, but how a plant virus induces a specific disease symptom still needs further research. A lily strain of Cucumber mosaic virus (CMV-HL) can induce discrete necrotic spots on infected Arabidopsis (Arabidopsis thaliana) plants; other CMV strains can induce similar spots, but they are not as distinct as those induced by CMV-HL. The CMV 2b protein (2b), a known RNA-silencing suppressor, is involved in viral movement and symptom induction. Using in situ proximity ligation assay immunostaining and the protoplast assays, we report here that CMV 2b interacts directly with Catalase3 (CAT3) in infected tissues, a key enzyme in the breakdown of toxic hydrogen peroxide. Interestingly, CAT3, normally localized in the cytoplasm (glyoxysome), was recruited to the nucleus by an interaction between 2b and CAT3. Although overexpression of CAT3 in transgenic plants decreased the accumulation of CMV and delayed viral symptom development to some extent, 2b seems to neutralize the cellular catalase contributing to the host defense response, thus favoring viral infection. Our results thus provide evidence that, in addition to altering the type of symptom by disturbing microRNA pathways, 2b can directly bind to a host factor that is important in scavenging cellular hydrogen peroxide and thus interfere specifically with that host factor, leading to the induction of a specific necrosis.

The tremendous evolutionary potential of RNA viruses allows them to thrive despite host defense mechanisms and endows them with properties such as emergence, host switching, and virulence. The frequency of mutant viruses after an infectious process results from the interplay between the error rate of the viral replicase, from purifying mechanisms acting after transcription on aberrant RNAs, and from the amplification dynamics of virus RNA positive (+) and negative (-) strands. Two extreme scenarios describing viral RNA amplification are the geometric growth, in which each RNA strand serves as template for the synthesis of complementary strands with the same efficiency, and the stamping machine, where a strand is reiteratively used as template to synthesize multiple copies of the complementary. The resulting mutation frequencies are completely different, being geometric growth largely more mutagenic than stamping machine. In this work we evaluate the contribution of geometric growth and stamping machine to the overall genome amplification of the plant (+)-strand RNA virus turnip mosaic virus (TuMV). By means of transfection experiments of Nicotiana benthamiana protoplasts with a TuMV cDNA infectious clone and by using strand-specific quantitative real-time PCR, we determined the amplification dynamics of viral (+) and (-) RNA during a single-cell infectious process. A mathematical model describing the amplification of each viral strand was fitted to the data. Analyses of the model parameters showed that TuMV (+) and (-) RNA amplification occurs through a mixed strategy with ∼93% of genomes produced via stamping machine and only ∼7% resulting from geometric growth.

Soybean 'Harosoy' is resistant to Cucumber mosaic virus soybean strain C (CMV-SC) and susceptible to CMV-S strain D (CMV-SD). Using enzyme-linked immunosorbent assay and Northern hybridization, we characterized the Harosoy resistance and found that CMV-SC did not spread systemically but was restricted to the inoculated leaves in Harosoy. Harosoy resistance was not controlled by either a dominant or recessive single gene. To dissect this system controlling long-distance movement of CMV in soybean, we constructed infectious cDNA clones of CMV-SC and CMV-SD. Using these constructs and the chimeric RNAs, we demonstrated that two viral components were required for systemic infection by the virus. The region including the entire 2b gene and the 5' region of RNA3 (mainly the 5' untranslated region) together were required. By quantitative trait locus (QTL) analysis using an F(2) population and the F(3) families derived from Harosoy and susceptible 'Nemashirazu', we also showed that at least three QTLs affected systemic infection of CMV in soybean. Our study on Harosoy resistance to CMV-SC revealed an interesting mechanism, in which multiple host and viral genes coordinately controlled viral systemic infection.

Nicotiana benthamiana has been described as non-host for Melon necrotic spot virus (MNSV). We investigated the basis of this resistance using the unique opportunity provided by strain MNSV-264, a recombinant virus that is able to overcome the resistance. Analysis of chimeric MNSV mutants showed that virulence in N. benthamiana is conferred by a 49 nucleotide section of the MNSV-264 3'-UTR, which acts in this host as a cap-independent translational enhancer (3'-CITE). Although the 3'-CITE of non-adapted MNSV-Mα5 is active in susceptible melon, it does not promote efficient translation in N. benthamiana, thus preventing expression of proteins required for virus replication. However, MNSV-Mα5 gains the ability to multiply in N. benthamiana cells if eIF4E from a susceptible melon variety (Cm-eIF4E-S) is supplied in trans. These data show that N. benthamiana resistance to MNSV-Mα5 results from incompatibility between the MNSV-Mα5 3'-CITE and N. benthamiana eIF4E in initiating efficient translation of the viral genome. Therefore, non-host resistance conferred by the inability of a host susceptibility factor to support viral multiplication may be a possible mechanism for this type of resistance to viruses., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Host-to-host transmission--a key step in plant virus infection cycles--is ensured predominantly by vectors, especially aphids and related insects. A deeper understanding of the mechanisms of virus acquisition, which is critical to vector-transmission, might help to design future virus control strategies, because any newly discovered molecular or cellular process is a potential target for hampering viral spread within host populations. With this aim in mind, an aphid membrane-feeding assay was developed where aphids transmitted two non-circulative viruses [cauliflower mosaic virus (CaMV) and turnip mosaic virus] from infected protoplasts. In this assay, virus acquisition occurs exclusively from living cells. Most interestingly, we also show that CaMV is less efficiently transmitted by aphids in the presence of oryzalin--a microtubule-depolymerising drug. The example presented here demonstrates that our technically simple "virus-acquisition phenotyping assay" (VAPA) provides a first opportunity to implement correlative studies relating the physiological state of infected plant cells to vector-transmission efficiency.

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-FnyΔ2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVY-SON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

Previous evidence has indicated that the P25 protein encoded by Potato virus X (PVX) inhibits either the assembly or function of the effector complexes in the RNA silencing-based antiviral defence system (Bayne et al., Cell-to-cell movement of Potato Potexvirus X is dependent on suppression of RNA silencing. Plant J.44, 471-482). This finding prompted us to investigate the possibility that P25 targets the Argonaute (AGO) effector nuclease of RNA silencing. Co-immunoprecipitation and Western blot analysis indicated that there is a strong interaction between P25 and AGO1 of Arabidopsis when these proteins are transiently co-expressed in Nicotiana benthamiana. P25 also interacts with AGO1, AGO2, AGO3 and AGO4, but not with AGO5 and AGO9. As an effective suppressor, the amount of AGO1 accumulated in the presence of P25 was dramatically lower than that infiltrated with HcPro, but was restored when treated with a proteasome inhibitor MG132. These findings are consistent with the idea that RNA silencing is an antiviral defence mechanism and that the counter-defence role of P25 is through the degradation of AGO proteins via the proteasome pathway. Further support for this idea is provided by the observation that plants treated with MG132 are less susceptible to PVX and its relative Bamboo mosaic virus.

The potential host range of mycoviruses is poorly understood because of the lack of suitable inoculation methods. Recently, successful transfection has been reported for somatically incompatible fungal isolates with purified virus particles of two mycoviruses, the partitivirus RnPV1-W8 (RnPV1) and the mycoreovirus RnMyRV3/W370 (MyRV3), from the white root rot fungus Rosellinia necatrix (class Sordariomycetes, subclass Xylariomycetidae). These studies examined and revealed the effect of the mycoviruses on growth and pathogenicity of R. necatrix. Here, we extended the experimental host range of these two mycoviruses using a transfection approach. Protoplasts of other phytopathogenic Sordariomycetous fungi-Diaporthe sp., Cryphonectria parasitica, Valsa ceratosperma (Sordariomycetidae), and Glomerella cingulata (Hypocreomycetidae)-were inoculated with RnPV1 and MyRV3 viral particles. The presence of double-stranded RNA viral genomes in regenerated mycelia of Diaporthe sp., C. parasitica, and V. ceratosperma confirmed both types of viral infections in these three novel host species. An established RnPV1 infection was confirmed in G. cingulata but MyRV3 did not infect this host. Horizontal transmission of both viruses from newly infected strains to virus-free, wild-type strains through hyphal anastomosis was readily achieved by dual culture; however, vertical transmission through conidia was rarely observed. The virulence of Diaporthe sp., C. parasitica, and V. ceratosperma strains harboring MyRV3 was reduced compared with their virus-free counterpart. In summary, our protoplast inoculation method extended the experimental host range of RnPV1-W8 and MyRV3 within the class Sordariomycetes and revealed that MyRV3 confers hypovirulence to the new hosts, as it does to R. necatrix.

Four dsRNA bands were extracted from Pleurotus ostreatus TD300 by the dsRNA isolation technique with sizes of 8.2 kb, 2.5 kb, 2.1 kb, and 1.1 kb, respectively. Four virus-eliminated methods, i. e. hyphal tips cut (HTC), protoplast regeneration (PR), single spore hybridization (SSH), and frozen and lyophilized (FL), were applied to prepare virus-eliminated strains, and one virus-eliminated strain was selected for each virus-elimination method. The virus-eliminated strains were named as HTC8, PR15, FL01, and SSH11, respectively. There were low concentration of 8.2 kb dsRNA remained in HTC8, as well as low concentration of 8.2 kb and 2.5 kb dsRNA remained in FL01. However, no dsRNA remained in PR15 and SSH11. The hyphal growth rate and laccase activity of the virus-eliminated strains increased, especially HTC8 and PR15, whose hyphal growth rate was higher by 22.73% and 18.18%, and laccase activities higher by 145.83% and 134.38% than that of the original strain, respectively. The conclusion is that hyphal tips cut and protoplast regeneration are suitable to prepare virus-eliminated strains of edible fungi.

A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro. To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro.

Though the duration of a single round of replication is an important biological parameter, it has been determined for only few viruses. Here, this parameter was determined for Cauliflower mosaic virus (CaMV) in transfected protoplasts from different hosts: the highly susceptible Arabidopsis and turnip, and Nicotiana benthamiana, where CaMV accumulates only slowly. Four methods of differing sensitivity were employed: labelling of (1) progeny DNA and (2) capsid protein, (3) immunocapture PCR,, and (4) progeny-specific PCR. The first progeny virus was detected about 21 h after transfection. This value was confirmed by all methods, indicating that our estimate was not biased by the sensitivity of the detection method, and approximated the actual time required for one round of CaMV replication. Unexpectedly, the replication kinetics were similar in the three hosts; suggesting that slow accumulation of CaMV in Nicotiana plants is determined by non-optimal interactions in other steps of the infection cycle.

Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation.

Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, is specifically transmitted by the sweet potato whitefly (Bemisia tabaci) in a semipersistent manner. LIYV infections result in a low virus titer in plants and protoplasts, impeding reverse genetic efforts to analyze LIYV gene/protein functions. We found that synergistic interactions occurred in mixed infections of LIYV and Turnip mosaic virus (TuMV) in Nicotiana benthamiana plants, and these resulted in enhanced accumulation of LIYV. Furthermore, we examined the ability of transgenic plants and protoplasts expressing only the TuMV P1/HC-Pro sequence to enhance the accumulation of LIYV. LIYV RNA and protein titers increased by as much as 8-fold in these plants and protoplasts relative to control plants. LIYV infections remained phloem-limited in P1/HC-Pro transgenic plants, suggesting that enhanced accumulation of LIYV in these plants was due primarily to increased replication efficiency, not to greater spread.

Plant virus diversity and the spatial distribution of viral strains or isolates are studied at many different scales: global, regional, local, and even within a single host in different organs or tissues. However, one level that has been totally lacking at the extremity of this scale is that of the single cell. The technical difficulties involved in isolating individual cells from infected plants, and the lack of an efficient diagnostic procedure allowing the specific detection of viral sequences with no major contamination from other cells, have precluded such single cell analysis to date. This paper describes the preparation of protoplasts from plants infected with Cauliflower mosaic virus (CaMV), and their decontamination and separation using a technique requiring no specialised equipment. Efficient single-cell nested-PCR procedures (both standard and high-resolution-melting) were developed to allow efficient amplification and analysis of viral sequences from isolated single cells. Moreover, the specific identification of two CaMV variants in different cells demonstrated a very low level of cross-contamination. This technique paves the way for the future development of numerous applications of broad interest in the study of viral diversity and population genetics of plant viruses at the cellular level.

The multiplicity of infection (MOI), i.e., the number of virus genomes that infect a cell, is a key parameter in virus evolution, as it determines processes such as genetic exchange among genomes, selection intensity on viral genes, epistatic interactions, and the evolution of multipartite viruses. In fact, the MOI level is equivalent to the virus ploidy during genome expression. Nevertheless, there are few experimental estimates of MOI, particularly for viruses with eukaryotic hosts. Here we estimate the MOI of Tobacco mosaic virus (TMV) in its systemic host, Nicotiana benthamiana. The progress of infection of two TMV genotypes, differently tagged with the green or red fluorescent proteins GFP and RFP, was monitored by determining the number of leaf cell protoplasts that showed GFP, RFP, or GFP and RFP fluorescence at different times postinoculation. This approach allowed the quantitative analysis of the kinetics of infection and estimation of the generation time and the number of infection cycles required for leaf colonization. MOI levels were estimated from the frequency of cells infected by only TMV-GFP or TMV-RFP. The MOI was high, but it changed during the infection process, decreasing from an initial level of about 6 to a final one of 1 to 2, with most infection cycles occurring at the higher MOI levels. The decreasing MOI can be explained by mechanisms limiting superinfection and/or by genotype competition within double-infected cells, which was shown to occur in coinfected tobacco protoplasts. To our knowledge, this is the first estimate of MOI during virus colonization of a eukaryotic host.

The sequence of Lettuce chlorosis virus (LCV) (genus Crinivirus) was determined and found to contain unique open reading frames (ORFs) and ORFs similar to those of other criniviruses, as well as 3' non-coding regions that shared a high degree of identity. Northern blot analysis of RNA extracted from LCV-infected plants identified subgenomic RNAs corresponding to six prominent internal ORFs and detected several novel LCV-single stranded RNA species. Virus replication in tobacco protoplasts was investigated and results indicated that LCV replication proceeded with novel crinivirus RNA accumulation kinetics, wherein viral genomic RNAs exhibited a temporally similar expression pattern early in the infection. This was noticeably distinct from the asynchronous RNA accumulation pattern previously observed for Lettuce infectious yellows virus (LIYV), the type member of the genus, suggesting that replication of the two viruses likely operate via dissimilar mechanisms.

Deletion and alanine-substitution mutants of the Tomato spotted wilt virus NSm protein were generated to identify domains involved in tubule formation, movement and symptomatology using a heterologous Tobacco mosaic virus expression system. Two regions of NSm, G(19)-S(159) and G(209)-V(283), were required for both tubule formation in protoplasts and cell-to-cell movement in plants, indicating a correlation between these activities. Three amino acid groups, D(154), EYKK(205-208) and EEEEE(284-288) were linked with long-distance movement in Nicotiana benthamiana. EEEEE(284-288) was essential for NSm-mediated long-distance movement, whereas D(154) was essential for tubule formation and cell-to-cell movement; indicating separate genetic controls for cell-to-cell and long-distance movement. The region I(57)-N(100) was identified as the determinant of foliar necrosis in Nicotiana benthamiana, and mutagenesis of HH(93-94) greatly reduced necrosis. These findings are likely applicable to other tospovirus species, especially those within the 'New World' group as NSm sequences are highly conserved.

During replication, Citrus tristeza virus (CTV) produces large amounts of two unusual subgenomic (sg) RNAs that are positive-stranded and 5' coterminal. Although these RNAs are produced in similar amounts and are similar in size, with LMT1 ( approximately 750 nt) only slightly larger than LMT2 ( approximately 650), we found that the similar sgRNAs are produced differently. We previously showed that the LMT1 RNA is produced by premature termination during genomic RNA synthesis. However, LMT2 production was found to correlate with virion assembly instead of RNA replication. The time course of accumulation of the LMT2 RNA occurred late, coinciding with virion accumulation. The long flexuous virions of CTV contain two coat proteins that encapsidate the virions in a polar manner. The major coat protein encapsidates approximately 97% of the virion, while the minor capsid protein encapsidates the remainder of the genome beginning in the 5' non-translated region with the transition zone at approximately 630 nucleotides from the 5' end. The section of the virion RNA that was encapsidated by CPm was identical in size to the LMT2 RNA, suggesting that the LMT2 RNA represented a portion of the viral RNA protected by CPm encapsidation. Mutations that abrogated encapsidation by CPm also abolished the accumulation of LMT2 RNA. Thus, these two unusual but similar RNAs are produced via different pathways, one from RNA replication and one processed by the virion assembly process. To our knowledge, this represents the first evidence of a viral RNA processed by the assembly mechanism.

The helicase-like domain of the Bamboo mosaic virus replicase catalyzes the release of 5'-gamma-phosphate from both ATP and 5'-triphosphated RNA by an identical set of catalytic residues with a presumably larger binding pocket for RNA. In this study, the peptidyl regions involved in RNA binding were mapped by reversible formaldehyde crosslinking and mass spectrometry. Eleven residues within these regions were examined by mutational analysis. H636A, Y704A, and K706A greatly diminished the enzymatic activities and were unable to support the viral replication in Nicotiana benthamiana protoplasts. K843A decreased activity toward the RNA substrate to 17% of WT, and approximately 20% replication efficiency was retained in protoplasts. R597A and K610A retained approximately 50 and approximately 90% of the enzymatic activities, respectively. However, replication in protoplasts of these mutants was extremely limited. Proteins with the mutations K603A, R628A, R645A, H794A, and R799A were present at levels 30-69% of WT in protoplasts. However, the fates of these mutations in plants were different. Viral cell-to-cell movement was limited by the K603A and R628A mutations, while systemic movement was restricted by R645A and H794A. The implications of the helicase-like domain in the viral replication and movement are discussed.

Interactions between microtubules and viruses play important roles in viral infection. The best-characterized examples involve transport of animal viruses by microtubules to the nucleus or other intracellular destinations. In plant viruses, most work to date has focused on interaction between viral movement proteins and the cytoskeleton, which is thought to be involved in viral cell-to-cell spread. We show here, in Cauliflower mosaic virus (CaMV)-infected plant cells, that viral electron-lucent inclusion bodies (ELIBs), whose only known function is vector transmission, require intact microtubules for their efficient formation. The kinetics of the formation of CaMV-related inclusion bodies in transfected protoplasts showed that ELIBs represent newly emerging structures, appearing at late stages of the intracellular viral life cycle. Viral proteins P2 and P3 are first produced in multiple electron-dense inclusion bodies, and are later specifically exported to transiently co-localize with microtubules, before concentrating in a single, massive ELIB in each infected cell. Treatments with cytoskeleton-affecting drugs suggested that P2 and P3 might be actively transported on microtubules, by as yet unknown motors. In addition to providing information on the intracellular life cycle of CaMV, our results show that specific interactions between host cell and virus may be dedicated to a later role in vector transmission. More generally, they indicate a new unexpected function for plant cell microtubules in the virus life cycle, demonstrating that microtubules act not only on immediate intracellular or intra-host phenomena, but also on processes ultimately controlling inter-host transmission., (© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd.)