Yeast Expressing a Phage Endolysin Reduces Endogenous in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from exhibits lytic activity against (CP) . The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast () was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log 4.

Coding-complete genome sequence of an isolate of papaya virus E in tomato.

An isolate of papaya virus E was identified in tomato fruits from Mexico. The coding-complete genome sequence was determined using high-throughput sequencing. The coding-complete genome is 13,412 nucleotides and contains 8 open reading frames.

Wheat Pore-Forming Toxin-Like Protein Confers Broad-Spectrum Resistance to Fungal Pathogens in .

Fusarium head blight (FHB), caused by the hemibiotrophic fungus , is one of the major threats to global wheat productivity. A wheat pore-forming toxin-like (PFT) protein was previously reported to underlie , the most widely used quantitative trait locus in FHB breeding programs worldwide. In the present work, wheat PFT was ectopically expressed in the model dicot plant .

Mexico: A Landscape of Viroid Origin and Epidemiological Relevance of Endemic Species.

Viroids are single-stranded, circular RNA molecules (234-406 nt) that infect a wide range of crop species and cause economic losses in agriculture worldwide. They are characterized by the existence of a population of sequence variants, attributed to the low fidelity of RNA polymerases involved in their transcription, resulting in high mutation rates. Therefore, these biological entities exist as .

Insights into the Transcriptional Reprogramming in Tomato Response to PSTVd Variants Using Network Approaches.

Viroids are the smallest 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 mechanisms of host viroid response, we implemented network approaches to identify master transcription regulators and their differentially expressed targets in tomato infected with mild and severe variants of PSTVd.

Phytoplasma Infection Blocks Starch Breakdown and Triggers Chloroplast Degradation, Leading to Premature Leaf Senescence, Sucrose Reallocation, and Spatiotemporal Redistribution of Phytohormones.

Witches'-broom (WB, excessive initiation, and outgrowth of axillary buds) is one of the remarkable symptoms in plants caused by phytoplasmas, minute wall-less intracellular bacteria. In healthy plants, axillary bud initiation and outgrowth are regulated by an intricate interplay of nutrients (such as sugars), hormones, and environmental factors. However, how these factors are involved in the induction of WB by phytoplasma is poorly understood.

Cloning and Sequencing of Viroids.

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.

Extraction and Purification of Viroids from Herbaceous Hosts.

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.

Detection and Characterization of Viroids via Biological Assays on Herbaceous Hosts.

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).

Rapid diagnostic detection of tomato apical stunt viroid based on isothermal reverse transcription-recombinase polymerase amplification.

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® Acceler8™ and AmplifyRP® XRT, expressed in the absence of any cross-reaction activity to all total RNA extracts obtained from plants infected with other pospiviroids.

Development and optimization of a pepino mosaic virus-based vector for rapid expression of heterologous proteins in plants.

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.

Identification and characterization of Miscanthus yellow fleck virus, a new polerovirus infecting Miscanthus sinensis.

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.

Plant Virus-Derived Vectors: Applications in Agricultural and Medical Biotechnology.

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.

Complete Genome Sequence of an American Isolate of Pepino Mosaic 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.

Optimized production of a biologically active Clostridium perfringens glycosyl hydrolase phage endolysin PlyCP41 in plants using virus-based systemic expression.

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.

Complete Genome Sequence of a Little Cherry Virus-2 Isolate from Sweet Cherry in China.

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 with close relationships to previously reported LChV-2 isolates.

Antimicrobial activity of bacteriophage derived triple fusion protein against .

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 () 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 Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus.

Pospiviroid Infection of Tomato Regulates the Expression of Genes Involved in Flower and Fruit Development.

Viroids are unencapsidated, single-stranded, covalently-closed circular, highly structured, noncoding RNAs of 239⁻401 nucleotides that cause disease in several economically important crop plants. In tomato ( cv. Rutgers), symptoms of pospiviroid infection include stunting, reduced vigor, flower abortion, and reduced size and number of fruits, resulting in significant crop losses.

Characterization of alfalfa virus F, a new member of the genus Marafivirus.

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.

Engineering resistance against viroids.

Viroids, the smallest infectious agents endowed with autonomous replication, are tiny single-stranded circular RNAs (∼250 to 400nt) 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.

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification.

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.

Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

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.

Complete nucleotide sequence of little cherry virus 1 (LChV-1) infecting sweet cherry in China.

Little cherry virus 1 (LChV-1), associated with little cherry disease (LCD), has a significant impact on fruit quality of infected sweet cherry trees. We report the full genome sequence of an isolate of LChV-1 from Taian, China (LChV-1-TA), detected by small-RNA deep sequencing and amplified by overlapping RT-PCR. The LChV-1-TA genome was 16,932 nt in length and contained nine open reading frames (ORFs), with sequence identity at the overall genome level of 76%, 76%, and 78% to LChV-1 isolates Y10237 (UW2 isolate), EU715989 (ITMAR isolate) and JX669615 (V2356 isolate), respectively.

Antimicrobial Activity of Bacteriophage Endolysin Produced in Nicotiana benthamiana Plants.

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.

In silico prediction and validation of potential gene targets for pospiviroid-derived small RNAs during tomato infection.

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.