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Phytophthora infestans, the causal agent of late blight disease of potatoes, is mainly controlled by the use of fungicides. Isolates that are resistant to commonly used fungicides have been reported. Also, several studies show that originally mefenoxam-sensitive isolates acquire resistance to this fungicide when exposed to sublethal concentrations. This phenomenon, termed “mefenoxam-acquired resistance,” has been observed in different Phytophthora species and seems to be unique to mefenoxam. In this study, we aimed to elucidate the molecular mechanism mediating this type of resistance as well as a possible regulatory process behind it. A combination of computational analyses and experimental approaches was used to identify differentially expressed genes with a potential association to the phenomenon. These genes were classified into seven functional groups. Most of them seem to be associated with a pleiotropic drug resistance (PDR) phenotype, typically involved in the expulsion of diverse metabolites, drugs, or other substances out of the cell. Despite the importance of RNA Polymerase I for the constitutive resistance of P. infestans to mefenoxam, our results indicate no clear interaction between this protein and the acquisition of mefenoxam resistance. Several small non-coding RNAs were found to be differentially expressed and specifically related to genes mediating the PDR phenotype, thus suggesting a possible regulatory process. We propose a model of the molecular mechanisms acting within the cell when P. infestans acquires resistance to mefenoxam after exposed to sublethal concentrations of the fungicide. This study provides important insights into P. infestans’ cellular and regulatory functionalities.

In this study 163 complete whole-genome sequences of the emerging pathogen grapevine red blotch virus (GRBV; genus

We present a taxonomic proposal for revision of the family Secoviridae, a taxon of plant viruses in the order Picornavirales. We propose the reorganization of the genus Sadwavirus to create three new subgenera and to update the classification of five existing species. The proposed subgenera are “Satsumavirus” (one species: Satsuma dwarf virus), “Stramovirus” (two species: Strawberry mottle virus and Black raspberry necrosis virus) and “Cholivirus” (two species: Chocolate lily virus A and Dioscorea mosaic associated virus).

Here, we describe the full-length genome sequence of a novel potyvirus, tentatively named “miscanthus sinensis mosaic virus” (MsiMV), isolated from Miscanthus sinensis (silver grass) held in a post entry quarantine facility following its initial import into Western Australia, Australia. The MsiMV genome encompasses 9604 nucleotides (nt) encoding a 3071 amino acids (aa) polyprotein with conserved sequence motifs. The MsiMV genome is most closely related to sorghum mosaic virus (SrMV) with 74% nt and 78.5% aa sequence identity to the SrMV polyprotein region. Phylogenetic analysis based on the polyprotein grouped MsiMV with SrMV, sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV). This is the first report of a novel monopartite ssRNA virus in Miscanthus sinensis related to members of the genus Potyvirus in the family Potyviridae.

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) ‘2a’ consisting of the full-length 2a gene (839 amino acids, aa), (ii) ‘Motifs’ covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) ‘GDD’ located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the ‘2a’ and ‘Motifs’ constructs interacted with 96 and 25 library constructs, respectively, while the ‘GDD’ construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants.

Grapevine red blotch virus (GRBV) is an emerging virus of significant viticultural importance throughout North America. Here, we report the development of a simple protocol for point-of-use detection of GRBV. Extraction of nucleic acids is not required; instead, the whole intact plant can simply be pricked with a sterile pipette tip, which is then incubated in sterile distilled water to provide the sample template in a loop-mediated isothermal amplification (LAMP) reaction. This method is 10,000 times more sensitive than conventional PCR, costs under a dollar per sample, and can be completed from sampling to readout in just over half an hour.

The distribution and diversity of grapevine red blotch virus (GRBV) and wild Vitis virus 1 (WVV1) (genus Grablovirus; family Geminiviridae) were determined in free-living Vitis spp. in northern California and New York from 2013 to 2017. Grabloviruses were detected by polymerase chain reaction in 28% (57 of 203) of samples from California but in none of the 163 samples from New York. The incidence of GRBV in free-living vines was significantly higher in samples from California counties with high compared with low grape production (χ2 = 83.09; P < 0.001), and in samples near (5 km) from vineyards (χ2 = 57.58; P < 0.001). These results suggested a directional spread of GRBV inoculum predominantly from vineyards to free-living Vitis spp. WVV1 incidence was also significantly higher in areas with higher grape production acreage (χ2 = 16.02; P < 0.001). However, in contrast to GRBV, no differential distribution of WVV1 incidence was observed with regard to distance from vineyards (χ2 = 0.88; P = 0.3513). Two distinct phylogenetic clades were identified for both GRBV and WVV1 isolates from free-living Vitis spp., although the nucleotide sequence variability of the genomic diversity fragment was higher for WWV1 (94.3 to 99.8% sequence identity within clade 1 isolates and 90.1 to 100% within clade 2 isolates) than GRBV (98.3% between clade 1 isolates and 96.9 to 100% within clade 2 isolates). Additionally, evidence for intraspecific recombination events was found in WVV1 isolates and confirmed in GRBV isolates. The prevalence of grabloviruses in California free-living vines highlights the need for vigilance regarding potential grablovirus inoculum sources in order to protect new vineyard plantings and foundation stock vineyards in California.

Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative polymerase chain reaction (qPCR) method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a pattern similar to that found for the greenhouse-grown plants but, most strikingly, close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.

In North America, uncultivated, free-living grapevines (Vitis spp.) frequently grow alongside their cultivated counterparts, thus increasing the potential for exchange of microbiota. For this study, we used high-throughput sequencing (HTS) of small RNAs to survey for virus populations in free-living grapevines of the Finger Lakes region of New York State. Of 32 grapevines analyzed, 23 were free-living vines, while the remaining 9 were commercially grown Vitis vinifera plants from the same region. In total, 18 (78.3%) of the free-living grapevines tested were positive for grapevine asteroid mosaic-associated virus (GAMaV) infection by HTS, with detection confirmed by seminested reverse-transcription PCR and sequencing of nine isolates. Phylogenetic analyses of an ungapped alignment of the New York GAMaV sequences (length: 2,334 nucleotides) with the five known full-length or close to full-length global sequences showed that the New York isolates were broadly grouped. Of the nine cultivated plants, eight were infected with both hop stunt viroid and grapevine yellow speckle viroid 1, three were singly infected with grapevine leafroll-associated virus 3, and one harbored GAMaV. This limited survey of free-living grapevines, one of the first to use HTS, has highlighted the high incidence of a virus associated with disease in commercial V. vinifera.

In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3′ untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites.

During screening of non-cultivated (wild) grapevine (Vitis sp.) from Napa County, California for the grapevine red blotch virus (GRBV; genus Glabrovirus, family Geminiviridae), an atypical polymerase chain reaction product pattern was observed. Rolling circle amplification followed by cloning and sequencing revealed the presence of a circular DNA characteristic of geminiviruses. The complete genome of nine isolates of the virus ranged from 3204 to 3278 nt in size. The genome most closely resembled that of GRBV in both sequence (57 to 59% identity) and organization. With limited sequence identity to described geminiviruses, this virus warrants designation as a new species, and the name 'Wild Vitis virus 1' is proposed.

Synthetic biology based diagnostic technologies have improved upon gold standard diagnostic methodologies by decreasing cost, increasing accuracy, and enhancing portability. However there has been little effort in adapting these technologies towards applications related to point-of-use monitoring of plant and crop health. Here, we take a step towards this vision by developing an approach that couples isothermal amplification of specific plant pathogen genomic sequences with customizable synthetic RNA regulators that are designed to trigger the production of a colorimetric output in cell-free gene expression reactions. We demonstrate our system can sense viral derived sequences with high-sensitivity and specificity, and can be utilized to directly detect viruses from infected plant material. Furthermore, we demonstrate that the entire system can operate using only body heat and naked-eye visual analysis of outputs. We anticipate these strategies to be important components of user-friendly and deployable diagnostic systems that can be configured to detect a range of important plant pathogens.

Grapevine red blotch virus (GRBV) is type member of the newly identified genus Grablovirus. It possesses a single-stranded circular DNA genome of around 3200 nucleotides encoding three open reading frames (ORFs) in both the virion sense, the V1 (CP), V2 and V3, and complementary sense, C1 (RepA), C2 and C3. As shown for members of the genus Mastrevirus, the C1 and C2 ORFs are predicted to fuse through splicing to form a replication-associated protein (Rep). Data obtained using high-throughput sequencing (RNA-Seq) of three RNA-enriched populations, extracted from GRBV-infected grapevine (Vitis vinifera), confirmed the presence of the predicted C1–C2 intron (nts 2288–2450), but in addition identified a larger virion-sense intron (nts 251–589) spanning the V2 ORF. Evidence for both introns in a number of isolates was supported by bioinformatic analysis of publicly available datasets (n=20). These observations were further supported by RT-PCR analyses in both GRBV-infected grapevine and transient expression assays where GRBV genome segments were agro-inoculated onto Nicotiana benthamiana. The donor site of the virion-sense intron is located within two small ORFs, V0 and V02, while the acceptor site is two-thirds along the V2 ORF. Splicing at these positions is predicted to delete the N terminus of the encoded V2 protein. Comparative analyses of full-length GRBV sequences and the related tentative grabloviruses Prunus geminivirus A and wild Vitis virus 1 support the existence of both introns and V0. The probable regulatory role of these introns in the GRBV infection cycle is discussed.

A survey for the presence of Grapevine virus E (GVE, genus Vitivirus, family Betaflexiviridae) in vineyards in New York and California was conducted using macroarray hybridization or reverse-transcription polymerase chain reaction (RT-PCR) assays. In New York, GVE was detected in 10 of 46 vines of Vitis labrusca, one V. riparia, and one Vitis hybrid. All GVE-infected New York vines were coinfected with Grapevine leafroll-associated virus-3. In California, GVE was detected in 8 of 417 vines of V. vinifera. All GVE-infected California vines were also coinfected by one of the leafroll-associated viruses and other vitiviruses. In order to assess the genetic diversity among GVE isolates, a viral cDNA was amplified by RT-PCR, and a 675-nucleotide region that included the 3' terminus of the coat protein gene, a short intergenic region, and the 5' terminus of the putative nucleic acid binding protein gene was sequenced. All 20 GVE isolates sequenced in this study were very closely related, with >98% nucleotide identity to the SA94 isolate from South Africa. These findings confirm the presence of GVE in major grape-growing regions of the United States and indicate a very low level of genetic diversity. © 2016 The American Phytopathological Society.

In the early 2000s an epidemic of cucumber mosaic virus (CMV) spread within the Midwestern and Eastern US affecting snap and dry bean (Phaseolus vulgaris L.) cultivation. Fifty one CMV isolates from this period were partially characterized from varied hosts by sequencing a section from each of the three genomic RNAs. Aside from one subgroup II strain from pepper, all isolates, including those from snap bean, fell within the IA subgroup. The nucleotide sequence diversity of virus populations sampled at multiple sites and at different years was significantly higher than that of a population from single site in a single year, although in general the number of polymorphisms was low (

Grapevine red blotch virus (GRBV) has a monopartite single-stranded DNA genome and is the type species of the genus Grablovirus in the family Geminiviridae. To address the etiological role of GRBV in the recently recognized red blotch disease of grapevine, infectious GRBV clones were engineered from the genome of each of the two previously identified phylogenetic clades for Agrobacterium tumefaciens-mediated inoculations of tissue culture-grown Vitis spp. plants. Following agroinoculation and one or two dormancy cycles, systemic GRBV infection was detected by multiplex polymerase chain reaction (PCR) in Vitis vinifera exhibiting foliar disease symptoms but not in asymptomatic vines. Infected rootstock genotype SO4 (V. berlandieri × V. riparia) exhibited leaf chlorosis and cupping, while infection was asymptomatic in agroinoculated 110R (V. berlandieri × V. rupestris), 3309C (V. riparia × V. rupestris), and V. rupestris. Spliced GRBV transcripts of the replicase-associated protein coding region accumulated in leaves of agroinfected vines, as shown by reverse-transcription PCR; this was consistent with systemic infection resulting from virus replication. Additionally, a virus progeny identical in nucleotide sequence to the infectious GRBV clones was recovered from agroinfected vines by rolling circle amplification, cloning, and sequencing. Concomitantly, subjecting naturally infected grapevines to microshoot tip culture resulted in an asymptomatic plant progeny that tested negative for GRBV in multiplex PCR. Altogether, our agroinoculation and therapeutic experiments fulfilled Koch’s postulates and revealed the causative role of GRBV in red blotch disease.

Grapevine red blotch disease has been recognized since 2008 as affecting North American grape production. The presence of the newly described Grapevine red blotch-associated virus (GRBaV) is highly correlated with the disease. To more effectively detect and monitor the presence of the virus, a sample processing strategy and multiplex polymerase chain reaction assay were developed. A total of 42 of 113 vine samples collected in or received from seven of the United States were shown to harbor the virus, demonstrating the virus is widely distributed across North America. Phylogenetic analyses of a viral replication-associated protein (Rep) gene fragment from the 42 isolates of GRBaV demonstrated distinct clades of the virus (1 and 2), with clade 1 showing the greatest variability. The full-length genome of six virus isolates was sequenced, and phylogenetic analyses of 14 whole genomes recapitulated results seen for the Rep gene. A comparison of GRBaV genomes revealed evidence of recombination underlying some of the variation seen among GRBaV genomes within clade 1. Phylogenetic analyses of coat and replicase-associated protein sequences among single-stranded DNA viruses showed GRBaV to group within the family Geminiviridae. This grouping is distinct from members of the families Nanoviridae and Circoviridae, with limited significant affinities to both recognized genera and novel plant-infecting, gemini-like viruses.

Crop-specific diagnostics to simultaneously detect a large number of pathogens provides an invaluable platform for the screening of vegetative material prior to its propagation. Here we report the use of what is to-date the largest published example of a crop-specific macroarray for the detection of 38 of the most prevalent or emergent viruses to infect grapevine. The reusable array consists of 1,578 virus-specific 60 to 70mer oligonucleotide probes and 19 plant and internal control probes spotted onto an 18 × 7 cm nylon membrane. In a survey of 99 grapevines from the United States and Europe, virus infections were detected in 46 selections of Vitis vinifera, V. labrusca, and interspecific hybrids. The majority of infected vines (30) was singly infected, while 16 were mixed-infected with viruses from two or more families. Representatives of the four main virus families Betaflexiviridae, Closteroviridae, Secoviridae, and Tymoviridae present in grapevines were found alone and in combination, with a notable bias in representation by members of the family Tymoviridae. This work demonstrates the utility of the macroarray platform for the multiplex detection of viruses in a single crop, its potential for characterizing grapevine virus associations, and usefulness for rapid diagnostics of introduced material in quarantine centers or in certification programs. © 2014 The American Phytopathological Society.

In analyzing grapevine clones infected with grapevine red blotch associated virus, we identified a small number of isometric particles of approximately 30 nm in diameter from an enriched fraction of leaf extract. A dominant protein of 25 kDa was isolated from this fraction using SDS-PAGE and was identified by mass spectrometry as belonging to grapevine asteroid mosaic associated virus (GAMaV). Using a combination of three methods RNA-Seq, sRNA-Seq, and Sanger sequencing of RT- and RACE-PCR products, we obtained a full-length genome sequence consisting of 6719 nucleotides without the poly(A) tail. The virus possesses all of the typical conserved functional domains concordant with the genus Marafivirus and lies evolutionarily between citrus sudden death associated virus and oat blue dwarf virus. A large shift in RNA-Seq coverage coincided with the predicted location of the subgenomic RNA involved in coat protein (CP) expression. Genus wide sequence alignments confirmed the cleavage motif LxG(G/A) to be dominant between the helicase and RNA dependent RNA polymerase (RdRp), and the RdRp and CP domains. A putative overlapping protein (OP) ORF lacking a canonical translational start codon was identified with a reading frame context more consistent with the putative OPs of tymoviruses and fig fleck associated virus than with those of marafiviruses. BLAST analysis of the predicted GAMaV OP showed a unique relatedness to the OPs of members of the genus Tymovirus .

Red blotch is an emerging disease of grapevine associated with grapevine red blotch-associated virus (GRBaV). The virus spreads with infected planting stocks but no vector of epidemiological significance has been conclusively identified. A vineyard block of red-blotch-affected Vitis vinifera ‘Cabernet franc’ clone 214 was observed in California, with a clustering of infected, symptomatic vines focused along one edge of the field proximal to a riparian habitat with free-living Vitis spp. No genetic heterogeneity was observed in a 587-nucleotide region of the GRBaV genome in a population of 44 Cabernet franc clone 214 isolates. By contrast, genetic differences were observed in isolates from other cultivars and clones growing in adjacent blocks. GRBaV was confirmed infecting four free-living vines, two of which were shown to be V. californica × V. vinifera hybrids. The genomes of three free-living GRBaV vine isolates and seven from V. vinifera cultivars were compared; free-living vine isolates were shown to be more similar to each other and a ‘Merlot’ isolate than to the other cultivated vine isolates. The finding that GRBaV is present in free-living Vitis spp. indicates the virus can be spread by natural (nonhuman-mediated) means, and we hypothesize that in-field spread of GRBaV is occurring.

The grapevine leafroll-associated viruses (GLRaVs) (Closteroviridae) represent an emerging threat to world grape production. One group of GLRaVs within the genus Ampelovirus, the GLRaV-4-like viruses (GLRaV-4LVs), contains a fragmented collection of seven viruses only two of which (GLRaV-Pr and GLRaCV) are fully sequenced. Here in reporting the sequence of GLRaV-5, a member of GLRaV-4LVs, we identify genomic elements common to the GLRaV-4LV group. Exclusive properties include a highly conserved p5 gene product and phylogenies for complete genes that, except for the p23 gene, are reliably monophyletic. In comparison with other members of the genus Ampelovirus, GLRaV-4LVs form a tight cluster for all genes analyzed. In addition, they all possess a conserved AlkB domain which is most similar to the more distantly related GLRaV-3, suggesting recombination. In silico RNA structural analyses revealed a conserved five stem-loop structure at the 3' untranslated region that extends to all GLRaV-4LVs, and the ampeloviruses Pineapple mealybug wilt-associated virus 1 and Pineapple mealybug wilt-associated virus 3. A conserved G-U rich stem loop was also found upstream of the ORF1a stop and 1b start codons. Taken together, this work allows for a more thorough contextualization of GLRaV-5 and the GLRaV-4LVs as a group within the genus Ampelovirus.

The uncapped and ORF-less subgenomic RNA5 is produced in subgroup II strains of cucumber mosaic virus (CMV), but not in subgroup I strains. Its initiation nucleotide (nt 1903) is in a 21 nt conserved sequence (Box1) that is absent in CMV subgroup I. Putative non-coding RNA structural elements surrounding Box1 in the plus and minus strand were identified in silico and by in vitro RNase probing. Four main stem–loop structures (SLM, SLL, SLK and SLJ) were identified between nt 1887 and 1999 of isolate R-CMV (subgroup II), with notable differences within SLM and SLL between the two strands. Mutation of a stem–loop within SLM, even when the predicted wild-type structure was maintained, showed significant reduction in RNA5 levels in planta. Three mutants containing 3–4 nt substitutions between positions −39 and +49 showed significantly reduced levels of RNA5, while another similar mutant at positions 80–83 had RNA5 levels comparable to wild-type. Deletion of Box1 resulted in similar levels of RNA3 and 4 as wild-type, while eliminating RNA5. Insertion of Box1 into a subgroup I isolate was not sufficient to produce RNA5. However, in a mutant with an additional 21 nt of R-CMV 3′ of Box1 (positions −1 to +41), low levels of RNA5 were detected. Taken together, these results have identified regions of the viral genome responsible for RNA5 production and in addition provide strong evidence for the existence of newly identified conserved structural elements in the 5′ part of the 3′ untranslated region.

Plant genetic engineering has promised researchers improved speed and flexibility with regard to the introduction of new traits into cultivated crops. A variety of approaches have been applied to produce virus-resistant transgenic plants, some of which have proven to be remarkably successful. Studies on transgenic resistance to Cucumber mosaic virus probably have been the most intense of any plant virus. Several effective strategies based on pathogen-derived resistance have been identified; namely, resistance mediated by the viral coat protein, the viral replicase, and post-transcriptional gene silencing. Techniques using non-pathogen-derived resistance strategies, some of which could offer broader resistance, generally have proven to be much less effective. Not only do the results obtained so far provide a useful guide to help focus on future strategies, but they also suggest that there are a number of possible mechanisms involved in conferring these resistances. Further detailed studies on the interplay between viral transgene-derived molecules and their host are needed in order to elucidate the mechanisms of resistance and pathogenicity.

International audience; Virus-resistant transgenic plants have been created primarily through the expression of viral sequences. It has been hypothesized that recombination between the viral transgene mRNA and the RNA of an infecting virus could generate novel viruses. As mRNA/viral RNA recombination can occur in virus-resistant transgenic plants, the key to testing this risk hypothesis is to compare the populations of recombinant viruses generated in transgenic and non-transgenic plants. This has been done with two cucumoviral systems, involving either two strains of cucumber mosaic virus (CMV), or CMV and the related tomato aspermy virus (TAV). Although the distribution of the sites of recombination in the CMV/CMV and TAV/CMV systems was quite different, equivalent populations of recombinant viruses were observed in both cases. These results constitute the first comparison of the populations of recombinants in transgenic and non-transgenic plants, and suggest that there is little risk of emergence of recombinant viruses in these plants, other than those that could emerge in non-transgenic plants

Poly-(β)-hydroxybutyric acid (PHB) is a biodegradable, thermoplastic biopolyester produced as a carbon-energy storage source by bacteria that are challenged by nutrient limitation. While production of PHB for industrial applications is generally accomplished via fermentation, in vitro enzymatic synthesis can be an attractive alternative. We now report a detailed analysis of the in vitro biosynthesis of PHB using the three-enzyme system from Wautersia eutropha . We have developed quantitative HPLC methodology which resolves CoA, acetyl-CoA, acetoacetyl-CoA, and β-hydroxybutyryl-CoA with baseline resolution and then utilized this methodology to elucidate the fluctuations in metabolite concentrations and the effect of each individual enzyme on the overall system. The experimental results were then used to design the first kinetic model for in vitro PHB biosynthesis. All previous modeling studies of PHB production have been designed to analyze in vivo biosyntheses, typically in bacteria but also in yeast and plants. From a biotechnological viewpoint, in vitro enzymatic synthesis has the advantage of obviating limitations related to cellular uptake and possible toxicity of substrates, thereby making it possible to utilize a much wider range of substrates for production of novel homopolymers or copolymers. Our results can therefore serve as a basis for further development of the potential of in vitro enzymatic syntheses of polyesters as a complement to the more widely used approach of in vivo production of these polymers.

In the early 2000s an epidemic of cucumber mosaic virus (CMV) spread within the Midwestern and Eastern US affecting snap and dry bean (Phaseolus vulgaris L.) cultivation. Fifty one CMV isolates from this period were partially characterized from varied hosts by sequencing a section from each of the three genomic RNAs. Aside from one subgroup II strain from pepper, all isolates, including those from snap bean, fell within the IA subgroup. The nucleotide sequence diversity of virus populations sampled at multiple sites and at different years was significantly higher than that of a population from single site in a single year, although in general the number of polymorphisms was low (11%). Complementary DNA (cDNA) clones of Bn57, a representative isolate from snap bean, were engineered for the production of infectious in vitro RNA transcripts initiated from a T7 promoter. Infections from these cDNAs resulted in symptoms consistent with those of the original field isolate, indicating that a satellite RNA is not involved in symptom expression in snap bean. These infectious clones were used to assess symptom determinants and the effects of virus infection on plant growth. Inoculations with pseudorecombinants derived from Bn57 and the non-bean infecting strain Fny confirmed RNA2 as a specific determinant for snap bean infection. Bn57, along with almost all isolates identified in this study contained the Y631 locus in the 2a protein, a determinant for systemic infection in bean. The presence of this locus extended to all non-bean hosts except two pepper infecting isolates. Infection by Bn57 in snap bean had a significant effect on pod number and mass with a 55 and 41 percent reduction in greenhouse assays, respectively. To our knowledge Bn57 is the first CMV strain isolated from P. vulgaris to be fully sequenced and cloned, providing a useful tool for analyses of CMV-host interactions.

Torradoviruses are an example of a group of recently discovered plant viruses. The first description of Tomato torrado virus, now the type member of the newly established genus Torradovirus within the family Secoviridae, was published in 2007 and was quickly followed by findings of other torradoviruses, initially all on tomato. Their characterization led to the development of tools that allowed recognition of still other torradoviruses, only very recently found on non-tomato crops, which indicates these viruses have a much wider host range and diversity than previously believed. This review describes the characteristics of this newly emerged group of plant viruses. It looks in detail at taxonomic relationships and specific characteristics in their genomes and encoded proteins. Furthermore, it discusses their epidemiology, including host range, semipersistent transmission by whitefly vectors, and impact on diverse cropping systems.

The single-stranded, positive-sense and tripartite RNA virus Cucumber mosaic virus (CMV) was used in this study as a method for monitoring the initial stages of virus infection following aphid transmission. The RNA2 of CMV was modified to incorporate, in a variety of arrangements, an open reading frame (ORF) encoding an enhanced green fluorescent protein (eGFP). The phenotypes of five engineered RNA2s were tested in Nicotiana tabacum, Nicotiana clevelandii and Nicotiana benthamiana. Only one construct (F4), in which the 2b ORF was truncated at the 3′ end and fused in-frame with the eGFP ORF, was able to systemically infect N. benthamiana plants, express eGFP and be transmitted by the aphid Myzus persicae. The utility of this construct was demonstrated following infection as early as one day post-transmission (dpt) continuing through to systemic infection. Comparisons of the inoculation sites in different petiole sections one to three dpt clearly showed that the onset of infection and eGFP expression always occurred in the epidermal or collenchymatous tissue just below the epidermis; an observation consistent with the rapid time frame characteristic of the non-persistent mode of aphid transmission.

Grapevine red blotch-associated virus is a recently discovered plant monopartite gemini-like virus found in North American grapevines. Leaf discoloration and a decrease in fruit quality are associated with its infection. Two of its six open reading frames (ORFs), V2 and V3, are of unknown function and share no obvious homology with plant or viral genes. Transient expression of these ORFs in fusion with the green fluorescent protein demonstrated that the V2 protein localizes in the nucleoplasm, Cajal bodies, and cytoplasm; and the V3 protein localizes in various unidentified subnuclear bodies. Additionally, the V2 protein is redirected to the nucleolus upon co-expression with the nucleolus and Cajal body-associated protein Fib2.

On constructing a cDNA library for potato, 'contaminating' sequences with a significant identity to Apple latent spherical virus (ALSV) were found. Determination of the remaining genome sequence indicated the presence of a bipartite virus with an RNA1 and 2 of 7034 and 3315 nucleotides, respectively, excluding a poly(A)tail. RNA1 encodes a single polyprotein (233 kDa) and shares highest amino acid identity with ALSV at 65%. Conserved amino acid motifs typical for helicase, protease and RNA-dependent polymerase (RdRp) functions are present. RNA2 encodes a single polyprotein (106 kDa) with amino acid identities to the flat apple isolate of Cherry rasp leaf virus (CRLV-FA) (97%) and ALSV (70%), suggesting this is a potato strain of CRLV (CRLV-pot). Phylogenetic analysis using the RdRp region shows that this virus falls within a group separate from the Comoviridae that includes members of the Sequiviridae and the taxonomically unassigned viruses ALSV, Strawberry mottle virus, Satsuma dwarf virus and Navel orange infectious mottling virus. Other regions of the genome have highest identities with both plant and animal infecting members of the picorna-like virus superfamily. The evolutionary context of CRLV-pot and related viruses is discussed. Similar viral sequences from an EST library of peppermint are also analysed.

Systemic movement of Cucumber green mottle mosaic virus (CGMMV) in cucumber plants was shown to be from photoassimilate source to sink, thus indicating phloem transport. Nevertheless, CGMMV was not detected by immunocytochemical procedures in the intermediary cell-sieve element complex in inoculated cotyledons, where photoassimilate loading occurs. In stem internodes, CGMMV was first localized in the companion cells of the external phloem and subsequently in all tissues except the medulla, therefore suggesting leakage of the virus from, and reloading into, the transport phloem during systemic movement. In systemically infected sink leaves, CGMMV was simultaneously detected in the xylem and phloem. Interestingly, CGMMV accumulated to high levels in the differentiating tracheids of young leaves implying that the xylem could be involved in the systemic movement of CGMMV. This possibility was tested using plants in which cell death was induced in a portion of the stem by steam treatment. At 24 degrees C, steam treatment effectively prevented the systemic movement of CGMMV, even though viral RNA was detected in washes of the xylem above the steamed internode suggesting that xylem circulation occurred. At 29 degrees C, CGMMV systemically infected steam-treated cucumber plants, indicating that CGMMV can move systemically via the xylem. Xylem transport of CGMMV was, however, less efficient than phloem transport in terms of the time required for systemic infection and the percentage of plants infected.

The principal aphid-borne viruses infecting Strawberry (Fragaria spp.) Strawberry crinkle virus (SCV), Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV) can cause serious crop losses. In this paper, a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) method is described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which can be used as an indicator of the effectiveness of the extraction and RT-PCR. In total, 18 strawberry isolates infected naturally were analysed by this method. Every combination of RNA virus was able to be detected and a full complement of all four viruses were found together in three isolates, all taken from wild strawberry (Fragaria chiloensis (L.) Duch.) in Chile. The upper detection limit for the four viruses was at an extract dilution of 1/200. The broad applicability of the RNA specific internal control primers—which produced a PCR fragment of the expected size in 25 of 27 plant species tested—combined with improvements, made in extraction methods described provides potentially a standard method for comparable RT-PCR analyses in a wide variety of plant species.

Strawberry mottle virus (SMoV) is probably the most important virus to infect strawberry (Fragaria spp.). All species of strawberry are susceptible to SMoV, resulting in severe losses both in fruit and runner yield. However, due to the absence of definitive symptoms in commercial varieties, the only effective means of detecting SMoV is by transmission to susceptible indicator plants. In this study, we describe a reverse transcription-polymerase chain reaction (RT-PCR) method for the detection of SMoV in Fragaria spp. with the use of primers specific for the noncoding regions (NCR) of both RNA1 and RNA2 of the virus. Using this method, all of 16 isolates from various geographical origins were positive for SMoV. Partial sequences of a 327-nt long coding region were obtained for the putative large coat protein of all isolates by RT-PCR using degenerate primers. Nucleotide identities between isolates ranged from 72.8 to 99.7%. A 546-nt sequence of the putative polymerase gene of nine isolates was obtained by RT-PCR and compared. Nucleotide identities ranged from 73.4 to 100%. There was a clear tendency for isolates to group according to their geographical origin. Sequence data obtained of the NCR show four completely conserved regions of 20 or more bases.

The plant-infecting Secoviridae family of viruses forms part of the Picornavirales order, an important group of non-enveloped viruses that infect vertebrates, arthropods, plants and algae. The impact of the secovirids on cultivated crops is significant, infecting a wide range of plants from grapevine to rice. The overwhelming majority are transmitted by ecdysozoan vectors such as nematodes, beetles and aphids. In this study, we have applied a variety of computational methods to examine the evolutionary traits of these viruses. Strong purifying selection pressures were calculated for the coat protein (CP) sequences of nine species, although for two species evidence of both codon specific and episodic diversifying selection were found. By using Bayesian phylogenetic reconstruction methods CP nucleotide substitution rates for four species were estimated to range from between 9.29×10(-3) to 2.74×10(-3) (subs/site/year), values which are comparable with the short-term estimates of other related plant- and animal-infecting virus species. From these data, we were able to construct a time-measured phylogeny of the subfamily Comovirinae that estimated divergence of ninety-four extant sequences occurred less than 1,000 years ago with present virus species diversifying between 50 and 250 years ago; a period coinciding with the intensification of agricultural practices in industrial societies. Although recombination (modularity) was limited to closely related taxa, significant and often unique similarities in the protein domains between secovirid and animal infecting picorna-like viruses, especially for the protease and coat protein, suggested a shared ancestry. We discuss our results in a wider context and find tentative evidence to indicate that some members of the Secoviridae might have their origins in insects, possibly colonizing plants in a number of founding events that have led to speciation. Such a scenario; virus infection between species of different taxonomic kingdoms, has significant implications for virus emergence.

Random amplified polymorphic DNA (RAPD) analysis was performed on 29 isolates of Botrytis cinerea Pers. ex Fr. isolated from table grapes (Vitis vinifera L.) and other crops in Chile with 29 decaprimers. No single primer was found to differentiate either the host or the geographical origin of each of the B. cinerea isolates tested. The DNA profiles obtained, particularly with primers OPA4 and OPA11, distinguished isolates of B. cinerea from other epiphyte fungi found on table grapes, including Alternaria alternata, Aspergillus niger, Cladosporium herbarum, Epiccocum nigrum, Rhizopus stolonifer, a Penicillium sp., and yeasts (Cryptococcus laurentii, Rhodotorula glutinis, and Saccharomyces cerevisiae). Regardless of host origin, primers OPA4 and OPA11 amplified a strong fragment of 1.2 kilobases (kb) and two fragments of 1.10 and 0.7 kb, respectively. These DNA fragments were obtained even when only one conidium of B. cinerea was in the test sample. Three main groups were clearly defined based on the genetic similarities found in additional RAPD analysis with 19 arbitrary decaprimers and 15 selected isolates of B. cinerea. The overall similarity coefficients (SC) between the groups obtained ranged from 0.326 to 0.891. Interestingly, all isolates from table grapes were included in group I (SC: 0.761 to 0.826), isolates from apple and tomato were in group II (SC: 0.739 to 0.848), while isolates from blueberry were either in group I (SC: 0.804) or III (SC: 0.673). Consequently, the genetic variability determined by RAPD analysis among these B. cinerea isolates suggested a possible host:pathogen relationship. However, further research is needed to clarify its pathological significance.

In a limited survey of commercial vineyards and a germplasm repository in Ontario County, NY, 20 vines of Vitis sp. were tested in fall and spring 2010 to 2012 for viruses using a double-antibody sandwich (DAS)-ELISA and macroarray with oligonucleotide probes for grapevine viruses ((3) and unpublished). The plants selected for analysis included those showing atypical growth including leaf deformation, yellowing, cupping or spotting, vein clearing, shortening of internodes, and reduced vigor. Arabis mosaic virus (ArMV; genus Nepovirus, family Secoviridae) was detected in leaf tissue and wood scrapings in two vines using the DAS-ELISA with antibodies from Bioreba (Reinach, Switzerland). The ArMV positive vines were from Vitis hybrid cultivars Noah and Geisenheim 26. ArMV was also detected in these two vines using the macroarray, with hybridization observed to 24 of 32 oligonucleotide probes specific to this virus. To confirm the identification of the virus, total RNAs were extracted from leaf tissues, hybridized with random hexamers, and reverse-transcribed using MMLV reverse transcriptase (Life Technologies, Grand Island, NY). Complementary DNAs were amplified by PCR using an IQ supermix (BioRad, Hercules, CA), and two sets of generic primers for nepoviruses (1,4). Thermocycler conditions were 94°C 5 min (1×); 94°C 30 s, 50°C 30 s, and 69°C 2 min (35×), and 72°C for 5 min. The PCR products were sequenced directly. Sequences from the 340-bp products obtained from cultivars Geisenheim 26 (GenBank Accession No. HE984333) and Noah (HE984334) using the Wei et al. primers (4) had 76 to 84% sequence identity to ArMV RNA1 GenBank accessions GQ369528 and AY303786. Sequences from the 301-bp products obtained from cultivars Geisenheim 26 (HE984335) and Noah (HE984336) using the Digiaro et al. primers (1) had 87 to 91% sequence identity to ArMV RNA2 GenBank accessions AY017339 and X81814. ArMV was mechanically transmitted from Geisenheim 26 to Nicotiana tabacum cultivar Xanthi NN. Inoculation gave rise to necrotic local lesions on the inoculated leaves of five plants in each of two experiments (10 of 10 plants total). The presence of ArMV in tobacco was confirmed by DAS-ELISA. Thus, the presence of ArMV in New York grapevines has been confirmed by the detection of the coat protein antigen, virus specific oligonucleotide probes, and the sequencing of portions of both genomic RNAs. There are limited reports of ArMV in North America and in grapevine in particular (2), but with a wide host range and seed and nematode transmissibility, ArMV has the ability to become more widespread among grapevine and other crops. References: (1) M. Digiaro, et al. J. Virol. Methods 141:34, 2007. (2) B. N. Milkus et al. Am. J. Enol. Vitic. 50:56, 1999. (3) J. Thompson et al. J. Virol. Methods 183:161, 2012. (4) T. Wei et al. J. Virol. Methods 153:16, 2008.

Grapevine leafroll-associated viruses (GLRaVs) are an emerging group of viruses that represent a significant threat to the global productivity and sustainability of the grapevine industry. Their control is achieved through the identification and elimination of infected vines, and the use of planting material derived from virus-tested, certified stocks. As such, much effort has been invested in developing reliable molecular diagnostic techniques. In this work, we report the development of a macroarray assay for the detection of the principal GLRaVs. In total 314 70-mer oligonucleotides specific to GLRaV-1, -2, -3, -4, -7, and GLRaV-4 strains 5, 6, 9 and Pr were spotted onto a 11 × 7 cm nylon membrane. Thirty-four grapevine samples from various origins were tested by the macroarray, RT-PCR and ELISA. Thirty were positive for virus infection using RT-PCR, 28 by ELISA and 25 by the macroarray. Mixed infections were identified by macroarray in two samples and confirmed by RT-PCR or ELISA. There were a few discrepancies between methods that were most likely due to differences in the sensitivity of detection, and in the case of the macroarray, limitations in the sequence data available for certain virus species in the design of the oligonucleotides. This work demonstrates the successful application of macroarray methodology using randomly primed and sequence-nonspecific amplified cDNAs derived from grapevine total RNA extracts, and provides a proof-of-principal for unbiased multiplex detection using a single robust platform.

An important property of some spherical plant viruses is their ability to reassemble in vitro from native capsid protein (CP) and RNA into infectious virus-like particles (VLPs). Virions of cucumber mosaic virus (CMV) are stabilized by protein–RNA interactions and the nucleic acid is essential for assembly. This study demonstrated that VLPs will form in the presence of both ssDNA and dsDNA oligonucleotides, and with a lower size limit of 20 nt. Based on urea disruption assays, assembled VLPs from CMV CP and RNA (termed ReCMV) exhibited a level of stability similar to that of virions purified from plants, whilst VLPs from CMV CP and a 20mer exhibited comparable or greater stability. Fluorescent labelling of VLPs was achieved by the encapsidation of an Alexa Fluor 488-labelled 45mer oligonucleotide (ReCMV-Alexa488-45) and confirmed by transmission electron and confocal microscopy. Using ssDNA as a nucleating factor, encapsidation of fluorescently labelled streptavidin (53 kDa) conjugated to a biotinylated oligonucleotide was observed. The biological activity and stability of ReCMV and ReCMV-Alexa488-45 was confirmed in infectivity assays and insect vector feeding assays. This work demonstrates the utility of CMV CP as a protein cage for use in the growing repertoire of nanotechnological applications.

International audience; Severe symptoms of cotton leaf curl disease (CLCuD) are caused by the association of a single-stranded circular DNA satellite (betasatellite) with a helper begomovirus. In this study, we analyzed 40 leaf samples (primarily cotton with CLCuD symptoms and other plants growing close by) from four sites between New Delhi and the Pakistan/India border, using rolling-circle amplification (RCA) and PCR. In total, the complete sequences of 12 different helper viruses, eight alphasatellites, and one betasatellite from five different plant species were obtained. A recombinant helper virus molecule found in okra and a novel alphasatellite-related DNA from croton are also described. This is the first report of the presence of both DNA components (helper virus and betasatellite) associated with resistance-breaking CLCuD in India, and it highlights the need for further work to combat its damage and spread.