Activation of peroxymonosulfate by sustainable biomass-based carbon nanotubes for controlling the spread of plant viruses in water environments.

With the increasing demand for water in hydroponic systems and agricultural irrigation, viral diseases have seriously affected the yield and quality of crops. By removing plant viruses in water environments, virus transmission can be prevented and agricultural production and ecosystems can be protected. But so far, there have been few reports on the removal of plant viruses in water environments. Herein, in this study, easily recyclable biomass-based carbon nanotubes catalysts were synthesized with varying metal activities to activate peroxymonosulfate (PMS). Among them, the magnetic 0.125Fe@NCNTs-1/PMS system showed the best overall removal performance against pepper mild mottle virus, with a 5.9 log10 removal within 1 min. Notably, the key reactive species in the 0.125Fe@NCNTs-1/PMS system is 1O2, which can maintain good removal effect in real water matrices (river water and tap water). Through RNA fragment analyses and label free analysis, it was found that this system could effectively cleave virus particles, destroy viral proteins and expose their genome. The capsid protein of pepper mild mottle virus was effectively decomposed where serine may be the main attacking sites by 1O2. Long viral RNA fragments (3349 and 1642 nt) were cut into smaller fragments (∼160 nt) and caused their degradation. In summary, this study contributes to controlling the spread of plant viruses in real water environment, which will potentially help protect agricultural production and food safety, and improve the health and sustainability of ecosystems.

Interplay between a polerovirus and a closterovirus decreases aphid transmission of the polerovirus.

Multi-infection of plants by viruses is very common and can change drastically infection parameters such as virus accumulation, distribution, and vector transmission. Sugar beet is an important crop that is frequently co-infected by the polerovirus beet chlorosis virus (BChV) and the closterovirus beet yellows virus (BYV), both vectored by the green peach aphid (Myzus persicae). These phloem-limited viruses are acquired while aphids ingest phloem sap from infected plants. Here we found that co-infection decreased transmission of BChV by ~50% but had no impact on BYV transmission. The drastic reduction of BChV transmission was due to neither lower accumulation of BChV in co-infected plants nor reduced phloem sap ingestion by aphids from these plants. Using the signal amplification by exchange reaction fluorescent in situ hybridization technique on plants, we observed that 40% of the infected phloem cells were co-infected and that co-infection caused redistribution of BYV in these cells. The BYV accumulation pattern changed from distinct intracellular spherical inclusions in mono-infected cells to a diffuse form in co-infected cells. There, BYV co-localized with BChV throughout the cytoplasm, indicative of virus-virus interactions. We propose that BYV-BChV interactions could restrict BChV access to the sieve tubes and reduce its accessibility for aphids and present a model of how co-infection could alter BChV intracellular movement and/or phloem loading and reduce BChV transmission.IMPORTANCEMixed viral infections in plants are understudied yet can have significant influences on disease dynamics and virus transmission. We investigated how co-infection with two unrelated viruses, BChV and BYV, affects aphid transmission of the viruses in sugar beet plants. We show that co-infection reduced BChV transmission by about 50% without affecting BYV transmission, despite similar virus accumulation rates in co-infected and mono-infected plants. Follow-up experiments examined the localization and intracellular distribution of the viruses, leading to the discovery that co-infection caused a redistribution of BYV in the phloem vessels and altered its repartition pattern within plant cells, suggesting virus-virus interactions. In conclusion, the interplay between BChV and BYV affects the transmission of BChV but not BYV, possibly through direct or indirect virus-virus interactions at the cellular level. Understanding these interactions could be crucial for managing virus propagation in crops and preventing yield losses.

Transcriptomic profiling reveals the complex interaction between a bipartite begomovirus and a cucurbitaceous host plant.

BACKGROUND: Begomoviruses are major constraint in the production of many crops. Upon infection, begomoviruses may substantially modulate plant biological processes. While how monopartite begomoviruses interact with their plant hosts has been investigated extensively, bipartite begomoviruses-plant interactions are understudied. Moreover, as one of the major groups of hosts, cucurbitaceous plants have been seldom examined in the interaction with begomoviruses. RESULTS: We profiled the zucchini transcriptomic changes induced by a bipartite begomovirus squash leaf curl China virus (SLCCNV). We identified 2275 differentially-expressed genes (DEGs), of which 1310 were upregulated and 965 were downregulated. KEGG enrichment analysis of the DEGs revealed that many pathways related to primary and secondary metabolisms were enriched. qRT-PCR verified the transcriptional changes of twelve selected DEGs induced by SLCCNV infection. Close examination revealed that the expression levels of all the DEGs of the pathway Photosynthesis were downregulated upon SLCCNV infection. Most DEGs in the pathway Plant-pathogen interaction were upregulated, including some positive regulators of plant defenses. Moreover, the majority of DEGs in the MAPK signaling pathway-plant were upregulated. CONCLUSION: Our findings indicates that SLCCNV actively interact with its cucurbitaceous plant host by suppressing the conversion of light energy to chemical energy and inducing immune responses. Our study not only provides new insights into the interactions between begomoviruses and host plants, but also adds to our knowledge on virus-plant interactions in general.

An Abscopal Effect on Lung Metastases in Canine Mammary Cancer Patients Induced by Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Nanoparticles and Anti-Canine PD-1.

Neoadjuvant intratumoral (IT) therapy could amplify the weak responses to checkpoint blockade therapy observed in breast cancer (BC). In this study, we administered neoadjuvant IT anti-canine PD-1 therapy (IT acPD-1) alone or combined with IT cowpea mosaic virus therapy (IT CPMV/acPD-1) to companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. CMC patients treated weekly with acPD-1 (n = 3) or CPMV/acPD-1 (n = 3) for four weeks or with CPMV/acPD-1 (n = 3 patients not candidates for surgery) for up to 11 weeks did not experience immune-related adverse events. We found that acPD-1 and CPMV/acPD-1 injections resulted in tumor control and a reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of treated dogs. In two metastatic CMC patients, CPMV/acPD-1 treatments resulted in the control and reduction of established lung metastases. CPMV/acPD-1 treatments were associated with altered gene expression related to TLR1-4 signaling and complement pathways. These novel therapies could be effective for CMC patients. Owing to the extensive similarities between CMC and human BC, IT CPMV combined with approved anti-PD-1 therapies could be a novel and effective immunotherapy to treat local BC and suppress metastatic BC.

Defense Responses Induced by Viral Movement Protein and Its Nuclear Localization Modulate Virus Cell-to-Cell Transport.

Movement proteins (MPs) encoded by plant viruses are essential for cell-to-cell transport of viral genomes through plasmodesmata. The genome of hibiscus green spot virus contains a module of two MP genes termed 'binary movement block' (BMB), encoding the proteins BMB1 and BMB2. Here, BMB1 is shown to induce a defense response in Nicotiana benthamiana plants that inhibits BMB-dependent virus transport. This response is characterized by the accumulation of reactive oxygen species, callose deposition in the cell wall, and upregulation of 9-LOX expression. However, the BMB1-induced response is inhibited by coexpression with BMB2. Furthermore, BMB1 is found to localize to subnuclear structures, in particular to Cajal bodies, in addition to the cytoplasm. As shown in experiments with a BMB1 mutant, the localization of BMB1 to nuclear substructures enhances BMB-dependent virus transport. Thus, the virus transport mediated by BMB proteins is modulated by (i) a BMB1-induced defense response that inhibits transport, (ii) suppression of the BMB1-induced response by BMB2, and (iii) the nuclear localization of BMB1 that promotes virus transport. Collectively, the data presented demonstrate multiple levels of interactions between viral pathogens and their plant hosts during virus cell-to-cell transport.

Transcriptional and hormonal profiling uncovers the interactions between plant developmental stages and RNA virus infection.

Arabidopsis thaliana is more susceptible to certain viruses during its later developmental stages. The differential responses and the mechanisms behind this development-dependent susceptibility to infection are still not fully understood. Here we explored the outcome of a viral infection at different host developmental stages by studying the response of A. thaliana to infection with turnip mosaic virus at three developmental stages: juvenile vegetative, bolting, and mature flowering plants. We found that infected plants at later stages downregulate cell wall biosynthetic genes and that this downregulation may be one factor facilitating viral spread and systemic infection. We also found that, despite being more susceptible to infection, infected mature flowering plants were more fertile (i.e. produce more viable seeds) than juvenile vegetative and bolting infected plants; that is, plants infected at the reproductive stage have greater fitness than plants infected at earlier developmental stages. Moreover, treatment of mature plants with salicylic acid increased resistance to infection at the cost of significantly reducing fertility. Together, these observations support a negative trade-off between viral susceptibility and plant fertility. Our findings point towards a development-dependent tolerance to infection.

Study of Endogenous Viruses in the Strawberry Plants.

Endogenous viral elements (EVEs) have been reported to exist widely in the genomes of eukaryotic organisms, and they are closely associated with the growth, development, genetics, adaptation, and evolution of their hosts. In this study, two methods-homologous sequence search and genome alignment-were used to explore the endogenous viral sequences in the genomes of Fragaria species. Results revealed abundant endogenous pararetroviruses (EPRVs) in the genomes of Fragaria species, including 786 sequences belonging to five known taxa such as Caulimovirus and other unclassified taxa. Differences were observed in the detected EPRVs between the two methods, with the homologous sequence search having a greater number of EPRVs. On the contrary, genome alignment identified various types and sources of virus-like sequences. Furthermore, through genome alignment, a 267-bp sequence with 95% similarity to the gene encoding the aphid-transmitted protein of Strawberry vein banding virus (Caulimovirus venafragariae) was discovered in the F. chiloensis genome, which was likely a recent insertion. In addition, the statistical analysis of the genome alignment results indicated a remarkably higher abundance of virus-like sequences in the genomes of polyploid strawberries compared with diploid ones. Moreover, the differences in virus-like sequences were observed between the genomes of Fragaria species and those of their close relatives. This study enriched the diversity of viruses that infect strawberries, and laid a theoretical foundation for further research on the origin of endogenous viruses in the strawberry genome, host-virus interactions, adaptation, evolution, and their functions.

Wuhan Sequence-Based Recombinant Antigens Expressed in E. coli Elicit Antibodies Capable of Binding with Omicron S-Protein.

The development of cross-reactive vaccines is one of the central aims of modern vaccinology. Continuous mutation and the emergence of new SARS-CoV-2 variants and subvariants create the problem of universal coronavirus vaccine design. Previously, the authors devised three recombinant coronavirus antigens, which were based on the sequence collected in 2019 (the Wuhan variant) and produced in an E. coli bacterial expression system. The present work has shown, for the first time, that these recombinant antigens induce the production of antibodies that clearly interact with produced in CHO full-length S-protein of the Omicron variant. The immunogenicity of these recombinant antigens was studied in formulations with different adjuvants: Freund's adjuvant, Al(OH)3 and an adjuvant based on spherical particles (SPs), which are structurally modified plant virus. All adjuvanted formulations effectively stimulated Omicron-specific IgG production in mice. These universal coronavirus antigens could be considered the main component for the further development of broad-spectrum coronavirus vaccines for the prevention of SARS-CoV-2 infection. The present work also provides evidence that the synthetic biology approach is a promising strategy for the development of highly cross-reactive vaccines. Moreover, it is important to note that the bacterial expression system might be appropriate for the production of antigenically active universal antigens.

Mutations in the WG and GW motifs of the three RNA silencing suppressors of grapevine fanleaf virus alter their systemic suppression ability and affect virus infectivity.

Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1BHel, as well as their fused form (1ABHel). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1BHel in their systemic RNA silencing suppression ability by co-infiltrating Nicotiana benthamiana 16c line plants with a GFP silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity in planta, and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1ABHel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1ABHel. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of NbDCL2, NbDCL4,, and NbRDR6 expression by 1BHel. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection in planta. Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate NbDCL2 and NbRDR6 expression. Finally, in silico protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology.

Recent advances and challenges in plant viral diagnostics.

Accurate and timely diagnosis of plant viral infections plays a key role in effective disease control and maintaining agricultural productivity. Recent advances in the diagnosis of plant viruses have significantly expanded our ability to detect and monitor viral pathogens in agricultural crops. This review discusses the latest advances in diagnostic technologies, including both traditional methods and the latest innovations. Conventional methods such as enzyme-linked immunosorbent assay and DNA amplification-based assays remain widely used due to their reliability and accuracy. However, diagnostics such as next-generation sequencing and CRISPR-based detection offer faster, more sensitive and specific virus detection. The review highlights the main advantages and limitations of detection systems used in plant viral diagnostics including conventional methods, biosensor technologies and advanced sequence-based techniques. In addition, it also discusses the effectiveness of commercially available diagnostic tools and challenges facing modern diagnostic techniques as well as future directions for improving informed disease management strategies. Understanding the main features of available diagnostic methodologies would enable stakeholders to choose optimal management strategies against viral threats and ensure global food security.

Endogenous nege-like viral elements in arthropod genomes reveal virus-host coevolution and ancient history of two plant virus families.

Negevirus is a recently proposed taxon of arthropod-infecting virus, which is associated with plant viruses of two families (Virgaviridae and Kitaviridae). Nevertheless, the evolutionary history of negevirus-host and its relationship with plant viruses remain poorly understood. Endogenous nege-like viral elements (ENVEs) are ancient nege-like viral sequences integrated into the arthropod genomes, which can serve as the molecular fossil records of previous viral infection. In this study, 292 ENVEs were identified in 150 published arthropod genomes, revealing the evolutionary history of nege-like viruses and two related plant virus families. We discovered three novel and eight strains of nege-like viruses in 11 aphid species. Further analysis indicated that 10 ENVEs were detected in six aphid genomes, and they were divided into four types (ENVE1-ENVE4). Orthologous integration and phylogenetic analyses revealed that nege-like viruses had a history of infection of over 60 My and coexisted with aphid ancestors throughout the Cenozoic Era. Moreover, two nege-like viral proteins (CP and SP24) were highly homologous to those of plant viruses in the families Virgaviridae and Kitaviridae. CP- and SP24-derived ENVEs were widely integrated into numerous arthropod genomes. These results demonstrate that nege-like viruses have a long-term coexistence with arthropod hosts and plant viruses of the two families, Virgaviridae and Kitaviridae, which may have evolved from the nege-like virus ancestor through horizontal virus transfer events. These findings broaden our perspective on the history of viral infection in arthropods and the origins of plant viruses. IMPORTANCE: Although negevirus is phylogenetically related to plant virus, the evolutionary history of negevirus-host and its relationship with plant virus remain largely unknown. In this study, we used endogenous nege-like viral elements (ENVEs) as the molecular fossil records to investigate the history of nege-like viral infection in arthropod hosts and the evolution of two related plant virus families (Virgaviridae and Kitaviridae). Our results showed the infection of nege-like viruses for over 60 My during the arthropod evolution. ENVEs highly homologous to viral sequences in Virgaviridae and Kitaviridae were present in a wide range of arthropod genomes but were absent in plant genomes, indicating that plant viruses in these two families possibly evolved from the nege-like virus ancestor through cross-species horizontal virus transmission. Our findings provide a new perspective on the virus-host coevolution and the origins of plant viruses.

Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana through Viral Delivery of SpCas9 and sgRNA.

Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in NbPDS. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8% to 13.2%) in the frequency of transgenerational gene editing. While the TRSV system had a preference for certain sgRNA sequences, co-inoculation of a TRSV vector carrying only Cas9 and a tobacco rattle virus vector carrying sgRNA successfully introduced systemic mutations with all five tested sgRNAs. Extensively gene-edited lateral shoots occasionally grew from plants inoculated with the virus vectors, of which the transgenerational gene editing frequency ranged up to 100%. This virus-mediated heritable gene editing method makes plant gene editing easy, requiring only the inoculation of non-transgenic plants with a virus vector(s) to obtain gene-edited individuals.

Small non-coding satellite RNAs - the 'game changers' at the virus-host plant interaction?

Satellite RNAs (satRNAs) are RNA molecules associated with many plant viruses and fully dependent on them for replication, encapsidation, and movement within the plant or transmission from plant to plant. Their classification is based on their length, functional protein-coding capacity, and RNA structure (whether linear or circular). They have been of interest for a long time as some of them, in particular systems, cause significant changes in the pathogenesis and epidemiology of plant viruses. The outcomes of how satRNAs affect pathogenesis depend on the components of the pathosystem: host plant species or variety, virus species or even strain, and the sequence of satRNA. These can be additionally affected by biotic and abiotic factors, for example, environmental conditions such as the presence of their vectors or ambient temperature. satRNAs may interfere with primary metabolism, signalling, plant defence [including post-transcriptional gene silencing (PTGS)], as well as the efficiency of virus transmission from plant to plant. In recent years, due to wider access to high-throughput technologies and the extension of studies on satRNAs to include the involvement of external factors in plant-virus-satRNA systems, we are gaining a broader view of the consequences of the presence of these small molecules in viral infections. This review presents the state of the art of satRNA interactions with the helper virus and host plant as well as the influence of satRNAs on the insect vector's behaviour. Moreover, areas requiring further research are identified and knowledge gaps indicated.

Composition and abundance of midgut plasma membrane proteins in two major hemipteran vectors of plant viruses, Bemisia tabaci and Myzus persicae.

Multiple species within the order Hemiptera cause severe agricultural losses on a global scale. Aphids and whiteflies are of particular importance due to their role as vectors for hundreds of plant viruses, many of which enter the insect via the gut. To facilitate the identification of novel targets for disruption of plant virus transmission, we compared the relative abundance and composition of the gut plasma membrane proteomes of adult Bemisia tabaci (Hemiptera: Aleyrodidae) and Myzus persicae (Hemiptera: Aphididae), representing the first study comparing the gut plasma membrane proteomes of two different insect species. Brush border membrane vesicles were prepared from dissected guts, and proteins extracted, identified and quantified from triplicate samples via timsTOF mass spectrometry. A total of 1699 B. tabaci and 1175 M. persicae proteins were identified. Following bioinformatics analysis and manual curation, 151 B. tabaci and 115 M. persicae proteins were predicted to localize to the plasma membrane of the gut microvilli. These proteins were further categorized based on molecular function and biological process according to Gene Ontology terms. The most abundant gut plasma membrane proteins were identified. The ten plasma membrane proteins that differed in abundance between the two insect species were associated with the terms "protein binding" and "viral processes." In addition to providing insight into the gut physiology of hemipteran insects, these gut plasma membrane proteomes provide context for appropriate identification of plant virus receptors based on a combination of bioinformatic prediction and protein localization on the surface of the insect gut.

An evaluation of nucleic acid-based molecular methods for the detection of plant viruses: a systematic review.

Precise and timely diagnosis of plant viruses is a prerequisite for the implementation of efficient management strategies, considering factors like globalization of trade and climate change facilitating the spread of viruses that lead to agriculture yield losses of billions yearly worldwide. Symptomatic diagnosis alone may not be reliable due to the diverse symptoms and confusion with plant abiotic stresses. It is crucial to detect plant viruses accurately and reliably and do so with little time. A complete understanding of the various detection methods is necessary to achieve this. Enzyme-linked immunosorbent assay (ELISA), has become more popular as a method for detecting viruses but faces limitations such as antibody availability, cost, sample volume, and time. Advanced techniques like polymerase chain reaction (PCR) have surpassed ELISA with its various sensitive variants. Over the last decade, nucleic acid-based molecular methods have gained popularity and have quickly replaced other techniques, such as serological techniques for detecting plant viruses due to their specificity and accuracy. Hence, this review enables the reader to understand the strengths and weaknesses of each molecular technique starting with PCR and its variations, along with various isothermal amplification followed by DNA microarrays, and next-generation sequencing (NGS). As a result of the development of new technologies, NGS is becoming more and more accessible and cheaper, and it looks possible that this approach will replace others as a favoured approach for carrying out regular diagnosis. NGS is also becoming the method of choice for identifying novel viruses. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-024-00863-0.

Identification of Virus-Derived Small Interfering RNAs (vsiRNAs) from Infected sRNA-Seq Samples.

Plants have developed sophisticated defense mechanisms to combat viral infections, prominently utilizing Dicer-like enzymes (DCL) for generating virus-derived small interfering RNAs (vsiRNAs) through RNA interference (RNAi). This intrinsic mechanism effectively impedes virus replication. Exploiting their potential, vsiRNAs have become a major focus area for comprehensive viral investigations in plants, integrating both bioinformatics and experimental strategies. This chapter introduces an up-to-date computational workflow optimized for identifying and comprehensively annotating vsiRNAs with the utilization of small RNA sequencing (sRNA-seq) data collected from virus-infected plants. The workflow detailed in this chapter centers on known plant-targeting viruses, providing step-by-step guidance to enhance vsiRNA analysis, ultimately advancing the comprehension of plant-virus interactions.

New insights into plasmodesmata: complex 'protoplasmic connecting threads'.

Intercellular communication in plants, as in other multicellular organisms, allows cells in tissues to coordinate their responses for development and in response to environmental stimuli. Much of this communication is facilitated by plasmodesmata (PD), consisting of membranes and cytoplasm, that connect adjacent cells to each other. PD have long been viewed as passive conduits for the movement of a variety of metabolites and molecular cargoes, but this perception has been changing over the last two decades or so. Research from the last few years has revealed the importance of PD as signaling hubs and as crucial players in hormone signaling. The adoption of advanced biochemical approaches, molecular tools, and high-resolution imaging modalities has led to several recent breakthroughs in our understanding of the roles of PD, revealing the structural and regulatory complexity of these 'protoplasmic connecting threads'. We highlight several of these findings that we think well illustrate the current understanding of PD as functioning at the nexus of plant physiology, development, and acclimation to the environment.

Improving grapevine virus diagnostics: Comparative analysis of three dsRNA enrichment methods for high-throughput sequencing.

The extraction of double stranded (ds) RNA is a common enrichment method for the study, characterization, and detection of RNA viruses. In addition to RNA viruses, viroids, and some DNA viruses, can also be detected from dsRNA enriched extracts which makes it an attractive method for detecting a wide range of viruses when coupled with HTS. Several dsRNA enrichment strategies have been developed. The oldest utilizes the selective binding properties of dsRNA to cellulose. More recent methods are based on the application of anti-dsRNA antibodies and viral proteins with a specific affinity for dsRNA. All three methods have been used together with HTS for plant virus detection and study. To our knowledge, this is the first comparative study of three alternative dsRNA enrichment methods for virus and viroid detection through HTS using virus-infected, and healthy grapevine test plants. Extracts were performed in triplicate using methods based on, the anti-dsRNA antibody mAb rJ2 (Millipore Sigma Canada Ltd, Oakville, ON, Canada), the B2 dsRNA binding protein, and ReliaPrep™ Resin (Promega Corporation, Madison, WI, USA). The results show that the workflows for all three methods are effectively comparable, apart from purification steps related to antibody and binding protein construct. Both the cellulose resin and dsRNA binding protein construct methods provide highly enriched dsRNA extracts suitable for HTS with the B2 method providing a 36× and the ReliaPrep™ Resin a 163× increase in dsRNA enrichment compared to the mAb rJ2 antibody. The overall consistency and cost effectiveness of the ReliaPrep™ cellulose resin-based method and the potentially simpler adaptation to robotics made it the method of choice for future transfer to a semi-automated workflow.

The role of reactive oxygen species in plant-virus interactions.

Reactive oxygen species (ROS) play a complex role in interactions between plant viruses and their host plants. They can both help the plant defend against viral infection and support viral infection and spread. This review explores the various roles of ROS in plant-virus interactions, focusing on their involvement in symptom development and the activation of plant defense mechanisms. The article discusses how ROS can directly inhibit viral infection, as well as how they can regulate antiviral mechanisms through various pathways involving miRNAs, virus-derived small interfering RNAs, viral proteins, and host proteins. Additionally, it examines how ROS can enhance plant resistance by interacting with hormonal pathways and external substances. The review also considers how ROS might promote viral infection and transmission, emphasizing their intricate role in plant-virus dynamics. These insights offer valuable guidance for future research, such as exploring the manipulation of ROS-related gene expression through genetic engineering, developing biopesticides, and adjusting environmental conditions to improve plant resistance to viruses. This framework can advance research in plant disease resistance, agricultural practices, and disease control.