A Review of Probe-Based Enrichment Methods to Inform Plant Virus Diagnostics.

Modern diagnostic techniques based on DNA sequence similarity are currently the gold standard for the detection of existing and emerging pathogens. Whilst individual assays are inexpensive to use, assay development is costly and carries risks of not being sensitive or specific enough to capture an increasingly diverse range of targets. Sequencing can provide the entire nucleic acid content of a sample and may be used to identify all pathogens present in the sample when the depth of coverage is sufficient. Targeted enrichment techniques have been used to increase sequence coverage and improve the sensitivity of detection within virus samples, specifically, to capture sequences for a range of different viruses or increase the number of reads from low-titre virus infections. Vertebrate viruses have been well characterised using in-solution hybridisation capture to target diverse virus families. The use of probes for genotyping and strain identification has been limited in plants, and uncertainty around sensitivity is an impediment to the development of a large-scale virus panel to use within regulatory settings and diagnostic pipelines. This review aims to compare significant studies that have used targeted enrichment of viruses to identify approaches to probe design and potential for use in plant virus detection and characterisation.

Integrating viruses into soil food web biogeochemistry.

The soil microbiome is recognized as an essential component of healthy soils. Viruses are also diverse and abundant in soils, but their roles in soil systems remain unclear. Here we argue for the consideration of viruses in soil microbial food webs and describe the impact of viruses on soil biogeochemistry. The soil food web is an intricate series of trophic levels that span from autotrophic microorganisms to plants and animals. Each soil system encompasses contrasting and dynamic physicochemical conditions, with labyrinthine habitats composed of particles. Conditions are prone to shifts in space and time, and this variability can obstruct or facilitate interactions of microorganisms and viruses. Because viruses can infect all domains of life, they must be considered as key regulators of soil food web dynamics and biogeochemical cycling. We highlight future research avenues that will enable a more robust understanding of the roles of viruses in soil function and health.

Bacterial homologs of innate eukaryotic antiviral defenses with anti-phage activity highlight shared evolutionary roots of viral defenses.

Prokaryotes have evolved a multitude of defense systems to protect against phage predation. Some of these resemble eukaryotic genes involved in antiviral responses. Here, we set out to systematically project the current knowledge of eukaryotic-like antiviral defense systems onto prokaryotic genomes, using Pseudomonas aeruginosa as a model organism. Searching for phage defense systems related to innate antiviral genes from vertebrates and plants, we uncovered over 450 candidates. We validated six of these phage defense systems, including factors preventing viral attachment, R-loop-acting enzymes, the inflammasome, ubiquitin pathway, and pathogen recognition signaling. Collectively, these defense systems support the concept of deep evolutionary links and shared antiviral mechanisms between prokaryotes and eukaryotes.

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.

Viral Recognition and Evasion in Plants.

Viruses, causal agents of devastating diseases in plants, are obligate intracellular pathogens composed of a nucleic acid genome and a limited number of viral proteins. The diversity of plant viruses, their diminutive molecular nature, and their symplastic localization pose challenges to understanding the interplay between these pathogens and their hosts in the currently accepted framework of plant innate immunity. It is clear, nevertheless, that plants can recognize the presence of a virus and activate antiviral immune responses, although our knowledge of the breadth of invasion signals and the underpinning sensing events is far from complete. Below, I discuss some of the demonstrated or hypothesized mechanisms enabling viral recognition in plants, the step preceding the onset of antiviral immunity, as well as the strategies viruses have evolved to evade or suppress their detection.

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.

Probing of plant transcriptomes reveals the hidden genetic diversity of the family Secoviridae.

Secoviruses are single-stranded RNA viruses that infect plants. In the present study, we identified 61 putative novel secoviral genomes in various plant species by mining publicly available plant transcriptome data. These viral sequences represent the genomes of 13 monopartite and 48 bipartite secovirids. The genome sequences of 52 secovirids were coding-complete, and nine were partial. Except for small open reading frames (ORFs) determined in waikaviral genomes and RNA2 of torradoviruses, all of the recovered genomes/genome segments contained a large ORF encoding a polyprotein. Based on genome organization and phylogeny, all but three of the novel secoviruses were assigned to different genera. The genome organization of two identified waika-like viruses resembled that of the recently identified waika-like virus Triticum aestivum secovirus. Phylogenetic analysis revealed a pattern of host-virus co-evolution in a few waika- and waika-like viruses and increased phylogenetic diversity of nepoviruses. The study provides a basis for further investigation of the biological properties of these novel secoviruses.

Functional role of geminivirus encoded proteins in the host: Past and present.

During plant-pathogen interaction, plant exhibits a strong defense system utilizing diverse groups of proteins to suppress the infection and subsequent establishment of the pathogen. However, in response, pathogens trigger an anti-silencing mechanism to overcome the host defense machinery. Among plant viruses, geminiviruses are the second largest virus family with a worldwide distribution and continue to be production constraints to food, feed, and fiber crops. These viruses are spread by a diverse group of insects, predominantly by whiteflies, and are characterized by a single-stranded DNA (ssDNA) genome coding for four to eight proteins that facilitate viral infection. The most effective means to managing these viruses is through an integrated disease management strategy that includes virus-resistant cultivars, vector management, and cultural practices. Dynamic changes in this virus family enable the species to manipulate their genome organization to respond to external changes in the environment. Therefore, the evolutionary nature of geminiviruses leads to new and novel approaches for developing virus-resistant cultivars and it is essential to study molecular ecology and evolution of geminiviruses. This review summarizes the multifunctionality of each geminivirus-encoded protein. These protein-based interactions trigger the abrupt changes in the host methyl cycle and signaling pathways that turn over protein normal production and impair the plant antiviral defense system. Studying these geminivirus interactions localized at cytoplasm-nucleus could reveal a more clear picture of host-pathogen relation. Data collected from this antagonistic relationship among geminivirus, vector, and its host, will provide extensive knowledge on their virulence mode and diversity with climate change.

Metabolites-induced co-evolutionary warfare between plants, viruses, and their associated vectors: So close yet so far away.

Agriculture and global food security encounter significant challenges due to viral threats. In the following decades, several molecular studies have focused on discovering biosynthetic pathways of numerous defensive and signaling compounds, as key regulators of plant interactions, either with viruses or their associated vectors. Nevertheless, the complexities of specialized metabolites mediated plant-virus-vector tripartite viewpoint and the identification of their co-evolutionary crossroads toward antiviral defense system, remain elusive. The current study reviews the various roles of plant-specialized metabolites (PSMs) and how plants use these metabolites to defend against viruses. It discusses recent examples of specialized metabolites that have broad-spectrum antiviral properties. Additionally, the study presents the co-evolutionary basis of metabolite-mediated plant-virus-insect interactions as a potential bioinspired approach to combat viral threats. The prospects also show promising metabolic engineering strategies aimed at discovering a wide range of PSMs that are effective in fending off viruses and their related vectors. These advances in understanding the potential role of PSMs in plant-virus interactions not only serve as a cornerstone for developing plant antiviral systems, but also highlight essential principles of biological control.

ICTV Virus Taxonomy Profile: Fimoviridae 2024.

Members of the family Fimoviridae are plant viruses with a multipartite negative-sense enveloped RNA genome (-ssRNA), composed of 4-10 segments comprising 12.3-18.5 kb in total, within quasi-spherical virions. Fimoviruses are transmitted to plants by eriophyid mites and induce characteristic cytopathologies in their host plants, including double membrane-bound bodies in the cytoplasm of virus-infected cells. Most fimoviruses infect dicotyledonous plants, and many cause serious disease epidemics. This is a summary of the ICTV Report on the family Fimoviridae, which is available at ictv.global/report/fimoviridae.

Plant viruses traveling without passport.

All plant viruses were thought to encode in its genome a movement protein that acts as a "passport," allowing active movement within the host. A new study in PLOS Biology characterizes the first plant virus that can colonize its host without encoding this protein.

Viruses shuttle between fungi and plants.

The intimate relationships between plants and fungi provide an opportunity for the shuttling of viruses. Dai et al. recently discovered that a virus undergoes cross-kingdom transmission, and naturally spreads to both plant and fungal populations. This finding expands our understanding of viral host range, evolution, transmission, and disease management.

Application of Integrated Computational Approaches in Prediction of Plant Virus Encoded miRNAs and Their Targeted Plant Genes.

This chapter presents a comprehensive approach to predict novel miRNAs encoded by plant viruses and identify their target plant genes, through integration of various ab initio computational approaches. The predictive process begins with the analysis of plant viral sequences using the VMir Analyzer software. VMir Viewer software is then used to extract primary hairpins from these sequences. To distinguish real miRNA precursors from pseudo miRNA precursors, MiPred web-based software is employed. Verified real pre-miRNA sequences with a minimum free energy of < -20 Kcal/mol, are further analyzed using the RNAshapes software. Validation of predictions involves comparing them with available Expressed Sequence Tags (ESTs) from the relevant plant using BlastN. Short sequences with lengths ranging from 19 to 25 nucleotides and exhibiting <5 mismatches are prioritized for miRNA prediction. The precise locations of these short sequences within pre-miRNA structures generated using RNAshapes are meticulously identified, with a focus on those situated on the 5' and 3' arms of the structures, indicating potential miRNAs. Sequences within the arms of pre-miRNA structures are used to predict target sites within the ESTs of the specific plant, facilitated by psRNA Target software, revealing genes with potential regulatory roles in the plant. To confirm the outcome of target prediction, results are individually submitted to the RNAhybrid web-based software. For practical demonstration, this approach is applied to analyze African cassava mosaic virus (ACMV) and East African cassava mosaic virus-Uganda (EACMV-UG) viruses, as well as the ESTs of Jatropha and cassava.

The role of rhizosphere phages in soil health.

While the One Health framework has emphasized the importance of soil microbiomes for plant and human health, one of the most diverse and abundant groups-bacterial viruses, i.e. phages-has been mostly neglected. This perspective reviews the significance of phages for plant health in rhizosphere and explores their ecological and evolutionary impacts on soil ecosystems. We first summarize our current understanding of the diversity and ecological roles of phages in soil microbiomes in terms of nutrient cycling, top-down density regulation, and pathogen suppression. We then consider how phages drive bacterial evolution in soils by promoting horizontal gene transfer, encoding auxiliary metabolic genes that increase host bacterial fitness, and selecting for phage-resistant mutants with altered ecology due to trade-offs with pathogen competitiveness and virulence. Finally, we consider challenges and avenues for phage research in soil ecosystems and how to elucidate the significance of phages for microbial ecology and evolution and soil ecosystem functioning in the future. We conclude that similar to bacteria, phages likely play important roles in connecting different One Health compartments, affecting microbiome diversity and functions in soils. From the applied perspective, phages could offer novel approaches to modulate and optimize microbial and microbe-plant interactions to enhance soil health.

Viroids of the Mediterranean Basin.

There has been substantial progress in the Mediterranean countries regarding research on viroids. Twenty-nine viroid species, all belonging to Pospiviroidae and Avsunviroidae genera, have been detected in the Mediterranean Basin. Not only have detection methods, such as reverse transcription-quantitative polymerase chain reaction and next-generation sequencing, been used for viroid detection, along with molecular hybridization techniques allowing for rapid detection, identification, and characterization of known and novel viroids in these countries, but eradication measures have also been taken that allowed for the efficient elimination of certain viroids in a number of Mediterranean countries. The eradication measures were followed as recommended by the European and Mediterranean Plant Protection Organization, which is known by its abbreviation, EPPO. The Mediterranean Region has been a niche for viroids since ancient times due to the warm climate and the socio-cultural conditions that facilitate viroid transmission among different host plant species.

Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for the Detection of Plant Viruses.

Isothermal nucleic acid amplification-based lateral flow testing (INAA-LFT) has emerged as a robust technique for on-site pathogen detection, providing a visible indication of pathogen nucleic acid amplification that rivals or even surpasses the sensitivity of real-time quantitative PCR. The isothermal nature of INAA-LFT ensures consistent conditions for nucleic acid amplification, establishing it as a crucial technology for rapid on-site pathogen detection. However, despite its considerable promise, the widespread application of isothermal INAA amplification-based lateral flow testing faces several challenges. This review provides an overview of the INAA-LFT procedure, highlighting its advancements in detecting plant viruses. Moreover, the review underscores the imperative of addressing the existing limitations and emphasizes ongoing research efforts dedicated to enhancing the applicability and performance of this technology in the realm of rapid on-site testing.

Salicylic acid-driven innate antiviral immunity in plants.

Pathogenic viruses are a constant threat to all organisms, including plants. However, in plants, a small group of cells (stem cells) protect themselves from viral invasion. Recently, Incarbone et al. uncovered a novel salicylic acid (SA) and RNAi mechanism of stem cell resistance, broadening our understanding of RNAi-mediated antiviral plant immunity.

Roles of ROS and redox in regulating cell-to-cell communication: Spotlight on viral modulation of redox for local spread.

Reactive oxygen species (ROS) are important signalling molecules that influence many aspects of plant biology. One way in which ROS influence plant growth and development is by modifying intercellular trafficking through plasmodesmata (PD). Viruses have evolved to use PD for their local cell-to-cell spread between plant cells, so it is therefore not surprising that they have found ways to modulate ROS and redox signalling to optimise PD function for their benefit. This review examines how intracellular signalling via ROS and redox pathways regulate intercellular trafficking via PD during development and stress. The relationship between viruses and ROS-redox systems, and the strategies viruses employ to control PD function by interfering with ROS-redox in plants is also discussed.