Conventional detection of viruses and virus-like diseases of plants is accomplished using a combination of molecular, serological, and biological indexing. These are the primary tools used by plant virologists to monitor and ensure trees are free of known viral pathogens. The biological indexing assay, or bioassay, is considered to be the “gold standard” as it is the only method of the three that can detect new, uncharacterized, or poorly characterized viral disease agents. Unfortunately, this method is also the most labor intensive and can take up to three years to complete. Next generation sequencing (NGS) is a technology with rapidly expanding possibilities including potential applications for the detection of plant viruses. In this study, comparisons are made between tree fruit testing by conventional and NGS methods, to demonstrate the efficacy of NGS. A comparison of 178 infected trees, many infected with several viral pathogens, demonstrated that conventional and NGS were equally capable of detecting known viruses and viroids. Comparable results were obtained for 170 of 178 of the specimens. Of the remaining eight specimens, some discrepancies were observed between viruses detected by the two methods, representing less than 5% of the specimens. NGS was further demonstrated to be equal or superior for the detection of new or poorly characterized viruses when compared with a conventional bioassay. These results validated both the effectiveness of conventional virus testing methods and the use of NGS as an additional or alternative method for plant virus detection.
Soybean consumption is increasing in many Western diets; however, recent reviews suggest that the prevalence of soy allergy can be as high as 0.5% for the general population and up to 13% for children. The immunoglobulin-E (IgE) binding of sera from six soy-sensitive adult human subjects to soybean proteins separated by 2D gel electrophoresis was studied. Synthetic peptide sets spanning the mature glycinin subunit A2 and A3 primary sequences were used to map the IgE-binding regions. Putative epitopes identified in this study were also localized on glycinin hexamer models using bioinformatics software. We identified linear IgE-binding epitopes of the major storage protein Gly m 6 by screening individual soy-sensitive patient sera. These epitopes were then further analysed by 3D in silico model localization and compared to other plant storage protein epitopes. Web-based software applications were also used to study the ability to accurately predict epitopes with mixed results. A total of nine putative IgE-binding epitopes were identified in the glycinin A3 (A3.1-A3.3) and A2 (A2.1-A2.6) subunits. Most patients' sera IgE bound to only one or two epitopes, except for one patient's serum which bound to four different A2 epitopes. Two epitopes (A3.2 and A2.4) overlapped with a previously identified epitope hot spot of 11S globulins from other plant species. Most epitopes were predicted to be exposed on the surface of the 3D model of the glycinin hexamer. Amino acid sequence alignments of soybean acidic glycinins and other plant globulins revealed one dominant epitope hot spot among the four reported hot spots. This study may be helpful for future development of soy allergy immunotherapy and diagnosis.
Cherry virus A (CVA) is a ubiquitous graft-transmissible virus that mainly infects Prunus spp. Next-generation sequencing was applied to 39 tree fruit specimens infected with CVA, and 75 full and 16 partial-length CVA genome sequences were assembled. Phylogenetic analysis of these and 11 previously sequenced CVA genomes resulted in six major clusters with no observable relationship between the host and the assembled genome sequences. Recombination analysis detected four recombinants. Consistent single-nucleotide polymorphism (SNP) patterns were observed between the 75 full-length genomes and their sequence clouds, which supports a quasispecies model for CVA evolution.
15-Acetyldeoxynivalenol (15-AcDONFusarium head blight of cereals and Fusarium ear rot of maize are caused by morphologically similar species (Fusarium graminearum and Fusarium culmorum, etc.) common throughout global agricultural regions. With few exceptions, Fusarium epidemics are characterized by cyclical and highly aggressive infection of commercial crops with economic impacts on food and feed industries that are immediate and far reaching. For example, losses associated with the most recent Fusarium outbreak in North America in the 1990s were estimated to range from 1.3 to 3.0 billion United States dollars (1).A toxin class commonly found within agricultural commodities infected by Fusarium are trichothecene mycotoxins. Trichothecenes represent a highly diverse group of over 180 sesquiterpenoid low molecular weight (typically 200 -500 Da) mycotoxins characterized by a tricyclic ring structure containing a double bond at C-9,10 and an epoxide group at C-13 (2). Regardless of size and structural composition, trichothecenes are potent inhibitors of eukaryotic protein synthesis with specific activity on ribosomal protein L3 within the 60 S subunit resulting in inhibition of peptidyltransferase activity (3, 4). Although the capacity to inhibit protein synthesis is regarded as central to trichothecene cytotoxicity (5, 6), adverse effects on eukaryotic cells may actually be attributed to dysregulation of cellular signaling and alterations in downstream gene expression (7). As a result, trichothecenes such as deoxynivalenol (DON), 2 15-acetyldeoxynivalenol (15-AcDON), and 3-acetyldeoxynivalenol (3-AcDON) (Fig. 1) are considered to be inherently hazardous feed-and foodborne contaminants (2,8).Numerous studies have demonstrated a correlation between in planta DON accumulation and Fusarium virulence in susceptible cultivars of wheat (9) and maize (10). Based on these findings, mechanisms that convey innate and acquired host plant resistance to DON and other trichothecene toxins have received considerable attention. To date, in planta trichothecene resistance has been achieved through mechanisms that alter targeted proteins within host cell ribosomes (11, 12), promote metabolic transformation to less toxic forms, e.g. DONglucosyl conjugate (13) or to 3-AcDON (14), and/or reduce intracellular concentrations to effectively limit mycotoxin exposure to sensitive cellular targets. Collectively, such research can be applied to impart novel mechanisms of trichothecene resistance in higher order plants.Yeast is well suited as a eukaryotic model organism to identify and validate mechanisms involved in host plant resistance to mycotoxins (12,13,15). Test systems based on yeast offer cost-effective convenience and flexibility as one can validate a wide range of novel detoxification mechanisms within a short period of time at a minimal cost using common/nonspecialized laboratory equipment. Assessment of mycotoxin resistance *
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