Responses of Passiflora spp. to cowpea aphid-borne mosaic virus reveal infection in asymptomatic plants and new species with probable immunity.

Passion fruit woodiness disease (PWD), caused by cowpea aphid-borne mosaic virus (CABMV), produces socioeconomic problems in Brazil. The objectives of this study were to i) evaluate the temporal progression of PWD, ii) identify Passiflora genotypes with resistance to CABMV, and iii) detect virus infection in asymptomatic plants by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in cases where standard RT-PCR detection failed. The experiment was conducted in a greenhouse using 128 genotypes belonging to 12 species and three hybrids (inter- and intraspecific) of Passiflora, evaluated at five time points after inoculation. Progression rates and disease severity were lower in P. cincinnata, P. gibertii, P. miersii, and P. mucronata than in P. edulis, P. alata, Passiflora sp., and hybrids. Of the genotypes tested, 20.31% were resistant, especially the accessions of P. suberosa, P. malacophylla, P. setacea, P. pohlii, and P. bahiensis, which remained asymptomatic throughout the experiment. The absence of symptoms does not imply immunity of plants to the virus, since RT-qPCR analysis confirmed infection by the virus in asymptomatic plants of P. cincinnata, P. gibertii, P. miersii, P. mucronata, P. setacea, P. malacophylla, and P. suberosa. Even after four inoculations, the virus was not detected by RT-qPCR in the upper leaves in plants of the species P. pohlii and P. bahiensis, indicating that these species are probably immune to CABMV.

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins.

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.

Development of a Platform for Noncovalent Coupling of Full Antigens to Tobacco Etch Virus-Like Particles by Means of Coiled-Coil Oligomerization Motifs.

Virus-like particles are excellent inducers of the adaptive immune response of humans and are presently being used as scaffolds for the presentation of foreign peptides and antigens derived from infectious microorganisms for subunit vaccine development. The most common approaches for peptide and antigen presentation are translational fusions and chemical coupling, but some alternatives that seek to simplify the coupling process have been reported recently. In this work, an alternative platform for coupling full antigens to virus-like particles is presented. Heterodimerization motifs inserted in both Tobacco etch virus coat protein and green fluorescent protein directed the coupling process by simple mixing, and the obtained complexes were easily taken up by a macrophage cell line.

RNA decay is an antiviral defense in plants that is counteracted by viral RNA silencing suppressors.

Exonuclease-mediated RNA decay in plants is known to be involved primarily in endogenous RNA degradation, and several RNA decay components have been suggested to attenuate RNA silencing possibly through competing for RNA substrates. In this paper, we report that overexpression of key cytoplasmic 5'-3' RNA decay pathway gene-encoded proteins (5'RDGs) such as decapping protein 2 (DCP2) and exoribonuclease 4 (XRN4) in Nicotiana benthamiana fails to suppress sense transgene-induced post-transcriptional gene silencing (S-PTGS). On the contrary, knock-down of these 5'RDGs attenuates S-PTGS and supresses the generation of small interfering RNAs (siRNAs). We show that 5'RDGs degrade transgene transcripts via the RNA decay pathway when the S-PTGS pathway is disabled. Thus, RNA silencing and RNA decay degrade exogenous gene transcripts in a hierarchical and coordinated manner. Moreover, we present evidence that infection by turnip mosaic virus (TuMV) activates RNA decay and 5'RDGs also negatively regulate TuMV RNA accumulation. We reveal that RNA silencing and RNA decay can mediate degradation of TuMV RNA in the same way that they target transgene transcripts. Furthermore, we demonstrate that VPg and HC-Pro, the two known viral suppressors of RNA silencing (VSRs) of potyviruses, bind to DCP2 and XRN4, respectively, and the interactions compromise their antiviral function. Taken together, our data highlight the overlapping function of the RNA silencing and RNA decay pathways in plants, as evidenced by their hierarchical and concerted actions against exogenous and viral RNA, and VSRs not only counteract RNA silencing but also subvert RNA decay to promote viral infection.

Over-expression of GmKR3, a TIR-NBS-LRR type R gene, confers resistance to multiple viruses in soybean.

KEY MESSAGE: That overexpression of GmKR3 enhances innate virus resistance by stimulating. Soybean mosaic virus (SMV) is found in many soybean production areas, and SMV infection is one of the prevalent viral diseases that can cause significant yield losses in soybean. In plants, resistance (R) genes are involved in pathogen reorganization and innate immune response activation. Most R proteins have nucleotide-binding site and leucine-rich repeat (NBS-LRR) domains, and some of the NBS-LRR type R proteins in dicots have Toll/Interleukin-1 Receptor (TIR) motifs. We report here the analysis of the over-expression of GmKR3, a soybean TIR-NBS-LRR type R gene on virus resistance in soybean. When over-expressed in soybean, GmKR3 enhanced the plant's resistance to several strains of SMV, the closely related potyviruses bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), and the secovirus bean pod mottle virus (BPMV). Importantly, over-expression of GmKR3 did not affect plant growth and development, including yield and qualities of the seeds. HPLC analysis showed that abscisic acid (ABA) content increased in the 35S:GmKR3 transgenic plants, and in both wild-type and 35S:GmKR3 transgenic plants in response to virus inoculation. Consistent with this observation, we found that the expression of two ABA catabolism genes was down-regulated in 35S:GmKR3 transgenic plants. We also found that the expression of Gm04.3, an ABA responsive gene encoding BURP domain-containing protein, was up-regulated in 35S:GmKR3 transgenic plants. Taken together, our results suggest that overexpression of GmKR3 enhanced virus resistance in soybean, which was achieved at least in part via ABA signaling.

Inheritance, fine-mapping, and candidate gene analyses of resistance to soybean mosaic virus strain SC5 in soybean.

Soybean mosaic virus (SMV) is one of the most devastating pathogens for soybeans in China. Among the country-wide 22 strains, SC5 dominates in Huang-Huai and Changjiang valleys. For controlling its damage, the resistance gene was searched through Mendelian inheritance study, gene fine-mapping, and candidate gene analysis combined with qRT-PCR (quantitative real-time polymerase chain reaction) analysis. The parents F1, F2, and RILs (recombinant inbred lines) of the cross Kefeng-1 (Resistance, R) × NN1138-2 (Susceptible, S) were used to examine the inheritance of SC5-resistance. The F1 was resistant and the F2 and RILs segregated in a 3R:1S and 1R:1S ratio, respectively, indicating a single dominant gene conferring the Kefeng-1 resistance. Subsequently, the genomic region conferring the resistance was found in "Bin 352-Bin353 with 500 kb" on Chromosome 2 using the phenotyping data of the 427 RILs and a high-density genetic map with 4703 bin markers. In the 500 kb genomic region, 38 putative genes are contained. The association analysis between the SNPs in a putative gene and the resistance phenotype for the 427 RILs prioritized 11 candidate genes using Chi-square criterion. The expression levels of these genes were tested by qRT-PCR. On infection with SC5, 7 out of the 11 genes had differential expression in Kefeng-1 and NN1138-2. Furthermore, integrating SNP-phenotype association analysis with qRT-PCR expression profiling analysis, Glyma02g13495 was found the most possible candidate gene for SC5-resistance. This finding can facilitate the breeding for SC5-resistance through marker-assisted selection and provide a platform to gain a better understanding of SMV-resistance gene system in soybean.

The P1N-PISPO trans-Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor.

UNLABELLED: The positive-sense RNA genome of Sweet potato feathery mottle virus (SPFMV) (genus Potyvirus, family Potyviridae) contains a large open reading frame (ORF) of 3,494 codons translatable as a polyprotein and two embedded shorter ORFs in the -1 frame: PISPO, of 230 codons, and PIPO, of 66 codons, located in the P1 and P3 regions, respectively. PISPO is specific to some sweet potato-infecting potyviruses, while PIPO is present in all potyvirids. In SPFMV these two extra ORFs are preceded by conserved G2A6 motifs. We have shown recently that a polymerase slippage mechanism at these sites could produce transcripts bringing these ORFs in frame with the upstream polyprotein, thus leading to P1N-PISPO and P3N-PIPO products (B. Rodamilans, A. Valli, A. Mingot, D. San Leon, D. B. Baulcombe, J. J. Lopez-Moya, and J.A. Garcia, J Virol 89:6965-6967, 2015, doi:10.1128/JVI.00337-15). Here, we demonstrate by liquid chromatography coupled to mass spectrometry that both P1 and P1N-PISPO are produced during viral infection and coexist in SPFMV-infected Ipomoea batatas plants. Interestingly, transient expression of SPFMV gene products coagroinfiltrated with a reporter gene in Nicotiana benthamiana revealed that P1N-PISPO acts as an RNA silencing suppressor, a role normally associated with HCPro in other potyviruses. Moreover, mutation of WG/GW motifs present in P1N-PISPO abolished its silencing suppression activity, suggesting that the function might require interaction with Argonaute components of the silencing machinery, as was shown for other viral suppressors. Altogether, our results reveal a further layer of complexity of the RNA silencing suppression activity within the Potyviridae family. IMPORTANCE: Gene products of potyviruses include P1, HCPro, P3, 6K1, CI, 6K2, VPg/NIaPro, NIb, and CP, all derived from the proteolytic processing of a large polyprotein, and an additional P3N-PIPO product, with the PIPO segment encoded in a different frame within the P3 cistron. In sweet potato feathery mottle virus (SPFMV), another out-of-frame element (PISPO) was predicted within the P1 region. We have shown recently that a polymerase slippage mechanism can generate the transcript variants with extra nucleotides that could be translated into P1N-PISPO and P3N-PIPO. Now, we demonstrate by mass spectrometry analysis that P1N-PISPO is indeed produced in SPFMV-infected plants, in addition to P1. Interestingly, while in other potyviruses the suppressor of RNA silencing is HCPro, we show here that P1N-PISPO exhibited this activity in SPFMV, revealing how the complexity of the gene content could contribute to supply this essential function in members of the Potyviridae family.

A new eIF4E1 allele characterized by RNAseq data mining is associated with resistance to potato virus Y in tomato albeit with a low durability.

Allele mining on susceptibility factors offers opportunities to find new sources of resistance among crop wild relatives for breeding purposes. As a proof of concept, we used available RNAseq data to investigate polymorphisms among the four tomato genes encoding translation initiation factors [eIF4E1 and eIF4E2, eIFiso4E and the related gene new cap-binding protein(nCBP)] to look for new potential resistance alleles to potyviruses. By analysing polymorphism among RNAseq data obtained for 20 tomato accessions, 10 belonging to the cultivated type Solanum lycopersicum and 10 belonging to the closest related wild species Solanum pimpinellifolium, we isolated one new eIF4E1 allele, in the S. pimpinellifolium LA0411 accession, which encodes a potential new resistance allele, mainly due to a polymorphism associated with an amino acid change within eIF4E1 region II. We confirmed that this new allele, pot12, is indeed associated with resistance to potato virus Y, although with a restricted resistance spectrum and a very low durability potential. This suggests that mutations occurring in eIF4E region II only may not be sufficient to provide efficient and durable resistance in plants. However, our study emphasizes the opportunity brought by RNAseq data to mine for new resistance alleles. Moreover, this approach could be extended to seek for putative new resistance alleles by screening for variant forms of susceptibility genes encoding plant host proteins known to interact with viral proteins.

Self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce IgG2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.

BACKGROUND: Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. FINDINGS: Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. CONCLUSIONS: His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-µm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid diagnosis of chilli veinal mottle virus.

Chilli veinal mottle virus (ChiVMV) causes significant economic loss to chilli cultivation in northeastern India, as well as in eastern Asia. In this study, we have developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid, sensitive and specific diagnosis of ChiVMV. Amplification could be visualized after adding SYBR Green I (1000×) dye within 60 min under isothermal conditions at 63 °C, with a set of four primers designed based on the large nuclear inclusion protein (NIb) domain of ChiVMV (isolate KC-ML1). The RT-LAMP method was 100 times more sensitive than one-step reverse transcription polymerase chain reaction (RT-PCR), with a detection limit of 0.0001 ng of total RNA per reaction.

Characterization and application of a common epitope recognized by a broad-spectrum C4 monoclonal antibody against capsid proteins of plant potyviruses.

A broad-spectrum monoclonal antibody (C4 MAb) against the capsid proteins (CPs) of plant potyviruses has been generated in previous studies. To clarify which epitope is recognized by this MAb, epitope mapping was performed via phage display library screening and amino acid substitution analysis. Subsequently, a 12-residue epitope in the core region of potyvirus CPs was identified and termed the C4 epitope (WxMMDGxxQxxY/F). This epitope contains tryptophan and tyrosine residues that are crucial for reacting with C4 MAb. The CP of Odontoglossum ringspot tobamovirus (ORSV) separately fused with the C4 epitope of Konjak mosaic potyvirus (KoMV), Zantedeschia mild mosaic potyvirus (ZaMMV), or Dasheen mosaic potyvirus (DsMV) was expressed in a bacterial system and purified. The results of indirect ELISA and Western blotting demonstrated that the C4 epitope of KoMV (Ko) fused to ORSV CP showed the strongest binding affinity to C4 MAb among the three viral epitope tags examined. The binding affinity between Ko tag (WTMMDGEEQIEY) and C4 MAb was determined. To examine the applicability of the Ko tag in planta, GFP and ORSV CP were transiently expressed in Nicotiana benthamiana, and both Ko-tagged proteins were specifically detected using C4 MAb. The Ko tag did not affect the silencing suppressor function of Tomato bushy stunt tombusvirus P19 in N. benthamiana. Furthermore, Ko-tagged EGFP could be successfully expressed, specifically detected and subsequently immunoprecipitated using C4 MAb in a mammalian cell system. Thus, the present study identified a common C4 epitope of potyviruses recognized by the broad-spectrum C4 and PTY 1 MAbs, and the results indicated that the newly designed Ko tag is suitable for application in bacterial, plant, and mammalian cell systems.

New food from a potato somatic hybrid: nutritional equivalence and safety assessment by a feeding study on rats.

BACKGROUND: Potato tubers from the STBd somatic hybrid line that exhibited improved tolerance to salinity and resistance to fungal and PVY infections were characterised. They were compared for their chemical composition to the Spunta variety produced by conventional agronomic practices. This study aimed to compare nutritional value and safety by feeding rats with STBd or commercial tubers added to the standard diet (20/80 w/w). RESULTS: The analysis of soluble sugar, fat, fibre and ash content of tubers did not reveal any significant differences between the hybrid line and the control Spunta variety. Small differences were observed in dry matter, starch and protein content of hybrid potatoes in comparison to controls. However, all values were within normal ranges reported in the literature. The feeding study on rats showed that overall health, weight gain, food consumption, morphological aspects and weights of organs were comparable between rat groups fed the STBd hybrid and the Spunta variety. CONCLUSION: Taken together, 28 days of consumption of STBd hybrid potato did not exert any adverse effect on rats compared with commercial Spunta potato. The STBd potato line was therefore considered to be as safe for food utilisation as the commercial variety.

Construction of a genetic map based on high-throughput SNP genotyping and genetic mapping of a TuMV resistance locus in Brassica rapa.

Brassica rapa is a member of the Brassicaceae family and includes vegetables and oil crops that are cultivated worldwide. The introduction of durable resistance against turnip mosaic virus (TuMV) into agronomically important cultivars has been a significant challenge for genetic and horticultural breeding studies of B. rapa. Based on our previous genome-wide analysis of DNA polymorphisms between the TuMV-resistant doubled haploid (DH) line VC40 and the TuMV-susceptible DH line SR5, we constructed a core genetic map of the VCS-13M DH population, which is composed of 83 individuals derived from microspore cultures of a F1 cross between VC40 and SR5, by analyzing the segregation of 314 sequence-characterized genetic markers. The genetic markers correspond to 221 SNPs and 31 InDels of genes as well as 62 SSRs, covering 1,115.9 cM with an average distance of 3.6 cM between the adjacent marker loci. The alignment and orientation of the constructed map showed good agreement with the draft genome sequence of Chiifu, thus providing an efficient strategy to map genic sequences. Using the genetic map, a novel dominant TuMV resistance locus (TuMV-R) in the VCS-13M DH population was identified as a 0.34 Mb region in the short arm of chromosome A6 in which four CC-NBS-LRR resistance genes and two pathogenesis-related-1 genes reside. The genetic map developed in this study can play an important role in the genetic study of TuMV resistance and the molecular breeding of B. rapa.

Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies.

Surface plasmon resonance (SPR)-based biosensors have been widely utilized for measuring interactions of a variety of molecules. Fewer examples include higher biological entities such as bacteria and viruses, and even fewer deal with plant viruses. Here, we describe the optimization of an SPR sensor chip for evaluation of the interaction of the economically relevant filamentous Potato virus Y (PVY) with monoclonal antibodies. Different virus isolates were efficiently and stably bound to a previously immobilized polyclonal antibody surface, which remained stable over subsequent injection regeneration steps. The ability of the biosensor to detect and quantify PVY particles was compared with ELISA and RT-qPCR. Stably captured virus surfaces were successfully used to explore kinetic parameters of the interaction of a panel of monoclonal antibodies with two PVY isolates representing the main viral serotypes N and O. In addition, the optimized biosensor proved to be suitable for evaluating whether two given monoclonal antibodies compete for the same epitope within the viral particle surface. The strategy proposed in this work can help to improve existing serologic diagnostic tools that target PVY and will allow investigation of the inherent serological variability of the virus and exploration for new interactions of PVY particles with other proteins.

Sequence variability in the HC-Pro coding regions of Korean soybean mosaic virus isolates is associated with differences in RNA silencing suppression.

Soybean mosaic virus (SMV), a member of the family Potyviridae, is an important viral pathogen affecting soybean production in Korea. Variations in helper component proteinase (HC-Pro) sequences and the pathogenicity of SMV samples from seven Korean provinces were compared with those of previously characterized SMV isolates from China, Korea and the United States. Phylogenetic analysis separated 16 new Korean SMV isolates into two groups. Fourteen of the new Korean SMV samples belonged to group II and were very similar to U.S. strain SMV G7 and Chinese isolate C14. One isolate in group II, A297-13, differed at three amino acid positions (L54F, N286D, D369N) in the HC-Pro coding sequence from severe isolates and SMV 413, showed very weak silencing suppressor activity, and produced only mild symptoms in soybean. To test the role of each amino acid substitution in RNA silencing and viral RNA accumulation, a series of point mutations was constructed. Substitution of N for D at position 286 in HC-Pro of SMV A297-12 significantly reduced silencing suppression activity. When the mutant HC-Pro of A297-13 was introduced into an infectious clone of SMV 413, accumulation of viral RNA was reduced to less than 3 % of the level of SMV 413 containing HC-Pro of A297-12 at 10 days post-inoculation (dpi) but increased to 40 % of SMV 413(HC-Pro A297-12) at 40 dpi. At 50 dpi RNA accumulation of SMV 413(HC-Pro A297-13) was similar to that of SMV 413(HC-Pro A297-12). However, at 50 dpi, the D at position 286 of HC-Pro in SMV 413(HC-Pro A297-13) was found to have reverted to N. The results showed that 1) a naturally occurring mutation in HC-Pro significantly reduced silencing suppression activity and accumulation of transgene and viral RNAs, and 2) that there was strong selection for revision to wild type when the mutation was introduced into an infectious clone of SMV.

Recombinants of bean common mosaic virus (BCMV) and genetic determinants of BCMV involved in overcoming resistance in common bean.

Bean common mosaic virus (BCMV) exists as a complex of strains classified by reactions to resistance genes found in common bean (Phaseolus vulgaris); seven BCMV pathotypes have been distinguished thus far, numbered I to VII. Virus genetic determinants involved in pathogenicity interactions with resistance genes have not yet been identified. Here, we describe the characterization of two novel field isolates of BCMV that helped to narrow down these genetic determinants interacting with specific P. vulgaris resistance factors. Based on a biological characterization on common bean differentials, both isolates were classified as belonging to pathotype VII, similar to control isolate US10, and both isolates exhibited the B serotype. The whole genome was sequenced for both isolates and found to be 98 to 99% identical to the BCMV isolate RU1 (pathotype VI), and a single name was retained: BCMV RU1-OR. To identify a genetic determinant of BCMV linked to the BCMV pathotype VII, the whole genome was also sequenced for two control isolates, US10 and RU1-P. Inspection of the nucleotide sequences for BCMV RU1-OR and US10 (both pathotype VII) and three closely related sequences of BCMV (RU1-P, RU1-D, and RU1-W, all pathotype VI) revealed that RU1-OR originated through a series of recombination events between US10 and an as-yet-unidentified BCMV parental genome, resulting in changes in virus pathology. The data obtained suggest that a fragment of the RU1-OR genome between positions 723 and 1,961 nucleotides that is common to US10 and RU1-OR in the P1-HC-Pro region of the BCMV genome may be responsible for the ability to overcome resistance in bean conferred by the bc-2(2) gene. This is the first report of a virus genetic determinant responsible for overcoming a specific BCMV resistance gene in common bean.

A Recombinant of Bean common mosaic virus Induces Temperature-Insensitive Necrosis in an I Gene-Bearing Line of Common Bean.

The I gene is a single, dominant gene conferring temperature-sensitive resistance to all known strains of Bean common mosaic virus (BCMV) in common bean (Phaseolus vulgaris). However, the closely related Bean common mosaic necrosis virus (BCMNV) induces whole plant necrosis in I-bearing genotypes of common bean, and the presence of additional, recessive genes is required to prevent this severe whole plant necrotic reaction caused by BCMNV. Almost all known BCMNV isolates have so far been classified as having pathotype VI based on their interactions with the five BCMV resistance genes, and all have a distinct serotype A. Here, we describe a new isolate of BCMV, RU1M, capable of inducing whole plant necrosis in the presence of the I gene, that appears to belong to pathotype VII and exhibits B-serotype. Unlike other isolates of BCMV, RU1M was able to induce severe whole plant necrosis below 30°C in bean cultivar Jubila that carries the I gene and a protective recessive gene bc-1. The whole genome of RU1M was cloned and sequenced and determined to be 9,953 nucleotides long excluding poly(A), coding for a single polyprotein of 3,186 amino acids. Most of the genome was found almost identical (>98%) to the BCMV isolate RU1-OR (also pathotype VII) that did not induce necrotic symptoms in 'Jubila'. Inspection of the nucleotide sequences for BCMV isolates RU1-OR, RU1M, and US10 (all pathotype VII) and three closely related sequences of BCMV isolates RU1P, RU1D, and RU1W (all pathotype VI) revealed that RU1M is a product of recombination between RU1-OR and a yet unknown potyvirus. A 0.8-kb fragment of an unknown origin in the RU1M genome may have led to its ability to induce necrosis regardless of temperature in beans carrying the I gene. This is the first report of a BCMV isolate inducing temperature-insensitive necrosis in an I gene containing bean genotype.

Viruses in maize and Johnsongrass in southern Ohio.

The two major U.S. maize viruses, Maize dwarf mosaic virus (MDMV) and Maize chlorotic dwarf virus (MCDV), emerged in southern Ohio and surrounding regions in the 1960s and caused significant losses. Planting resistant varieties and changing cultural practices has dramatically reduced virus impact in subsequent decades. Current information on the distribution, diversity, and impact of known and potential U.S. maize disease-causing viruses is lacking. To assess the current reservoir of viruses present at the sites of past disease emergence, we used a combination of serological testing and next-generation RNA sequencing approaches. Here, we report enzyme-linked immunosorbent assay and RNA-Seq data from samples collected over 2 years to assess the presence of viruses in cultivated maize and an important weedy reservoir, Johnsongrass (Sorghum halepense). Results revealed a persistent reservoir of MDMV and two strains of MCDV in Ohio Johnsongrass. We identified sequences of several other grass-infecting viruses and confirmed the presence of Wheat mosaic virus in Ohio maize. Together, these results provide important data for managing virus disease in field corn and sweet corn maize crops, and identifying potential future virus threats.

Development of nanobodies against the coat protein of maize chlorotic mottle virus.

Maize lethal necrosis (MLN) is a maize disease caused by the maize chlorotic mottle virus (MCMV), a potyvirus which causes yield losses of 30-100%. The present study aimed to isolate nanobodies against the MCMV coat protein (CP) for the diagnosis of MLN. MCMV CP expressed in Escherichia coli was used for llama immunization. VHH (i.e. variable heavy domain of heavy chain) gene fragments were prepared from blood drawn from the immunized llama and used to generate a library in E. coli TG1 cells. MCMV specific nanobodies were selected by three rounds of phage display and panning against MCMV CP. The selected nanobodies were finally expressed in E. coli WK6 cells and purified. Eleven MCMV-specific nanobodies were identified and shown to detect MCMV in infected maize plants. Thus, our results show that nanobodies isolated from llama immunized with MCMV CP can distinguish infected and healthy maize plants, potentially enabling development of affordable MCMV detection protocols.

A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia.

A suspected virus disease was identified from an arborescent Brugmansia x candida Pers. (syn. Datura candida Pers.) tree. The causal agent was aphid transmissible at low rates. Viral particles were purified from infected tobacco tissue, analyzed, and purified virions were inoculated into healthy tobacco plants to recreate the symptoms. The virions had a mean length of 720-729 nm, and infected cells contained inclusion bodies typical of potyvirus infections. Analysis of infected tissues and purified virions with a panel of potyvirus-specific antibodies confirmed identification as a potyvirus. Viral host range, dilution end point, thermal tolerance and aphid transmission characteristics were examined. The viral genome (9761 nt) is typical of potyviruses, with the closest related potyvirus being pepper mottle virus, at 72 % nt sequence identity. Based on conventions for naming novel potyviruses, the virus was determined to be a member of a previously undescribed species, tentatively named "Brugmansia mosaic virus" (BruMV).