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Viroids are minimal pathogens of angiosperms, consisting of non-coding RNAs that cause severe diseases in agronomic crops. Symptoms associated with viroid infection are linked to developmental alterations due to genetic regulation. To understand the global molecular mechanisms of host response, we implemented an omics approach to identify master transcription regulators (MTRs) and their differentially expressed targets in tomato infected with mild and severe variants of PSTVd. Our approach integrates root and leaf transcriptomic data, gene regulatory network analysis, and identification of affected biological processes. Overall, our results reveal that specific bHLH, MYB, and ERF transcription factors regulate genes involved in molecular mechanisms underlying critical signaling pathways. Functional enrichment of regulons shows that bHLH-MTRs are linked to metabolism and plant defense, while MYB-MTRs are involved in signaling regulation and hormone-related processes. Strikingly, a member of the bHLH-TF family have a potential specific role as a microprotein involved in the post-translational regulation of hormone signaling events. For the severe variant, ERF-MTRs are characteristic, while ZNF-TF, tf3a-TF, BZIP-TFs, and NAC-TF act as unique MTRs. Altogether, our results lay a foundation for further research on the PSTVd and host genome interaction, providing evidence for identifying potential key genes that influence symptom development in tomato plants.

Major advances in our understanding of plant viral genome expression strategies and the interaction of a virus with its host for replication and movement, induction of disease, and resistance responses have been made through the generation of infectious molecules from cloned viral sequences. Autonomously replicating viral vectors derived from infectious clones have been exploited to express foreign genes in plants. Applications of virus-based vectors include the production of human/animal therapeutic proteins in plant cells and the specific study of plant biochemical processes, including those that confer resistance to pathogens. Additionally, virus-induced gene silencing, which is RNA mediated and triggered through homology-dependent RNA degradation mechanisms, has been exploited as an efficient method to study the functions of host genes in plants and to deliver small RNAs to insects. New and exciting strategies for vector engineering, delivery, and applications of plant virus–based vectors are the subject of this review.

Protocols for extraction and purification of viroid RNAs from the tissues of infected herbaceous plant hosts are numerous. They range from lengthy, traditional protocols that require large amounts of starting tissue and take several days to perform to those based on column chromatography which is more efficient and can be performed with smaller amounts of infected tissue. The goal of all protocols is to enrich for RNA fractions that contain viroid RNAs, and the RNA extraction procedure is chosen and adjusted for the downstream method used for detection and characterization. Removal of inhibitors/impurities is generally not an issue for herbaceous hosts unless they contain and inordinate amounts of polysaccharides, tannins, and phenols. Subsequent purification of viroid circular and linear RNAs is performed using denaturing polyacrylamide gel electrophoresis. In this chapter, a specific method routinely used for viroid purification from herbaceous hosts and problems that may be encountered is described and is intended as a reference for beginners in the field.

Protocols for extraction and purification of viroid RNAs from the tissues of infected herbaceous plant hosts are numerous. They range from lengthy, traditional protocols that require large amounts of starting tissue and take several days to perform to those based on column chromatography which is more efficient and can be performed with smaller amounts of infected tissue. The goal of all protocols is to enrich for RNA fractions that contain viroid RNAs, and the RNA extraction procedure is chosen and adjusted for the downstream method used for detection and characterization. Removal of inhibitors/impurities is generally not an issue for herbaceous hosts unless they contain and inordinate amounts of polysaccharides, tannins, and phenols. Subsequent purification of viroid circular and linear RNAs is performed using denaturing polyacrylamide gel electrophoresis. In this chapter, a specific method routinely used for viroid purification from herbaceous hosts and problems that may be encountered is described and is intended as a reference for beginners in the field.

The characterization of the elusive disease agent of the potato spindle tuber disease, potato spindle tuber viroid (PSTVd), was aided by the ability to obtain large amounts of infected tomato tissue in a simple bioassay where PSTVd was easily mechanically transmissible to an alternate herbaceous host in which it thrived and produced dramatic symptoms in a relatively short period (Diener, Viroids. Handbook of plant virus infections: comparative diagnosis. Elsevier/North-Holland, Amsterdam, pp 913-934, 1981; Diener, Virology 45:411-428, 1971; Raymer and O'Brien, Am Pot J, 39:401-408, 1962). Reactions in the primary, or secondary, herbaceous indicator host can range from asymptomatic to severe depending upon the viroid strain, host species, and environmental conditions and can provide evidence of a viroid infection, but do not permit identification of the viroid in question. Further characterization by molecular hybridization, RT-PCR, and sequence analysis is used to determine the etiology of the disease agent. In this chapter, methods are described for mechanical inoculation of viroids to herbaceous hosts to determine the viroid nature of diseases and the experimental host range of the viroid or to shorten the time required for obtaining relatively large amounts of viroid for subsequent purification and characterization.

Determining the sequence identity of viroid RNAs present in symptomatic or asymptomatic plant tissues is critical to obtain knowledge of their distribution. It enables the development of tools for diagnostics and for studying the basic biology of viroids. With the advent of cDNA-based methods for cloning RNAs and cloning strategies that do not require prior knowledge of the viroid sequence, characterization of several newly discovered viroids has rapidly expanded our knowledge of these unusual pathogenic RNAs. This chapter describes two methods, using random primers or viroid-specific primers, to generate complementary DNA (cDNA) copies of viroid RNAs for subsequent cloning and sequence analysis.

Determining the sequence identity of viroid RNAs present in symptomatic or asymptomatic plant tissues is critical to obtain knowledge of their distribution. It enables the development of tools for diagnostics and for studying the basic biology of viroids. With the advent of cDNA-based methods for cloning RNAs and cloning strategies that do not require prior knowledge of the viroid sequence, characterization of several newly discovered viroids has rapidly expanded our knowledge of these unusual pathogenic RNAs. This chapter describes two methods, using random primers or viroid-specific primers, to generate complementary DNA (cDNA) copies of viroid RNAs for subsequent cloning and sequence analysis.

The characterization of the elusive disease agent of the potato spindle tuber disease, potato spindle tuber viroid (PSTVd), was aided by the ability to obtain large amounts of infected tomato tissue in a simple bioassay where PSTVd was easily mechanically transmissible to an alternate herbaceous host in which it thrived and produced dramatic symptoms in a relatively short period (Diener, Viroids. Handbook of plant virus infections: comparative diagnosis. Elsevier/North-Holland, Amsterdam, pp 913-934, 1981; Diener, Virology 45:411-428, 1971; Raymer and O'Brien, Am Pot J, 39:401-408, 1962). Reactions in the primary, or secondary, herbaceous indicator host can range from asymptomatic to severe depending upon the viroid strain, host species, and environmental conditions and can provide evidence of a viroid infection, but do not permit identification of the viroid in question. Further characterization by molecular hybridization, RT-PCR, and sequence analysis is used to determine the etiology of the disease agent. In this chapter, methods are described for mechanical inoculation of viroids to herbaceous hosts to determine the viroid nature of diseases and the experimental host range of the viroid or to shorten the time required for obtaining relatively large amounts of viroid for subsequent purification and characterization.

Hop stunt viroid (HSVd), a member of the family Pospiviroidae, infects a large number of plants and causes substantial economic losses. In this study, HSVd was detected in dappled fruits of sweet cherry and then molecularly characterized. We sequenced 164 cDNA clones from six sweet cherry cultivars in the Shandong Province of China and identified 23 HSVd sequence variants, which ranged in size between 293 and 303 nucleotides (nt) and shared 97~99% sequence identity with each other. Characterization of HSVd variants revealed that three were predominant in sweet cherry trees. Phylogenetic analysis showed that most of the HSVd variants isolated from sweet cherry were clustered within the plum-type group and that two variants were clustered with the recombinant P-C group, regardless of geographic origin. This study identified 23 HSVd variants and provided the molecular characterization of viroid infection (HSVd) in sweet cherry in China.

The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene (TF) encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Staphylococcus simulans Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The TF gene was expressed in Nicotiana benthamiana plants using the non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus (AltMV)-based pGD5TGB1L8823-MCS-CP3 vector, and in Escherichia coli using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a TF gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 (TF gene) to 0.93 (TFnt gene). Incorporation of the TFnt gene into the pEAQ-HT vector, followed by transient expression in N. benthamiana, led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive Staphylococcus aureus 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against S. aureus 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC50 25 µg/ml) and 60% (IC50 71 µg/ml) growth inhibition against S. aureus 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.

Tomato apical stunt viroid (TASVd) is a serious pathogen of tomato. Pathways for introduction include tomato seedlings, tomato seeds and ornamentals. If spread to tomato, considerable losses could result. TASVd is spread easily through plant sap, e.g. during pruning and propagation, and there is some evidence of insect transmission in the greenhouse. No symptoms appear on infected ornamental solanaceous plants, but these plants can act as a reservoir for the spread of viroids in tomato production, especially in greenhouse conditions. TASVd outbreaks in tomato are rare although it has occurred in several countries in Asia, Africa and Europe. The economic impact of TASVd in tomato production is not known, but heavy yield losses may result from infection with certain strains. This viroid has not been reported as an invasive species.

Pear blister canker disease, first reported in France in the 1960's, is caused by pear blister canker viroid (PBCVd). Diseases can occur in several cultivars of pear (Pyrus communis), its major host, but most pear cultivars are tolerant and do not produce symptoms of infection. PBCVd was initially reported in pear and quince, followed by wild pear and nashi and can experimentally infect species in other genera (Chaenomeles, Cydonia, Sorbus, Malus). Transmission through mechanical inoculation from pruning tools and grafting to infective propagative materials is a potential pathway for spread; no animal vector is known and it is not known to be seed transmitted. PBCVd has been reported in several European countries, Malta, Tunisia, Turkey, Australia, Japan, China, and the Americas; the geographic distribution may be underestimated because of symptomless infections in certain hosts. PBCVd was placed on the A1 list in Canada in 1995, listed as a quarantine pest in the United States in 1989, is regarded as quarantine pathogen for Australia, and is listed in a certification scheme to produce clean Pyrus and Cydonia sp. planting material in an OEPP/EPPO Bulletin in 1999 (OEPP/EPPO, 1999).

Tomato apical stunt viroid (TASVd) is a serious threat to tomato plants that can cause a considerable yield loss. In the present study, two isothermal molecular diagnostic assays based on reverse transcription-recombinase polymerase amplification (RT-RPA) utilizing the AmplifyRP® platform for plant pathogen detection were developed. The results of this research demonstrated distinct specificity of both developed assays, AmplifyRP® Acceler8TM and AmplifyRP® XRT, expressed in the absence of any cross-reaction activity to all total RNA extracts obtained from plants infected with other pospiviroids. The RT-RPA assays detected viroid RNA in 81- and 27-fold dilutions of the original TASVd-infected crude extract for AmplifyRP® Acceler8TM and AmplifyRP® XRT, respectively. The sensitivity tests in serial water dilutions showed the ability of AmplifyRP® Acceler8TM and AmplifyRP® XRT to detect 8 and 80 fg of pure TASVd RNA transcript, respectively. The influence of crude extract on viroid RNA transcript detection was also examined and a decrease of sensitivity of approximately 100-fold for both RT-RPA assays was revealed. To our knowledge, this is the first report describing development of RT-RPA assays to detect TASVd in plants using the AmplifyRP® platform that can be further employed both in laboratory conditions and in the field for on-site diagnosis.

Plant-virus-derived vectors are versatile tools with multiple applications in agricultural and medical biotechnology. In this study, we developed pepino mosaic virus (PepMV) (family Alphaflexiviridae; genus Potexvirus) into a vector for heterologous protein expression in plants. PepMV was initially cloned in a step-wise manner, fully sequenced and the full-length infectious clone was tested for infectivity in Nicotiana benthamiana. Initial infectious clones resulted in poor replication of PepMV and lack of systemic movement. Mutations in the viral sequence affected systemic infection. Two suspected mutations were altered to restore systemic infectivity. PepMV infection was apparent as early as 4 days post agroinfiltration (dpa) inoculation in N. benthamiana. A multiple cloning site was inserted into the PepMV genome for introduction and expression of foreign genes. Several modifications to the wild-type vector were made, such as a replacing the native subgenomic promoter (SGP) with a heterologous SGP, and introduction of translational enhancers and terminators, to improve heterologous expression of the foreign gene-of-interest. GFP was used as a reporter for monitoring virus infection and protein production. Strong GFP expression was observed as early as 4 dpa with a translational enhancer. The PepMV-based vector produces rapid expression of the foreign gene in comparison to two other potexvirus-based vectors. GFP production was monitored over time and optimal protein production was recorded between 5 and 7 dpa. GFP protein levels reached up to 4% and decreased to 0.5% total soluble protein at 7 and 14 dpa, respectively. Future studies will evaluate this virus-based vector for large-scale production of pharmaceutical compounds. KEY POINTS: • A pepino mosaic virus isolate was developed into a plant-based expression vector. • Expression levels of the heterologous protein were comparable or exceeded previously developed viral vectors. • Protein levels in plants were highest between 5 and 7 days and decreased gradually.

Miscanthus sinensis is a grass used for sugarcane breeding and bioenergy production. Using high throughput sequencing technologies, we identified a new viral genome in infected M. sinensis leaf tissue displaying yellow fleck symptoms. This virus is most related to members of the genus Polerovirus in the family Luteoviridae. The canonical ORFs were computationally identified, the P3 coat protein was expressed, and virus-like particles were purified and found to conform to icosahedral shapes, characteristic of the family Luteoviridae. We propose the name Miscanthus yellow fleck virus for this new virus.

Pepino mosaic virus (PepMV) is a widely distributed tomato virus. The complete genome sequence of the PepMV isolate US3 from infected tomato fruit was determined. The genome is 6,410 nucleotides long and has a poly(A) tail. US3 shares the highest similarity with strains belonging to the European genotype.

Background Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and a cause of necrotic enteritis in poultry. It is controlled using antibiotics, widespread use of which may lead to development of drug-resistant bacteria. Bacteriophage-encoded endolysins that degrade peptidoglycans in the bacterial cell wall are potential replacements for antibiotics. Phage endolysins have been identified that exhibit antibacterial activities against several Clostridium strains. Results An Escherichia coli codon-optimized gene encoding the glycosyl hydrolase endolysin (PlyCP41) containing a polyhistidine tag was expressed in E. coli. In addition, The E. coli optimized endolysin gene was engineered for expression in plants (PlyCP41p) and a plant codon-optimized gene (PlyCP41pc), both containing a polyhistidine tag, were expressed in Nicotiana benthamiana plants using a potato virus X (PVX)-based transient expression vector. PlyCP41p accumulated to ~ 1% total soluble protein (100μg/gm f. wt. leaf tissue) without any obvious toxic effects on plant cells, and both the purified protein and plant sap containing the protein lysed C. perfringens strain Cp39 in a plate lysis assay. Optimal systemic expression of PlyCP41p was achieved at 2 weeks-post-infection. PlyCP41pc did not accumulate to higher levels than PlyCP41p in infected tissue. Conclusion We demonstrated that functionally active bacteriophage PlyCP41 endolysin can be produced in systemically infected plant tissue with potential for use of crude plant sap as an effective antimicrobial agent against C. perfringens.

The first complete genome sequence of a little cherry virus-2 (LChV-2-TA) isolate from China was determined using small RNA deep sequencing combined with overlapping reverse transcriptase PCR (RT-PCR). Phylogenetic analysis revealed that LChV-2-TA grouped in a well-supported cluster with members of the genus Ampelovirus with close relationships to previously reported LChV-2 isolates.

Maize rayado fino virus (MRFV) causes severe yield losses in maize (Zea mays) in Latin America with reductions of 40–50% on indigenous genotypes and 100% in some new cultivars. A collection of 20 different maize genotypes were obtained from the Center for the Improvement of Maize and Wheat (CIMMYT, México), Estación Experimental Fabio Baudrit (EEFB-Alajuela), and the Consejo Nacional de la Producción (CNP-Costa Rica). Enzyme-linked immunosorbent assay (ELISA) and symptom severity were used to evaluate the responses of the maize genotypes to MRFV infection under screenhouse conditions. Two genotypes from the CIMMYT germ plasm collection (accession nos. 2980-93 and 3974) showed mild and delayed symptoms and low MRFV concentrations as determined by ELISA.

In early 2007, severe yellowing and chlorosis symptoms were observed in field-grown and greenhouse tomato (Solanum lycopersicum L.) plants in Costa Rica. Symptoms resembled those of the genus Crinivirus (family Closteroviridae), and large populations of whiteflies, including the greenhouse whitefly Trialeurodes vaporariorum (Westwood), were observed in the fields and on symptomatic plants. Total RNA was extracted from silica gel-dried tomato leaf tissue of 47 representative samples (all were from symptomatic plants) using TRI Reagent (Molecular Research Inc., Cincinnati, OH). Reverse transcription (RT)-PCR reactions were performed separately with each of the four primer sets with the Titan One-Tube RT-PCR Kit (Roche Diagnostics Corp., Chicago IL). Specific primers used for the detection of the criniviruses, Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV), were primer pair ToCV-p22-F (5′-ATGGATCTCACTGGTTGCTTGC-3′) and ToCV-p22-R (5′-TTATATATCACTCCCAAAGAAA-3′) specific for the p22 gene of ToCV RNA1 (1), primer pair ToCVCPmF (5′-TCTGGCAGTACCCGTTCGTGA-3′) and ToCVCPmR (5′-TACCGGCAGTCGTCCCATACC-3′) designed to be specific for the ToCV CPm gene of ToCV RNA2 (GenBank Accession No. AY903448) (2), primer pair ToCVHSP70F (5′-GGCGGTACTTTCGACACTTCTT-3′) and ToCVHSP70R (5′-ATTAACGCGCAAAACCATCTG-3′) designed to be specific for the Hsp70 gene of RNA2 of ToCV (GenBank Accession No. EU284744) (1), and primer pair TICV-CP-F and TICV-CP-R specific for the coat protein gene of TICV (1). Amplified DNA fragments (582 bp) were obtained from nine samples, four from the greenhouse and five from the open field, with the ToCV-p22 specific primers and were cloned into the pCRII TOPO cloning vector (Invitrogen, Carlsbad, CA). Nucleotide sequence analysis of all purified RT-PCR products verified their identity as ToCV, sharing 99.5 to 100% sequence identity among themselves and 96% to 98% sequence identity with previously reported ToCV p22 sequences from Florida (Accession No. AY903447), Spain (Accession No. DQ983480), and Greece (Accession No. EU284745). The presence of ToCV in the samples was confirmed by additional amplification and sequence analysis of the CPm (449-bp fragment) and Hsp70 (420-bp fragment) genes of ToCV RNA2 and sharing 98 to 99% sequence homology to Accession Nos. AY903448 and EU284774, respectively. One representative sequence of the p22 gene of the Costa Rican isolate was deposited at GenBank (Accession No. FJ809714). No PCR products were obtained using either the TICV-specific primers nor from healthy tomato tissue. The ToCV-positive samples were collected from a region in the Central Valley around Cartago, Costa Rica. To our knowledge, this is the first report of ToCV in Costa Rica. The economic impact on tomato has not yet been determined. Studies are underway to determine the incidence of ToCV in Costa Rica field-grown and greenhouse tomatoes. References: (1) A. R. A. Kataya et al. Plant Pathol. 57:819, 2008. (2) W. M. Wintermantel et al. Arch. Virol. 150:2287, 2005.

We describe the design and performance of the near-infrared (1.51--1.70 micron), fiber-fed, multi-object (300 fibers), high resolution (R = lambda/delta lambda ~ 22,500) spectrograph built for the Apache Point Observatory Galactic Evolution Experiment (APOGEE). APOGEE is a survey of ~ 10^5 red giant stars that systematically sampled all Milky Way populations (bulge, disk, and halo) to study the Galaxy's chemical and kinematical history. It was part of the Sloan Digital Sky Survey III (SDSS-III) from 2011 -- 2014 using the 2.5 m Sloan Foundation Telescope at Apache Point Observatory, New Mexico. The APOGEE-2 survey is now using the spectrograph as part of SDSS-IV, as well as a second spectrograph, a close copy of the first, operating at the 2.5 m du Pont Telescope at Las Campanas Observatory in Chile. Although several fiber-fed, multi-object, high resolution spectrographs have been built for visual wavelength spectroscopy, the APOGEE spectrograph is one of the first such instruments built for observations in the near-infrared. The instrument's successful development was enabled by several key innovations, including a "gang connector" to allow simultaneous connections of 300 fibers; hermetically sealed feedthroughs to allow fibers to pass through the cryostat wall continuously; the first cryogenically deployed mosaic volume phase holographic grating; and a large refractive camera that includes mono-crystalline silicon and fused silica elements with diameters as large as ~ 400 mm. This paper contains a comprehensive description of all aspects of the instrument including the fiber system, optics and opto-mechanics, detector arrays, mechanics and cryogenics, instrument control, calibration system, optical performance and stability, lessons learned, and design changes for the second instrument., 81 pages, 67 figures, PASP, accepted

The increasing spread of antibiotic-resistant pathogens has raised the interest in alternative antimicrobial treatments. In our study, the functionally active gram-negative bacterium bacteriophage CP933 endolysin was produced in Nicotiana benthamiana plants by a combination of transient expression and vacuole targeting strategies, and its antimicrobial activity was investigated. Expression of the cp933 gene in E. coli led to growth inhibition and lysis of the host cells or production of trace amounts of CP933. Cytoplasmic expression of the cp933 gene in plants using Potato virus X-based transient expression vectors (pP2C2S and pGR107) resulted in death of the apical portion of experimental plants. To protect plants against the toxic effects of the CP933 protein, the cp933 coding region was fused at its Nterminus to an N-terminal signal peptide from the potato proteinase inhibitor I to direct CP933 to the delta-type vacuoles. Plants producing the CP933 fusion protein did not exhibit the severe toxic effects seen with the unfused protein and the level of expression was 0.16 mg/g of plant tissue. Antimicrobial assays revealed that, in contrast to gram-negative bacterium E. coli (BL21(DE3)), the gram-positive plant pathogenic bacterium Clavibacter michiganensis was more susceptible to the plant-produced CP933, showing 18% growth inhibition. The results of our experiments demonstrate that the combination of transient expression and protein targeting to the delta vacuoles is a promising approach to produce functionally active proteins that exhibit toxicity when expressed in plant cells.

Viral infections of alfalfa are widespread in major cultivation areas and their impact on alfalfa production may be underestimated. A new viral species, provisionally named alfalfa virus F (AVF), was identified using a virion-associated nucleic acid (VANA) metagenomics-based approach in alfalfa (Medicago sativa L.) samples collected in Southern France. The nucleotide sequence of the viral genome was determined by de-novo assembly of VANA reads and by 5'/3' RACE with viral RNA extracted from enriched viral particles or with total RNA, respectively. The virus shares the greatest degree of overall sequence identity (similar to 78%) with Medicago sativa marafivirus 1 (MsMV1) recently deduced from alfalfa transcriptomic data. The tentative nucleotide sequence of the AVF coat protein shares similar to 83% identity with the corresponding region of MsMV1. A sequence search of the predicted single large ORF encoding a polyprotein of 235kDa in the Pfam database resulted in identification of five domains, characteristic of the genus Marafivirus, family Tymoviridae. The AVF genome also contains a conserved "marafibox" , a 16-nt consensus sequence present in all known marafi-viruses. Phylogenetic analysis of the complete nucleotide sequences of AVF and other viruses of the family Tymoviridae grouped AVF in the same cluster with MsMV1. In addition to 5' and 3' terminal extensions, the identity of the virus was confirmed by RT-PCRs with primers derived from VANA-contigs, transmission electron microscopy with virus-infected tissues and transient expression of the viral coat protein gene using a heterologous virus-based vector. Based on the criteria demarcating species in the genus Marafivirus that include overall sequence identity less than 80% and coat protein identity less than 90%, we propose that AVF represents a distinct viral species in the genus Marafivirus, family Tymoviridae.

Viroids are small, covalently closed, circular non-coding RNA pathogens of flowering plants. It is proposed that the symptoms of viroid pathogenesis result from a direct interaction between the viroid genomic RNA and unknown host plant factors. Using a comparative genomic approach we took advantage of the detailed annotation of the Arabidopsis thaliana (Arabidopsis) genome to identify sequence homologies between putative viroid-derived small RNAs (vd-sRNAs) and coding regions in the plant genome. A pool of sequence homologies among 29 species of the Pospiviroidae family and the Arabidopsis genome was analyzed. Using this strategy we identified putative host gene targets that may be involved in symptom expression in viroid-infected plants. In this communication, we report the in silico prediction and the experimental validation of pospiviroid-derived sRNAs conserved in the lower strand of the pathogenicity domain of seven viroid species infecting tomato; those vd-sRNAs targeted for cleavage the host mRNA encoding a conserved tomato WD40-repeat protein (SolWD40-repeat; SGN_U563134). Analysis of SolWD40-repeat expression indicated that this gene is down-regulated in tomato plants infected with tomato planta macho viroid (TPMVd). Furthermore, 5′ RLM-RACE revealed that the SolWD40-repeat mRNA is cleaved at the predicted target site showing complementarity to a corresponding TPMVd-sRNA identified in silico. Our approach proved to be useful for the identification of natural host genes containing sequence homologies with segments of the Pospiviroidae genome. Using this strategy we identified a functionally conserved gene in Arabidopsis and tomato, whose expression was modified during viroid infection in the host genome; regulation of this gene expression could be guided by vd-sRNA:mRNA complementarity, suggesting that the comparison of the Arabidopsis genome to viroid sequences could lead to the identification of unexpected interactions between viroid RNAs and their host.

Viral-based nanoplatforms rely on balancing the delicate array of virus properties to optimally achieve encapsidation of foreign materials with various potential objectives. We investigated the use of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) as a multifunctional nanoplatform and their potential application as protein cages. MRFV-VLPs are composed of two serologically related, carboxy co-terminal coat proteins (CP1 and CP2) which are capable of self-assembling in Nicotiana benthamiana plants into 30nm particles with T=3 symmetry. The N-terminus of CP1 was targeted for genetic modification to exploit the driving forces for VLP assembly, packaging and retention of RNA in vivo and in vitro. The N-terminus of MRFV-CP1 contains a peptide sequence of 37 amino acids which has been predicted to have an alpha-helical structure, is rich in hydrophobic amino acids, facilitates CP-RNA interactions, and is not required for self-assembly. Amino acid substitutions were introduced in the 37 amino acid N-terminus by site-directed mutagenesis and the mutant VLPs produced in plants by a Potato virus X (PVX)-based vector were tested for particle stability and RNA encapsidation. All mutant CPs resulted in production of VLPs which encapsidated non-viral RNAs, including PVX genomic and subgenomic (sg) RNAs, 18S rRNA and cellular and viral mRNAs. In addition, MRFV-VLPs encapsidated GFP mRNA when was expressed in plant cells from the pGD vector. These results suggest that RNA packaging in MRFV-VLPs is predominantly driven by electrostatic interactions between the N-terminal 37 amino acid extension of CP1 and RNA, and that the overall species concentration of RNA in the cellular pool may determine the abundance and species of the RNAs packaged into the VLPs. Furthermore, RNA encapsidation is not required for VLPs stability, VLPs formed from MRFV-CP1 were stable at temperatures up to 70°C, and can be disassembled into CP monomers, which can then reassemble in vitro into complete VLPs either in the absence or presence of RNAs.

The aim of this work was to detect plant infections caused by Tomato chlorosis virus (ToCV) and begomovirus in tomato plantlets, and in growing weeds around nursery greenhouses. During one year, starting in April 2008, 168 leaf tissue samples were collected, 90 tomato plantlets and 78 weeds from three different nurseries in Cartago province, Costa Rica. Reverse transcription and real time polymerase chain reaction (qRT-PCR) was used to determine that 18,9% of tomato plantlets and 7,7% of weeds were infected with ToCV virus. Begomoviruses were detected using Dot Blot hybridization and non-radioactive probe. Next, hybridization results were confirmed using Rolling Circle Amplification (RCA) followed by PCR, using universal primers. None tomato plantlet resulted positive when tested, but there were six weeds infected; in fact, Phytolacca icosandra and Brassica sp. were both coinfected with ToCV virus. These results suggest that ToCV infected tomato plantlets when commercialized, could serve as way of virus introduction to other country regions. Finally, weeds growing around greenhouses have shown to be potential viral sources of ToCV and begomovirus. El objetivo de este trabajo fue detectar infecciones causadas por el Tomato chlorosis virus (ToCV) y begomovirus en plántulas de tomate, y en las arvenses de los alrededores de los invernaderos. En el período de un año, a partir de abril de 2008, se recolectaron 168 muestras de tejido foliar, 90 de plántulas de tomate y 78 de arvenses en tres distintos semilleros en la provincia de Cartago, Costa Rica. Por medio de una transcripción reversa ligada a una reacción en cadena de la polimerasa en tiempo real (qRT-PCR), se determinaron infecciones con ToCV en un 18,9% de plántulas de tomate y en un 7,7% de las arvenses. Los begomovirus se detectaron por medio de hibridación con sonda de ADN no radioactiva. Los resultados de hibridación se con rmaron con una amplificación por el círculo rodante (RCA), seguida de una reacción en cadena de la polimerasa (PCR), utilizando dos pares de iniciadores degenerados. Ninguna muestra de tomate resultó positiva para los virus estudiados, mientras que seis arvenses estaban infectadas; dos de ellas Phytolacca icosandra y Brassica sp., mostraron coinfección con el ToCV. Los resultados sugieren que si las plántulas de tomate infectadas con ToCV se comercializan, este podría ser introducido en otras regiones del país. Las arvenses alrededor de los invernaderos mostraron ser potenciales fuentes de inóculo tanto del ToCV como de begomovirus.

UMR BFP - Equipe Virologie; Aside from potato spindle tuber viroid, the genus Pospiviroid contains several agents reported to naturally infect solanaceous crops (e.g., tomato, potato, pepper) or ornamental plants (e.g., Petunia hybrida, Solanum spp., Brugmansia spp.). The present chapter focuses on the following so-called solanaceous pospiviroids: Columnea latent viroid, tomato apical stunt viroid, tomato chlorotic dwarf viroid, tomato planta macho viroid, including the related Mexican papita viroid, and pepper chat fruit viroid. It briefly presents information on the taxonomy, biology (host range, symptomatology, transmission mechanisms and epidemiology), significance (distribution, impact), and the diagnosis and control of these agents.

Little or no naturally occurring durable resistance to viroids has been found in most viroid host species. Therefore, efforts to introduce resistance into these plant hosts have been made. These efforts include strategies that incorporate viroid-specific antisense RNAs, sense and antisense viroid RNAs combined with hammerhead ribozymes, small viroid-derived hairpin RNAs, and a double-stranded RNA-specific ribonuclease, into transgenic plants. The results of those efforts, some of which are promising, and the challenges associated with creating viroid resistant plants are summarized in this chapter.

The economic significance of viroids in ornamental crops is less obvious than in food crops. The most serious direct impact known is for chrysanthemum stunt viroid in chrysanthemum and, to a lesser extent, for chrysanthemum chlorotic mottle viroid in the same crop. However, the majority of viroid infections in ornamental crops are not accompanied by a disease. Nonetheless, even asymptomatic infections may result in indirect impact related to trade effects. For example, infected ornamental plants may be economically significant by acting as a source of viroid inoculum for other crops suffering severe losses when infected by viroids.

[EN] Viroids, the smallest infectious agents endowed with autonomous replication, are tiny single-stranded circular RNAs (similar to 250 to 400 nt) without protein-coding ability that, despite their simplicity, infect and often cause disease in herbaceous and woody plants of economic relevance. To mitigate the resulting losses, several strategies have been developed, the most effective of which include: firstly, search for naturally resistant cultivars and breeding for resistance, secondly, induced resistance by pre-infection with mild strains, thirdly, ribonucleases targeting double-stranded RNAs and catalytic antibodies endowed with intrinsic ribonuclease activity, fourthly, antisense, and sense, RNAs, fifthly, catalytic antisense RNAs derived from hammerhead ribozymes, and sixthly, hairpin RNAs and artificial small RNAs for RNA interference. The mechanisms underpinning these strategies, most of which have been implemented via genetic transformation, together with their present results and future potential, are the subject of this review., We apologize to colleagues whose work has not been quoted for space limitations. Work in RF laboratory has been supported by the Spanish Ministerio de Economia y Competitividad (grant BFU2014-5681.2-13).

Crop losses due to viroid infection occur in vegetable and field crops worldwide. Several viroids in the family Pospiviroidae infect these crops and economic losses range from minimal to severe depending upon the viroid/host combination, the host cultivar, the viroid strain and the environmental conditions. In this chapter, the impact of viroids in potato, tomato, cucumber, hop, and sweet pepper crops is summarized for each vegetable or field crop host and is followed by potential means of control to reduce losses caused by viroids.

Viroids cause economically important damage to crops and the potential for seed, pollen, and insect transmission is of considerable importance in the spread of viroid diseases. Viroid transmission through true seed and pollen has been confirmed for several viroid/host combinations; however, the seed transmission rate reported for members of the families Pospiviroidae and Avsunviroidae is quite variable in the same and different hosts. Therefore, a greater understanding of viroid movement into the developing seed is needed to shed light on the transmission efficiencies observed. The impact of viroid seed transmission on growers and international seed trade as a potential pathway for pathogen introduction is a serious issue that needs to be addressed. Additionally, some pospiviroids have been shown to be insect-transmitted either through encapsidation in an insect-transmitted virus or through mechanical inoculation by insects visiting plants to obtain pollen.

Potato spindle tuber viroid (PSTVd) is a small plant pathogenic circular RNA that does not encode proteins, replicates autonomously, and traffics systemically in infected plants. Long-distance transport occurs by way of the phloem; however, one report in the literature describes the presence of viroid RNA in the xylem ring of potato tubers. In this study, a modified method based on an EDTA-mediated phloem exudation technique was applied for detection of PSTVd in the phloem of infected tomato plants. RT-PCR, nucleic acid sequencing, and Southern blot analyses of RT-PCR products verified the presence of viroid RNA in phloem exudates. In addition, the guttation fluid collected from the leaves of PSTVd-infected tomato plants was analyzed revealing the absence of viroid RNA in the xylem sap. To our knowledge, this is the first report of PSTVd RNA detection in phloem exudates obtained by the EDTA-mediated exudation technique.

Infection of tomato by potato purple top (PPT) phytoplasma causes disruption of gibberellin (GA) homeostasis in the plant host. Such pathologically-induced GA deficiency can be partially reversed by exogenous application of GA. This study was designed to explore the role of GA in tomato defence response against phytoplasmal disease, and to determine whether pretreatment with GA would protect healthy tomato seedlings from subsequent phytoplasmal infection and disease development. Our results revealed that, following exogenous GA application and subsequent PPT phytoplasma graft inoculation, there was an apparently coordinated down-regulation of the gene encoding a key GA signalling component and growth repressor known as DELLA protein (GAI) and up-regulation of genes involved in salicylic acid (SA) synthesis (ICS1), signalling (NIM1) and downstream defence responses (PRP-1). Our results also indicated that differential regulation of the above genes was correlated with an increase in activities of defence-related enzymes β-1,3-glucanase and chitinase. The data presented in this communication provide evidence to suggest that GA may act via DELLA and SA signalling pathways to modulate host defence in response to PPT phytoplasma infection. Although the GA pretreatment-induced defence was not sufficient to prevent a systemic infection, it reduced phytoplasma titre and significantly attenuated disease symptoms. While the actual molecular mechanism underlying the GA-induced plant defence remains elusive, findings from the current study open new opportunities for in-depth studies of the functional role of the GA signalling network during defence response against phytoplasmal infection.

The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP(®) Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP(®) Acceler8™ assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection.

Recent outbreaks and continued spread of diseases caused by phytoplasmas in potato, tomato and other vegetable crops in the USA accentuates the need for practical strategies to mitigate the impact of the phytoplasmal diseases. The purpose of this study was to investigate whether exogenous application of salicylic acid (SA) on healthy tomato seedlings would enhance the plants' defence against a subsequent potato purple top (PPT) phytoplasma infection. Our results showed that a twice-applied SA pretreatment, at 4 and 2 days before phytoplasma inoculation, significantly decreased the rate of PPT phytoplasma infection and reduced disease symptoms. At 40 days postinoculation (dpi), while 94% of the PPT phytoplasma-inoculated control plants exhibited characteristic PPT symptoms, 53% of the SA-pretreated, PPT phytoplasma-inoculated plants remained symptom-free with no molecular evidence of phytoplasma infection. The remaining 47% of SA-treated plants became infected, but symptoms were much milder and the average phytoplasma titre was more than 300 times lower compared with that in control plants. Real-time qRT-PCR assays revealed that SA-pretreatment caused transcriptional reprogramming of three defence-related genes. Up-regulations of a WRKY-type transcriptional factor gene (LeWRKY1) and a mitogen-activated protein kinase gene (LeMPK3) in an early stage following graft inoculation, and a sustained higher level expression of a downstream pathogenesis-related protein gene (LePRP1), were observed in SA-pretreated, PPT phytoplasma-inoculated plants. With the dosage and treatment regime implemented in this study, no noticeable phytotoxic effects or other negative impact on vegetative growth or reproductive development was observed in SA-treated plants throughout our experiment. Our findings encourage field trials of SA pretreatment as a possible approach to protect crops from phytoplasmal diseases.