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.

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.
OBJECTIVE: 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.

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.

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.

Honey bees are economically important insects. However, they face multiple biotic and abiotic stresses, such as diseases, pesticides, climate change, and pests, which cause the loss of honey bee colonies worldwide. Among these factors, viruses have been identified as the major cause of colony loss. Research on honey bee viruses in Uzbekistan is limited. This study investigated the viruses affecting honey bees in Uzbekistan. Virome analysis was conducted for each sample using high-throughput sequencing and bioinformatics. Nine honey bee viruses have been identified: the acute bee paralysis virus, aphid lethal paralysis virus, Apis rhabdovirus 1 and 2, black queen cell virus, deformed wing virus, Lake Sinai virus 10, sacbrood virus, and Hubei partiti-like virus 34. Additionally, 15 plant viruses were identified, 7 of which were novel. This study is the first virome analysis of Uzbekistan honey bees and provides a foundation for understanding the viruses affecting honey bees and plants in Uzbekistan.

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.

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.

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.

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.

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 have 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.