Search Results (50)

Tomato brown rugose fruit virus (ToBRFV) is a recently emerged viral pathogen in the Tobamovirus genus first observed in 2014 in the Middle East that has since spread worldwide, causing significant losses in greenhouse tomato production. ToBRFV is easily mechanically transmitted and can escape the durable Tm-2² resistance gene, facilitating its global spread. Seed companies have identified novel sources of resistance and introduced these resistance traits into commercial cultivars. The identity, number, and mechanisms of these putative novel resistance genes are largely unknown but could be exerting selective pressures on ToBRFV. Here, we report 15 new ToBRFV genomic sequences from Canadian greenhouse production systems in susceptible and novel resistant or tolerant cultivars collected since 2023. We combined these sequences with five other Canadian ToBRFV genomes previously deposited in Genbank and a further five consensus sequences derived from metagenomic-based wastewater monitoring sequence data and conducted phylogenetic analysis. Most Canadian sequences grouped together when compared with 332 publicly available international sequences, but several isolates appeared distantly related, suggesting multiple introductions to Canadian production systems. High sequence identity between samples suggest movement of ToBRFV between independent greenhouses, highlighting areas where biosecurity can be improved. Several novel non-synonymous polymorphisms identified in the p126 and movement protein (MP) open reading frames (ORFs) were unique to Canadian sequences and associated with infection of novel resistant tomato cultivars. Many polymorphisms in the p126 ORF are located in a region of the protein associated with Tm-1 resistance-breaking isolates of tomato mosaic virus and ToBRFV, but have not been previously reported. Four novel polymorphisms in MP were also identified and do not appear to be associated with sites previously identified as interacting with Tm-2² and could be related to other unknown resistance genes. Together, these results confirm the difficulties in preventing the transmission of ToBRFV, identify putative adaptations to novel and existing resistance genes, and emphasize the urgent need for the cloning and characterization of these new sources of resistance to ToBRFV. Full article

Groundnut bud necrosis orthotospovirus (GBNV), a tripartite single-stranded RNA virus, poses a significant threat to United States agriculture. GBNV is a quarantine pathogen, and its introduction could lead to severe damage to economically important crops, such as groundnuts, tomatoes, potatoes, peas, and soybeans. For the rapid and accurate detection of GBNV at points of entry, TaqMan reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) assays were developed and the results validated using conventional reverse transcriptase–polymerase chain reaction (RT-PCR) followed by Sanger sequencing. These assays target highly conserved regions of the nucleocapsid (NP) and movement (MP) proteins within the viral genome. Multiplex GBNV detection assays targeting the NP and MP genes, as well as an internal control plant gene, ACT11, showed efficiency rates between 90% and 100% and R² values of 0.98 to 0.99, indicating high accuracy and precision. Moreover, there was no significant difference in sensitivity between multiplex and singleplex assays, ensuring reliable detection across various plant tissues. This rapid, sensitive, and specific diagnostic assay will provide a valuable tool at ports of entry to prevent the entry of GBNV into the United States. Full article

Pepper golden mosaic virus (PepGMV) is a bipartite begomovirus of pepper and tomato from North America. In ‘Anaheim’ pepper plants PepGMV-Mo strain (Mo) causes systemic yellow foliar mosaic symptoms, while PepGMV-D strain (D) causes distortion of 1st–6th expanding leaves, and asymptomatic infection of subsequently developing leaves, like other known ‘recovery’ phenotypes. Infections established with DNA-A Mo and D components expressing red-shifted green fluorescent protein in place of coat protein and in situ hybridization, showed PepGMV-Mo localized to phloem and mesophyll cells, while -D was mesophyll restricted. Alignment of PepGMV-Mo and -D DNA-B components revealed three indels upstream of the BC1 gene that encodes the movement protein (MP). To determine if this non-coding region (*BC1) D-strain MP putative promoter contributed to ‘recovery’, plants were inoculated with chimeric DNA-B Mo/D components harboring reciprocally exchanged *BC1, and wild-type DNA-A Mo and D components. Symptoms were reminiscent but not identical to wild-type -Mo or -D infection, respectively, suggesting ‘recovery’ cannot be attributed solely to the *BC1. Both BC1 and D*BC1 were targeted by post-transcriptional gene silencing; however, ‘recovered’ leaves accumulated fewer transcripts and 21–24 nt vsiRNAs. Thus, inefficient in planta movement of PepGMV-D is associated with a non-pepper-adapted ‘defective’ BC1 that facilitates hyper-efficient PTGS, leading to BC1 transcript degradation that in turn limits virus spread, thereby recapitulating disease ‘tolerance’. Full article

The size of viral genomes is limited, thus the majority of encoded proteins possess multiple functions. The main function of tobamoviral movement protein (MP) is to perform plasmodesmata gating and mediate intercellular transport of the viral RNA. MP is a remarkable example of a protein that, in addition to the initially discovered and most obvious function, carries out numerous activities that are important both for the manifestation of its key function and for successful and productive infection in general. Briefly, MP binds the viral genome, delivers it to the plasmodesmata (PD) and mediates its intercellular transfer. To implement the transport function, MP interacts with diverse cellular factors. Each of these cellular proteins has its own function, which could be different under normal conditions and upon viral infection. Here, we summarize the data available at present on the plethora of cellular factors that were identified as tobamoviral MP partners and analyze the role of these interactions in infection development. Full article

Background: Variants within factor VIII (F8) are associated with sex-linked hemophilia A and thrombosis, with gene therapy approaches being available for pathogenic variants. Many variants within F8 remain variants of uncertain significance (VUS) or are under-explored as to their connections to phenotypic outcomes. Methods: We assessed data on F8 expression while screening the UniProt, ClinVar, Geno2MP, and gnomAD databases for F8 missense variants; these collectively represent the sequencing of more than a million individuals. Results: For the two F8 isoforms coding for different protein lengths (2351 and 216 amino acids), we observed noncoding variants influencing expression which are also associated with thrombosis risk, with uncertainty as to differences in females and males. Variant analysis identified a severe stratification of potential annotation issues for missense variants in subjects of non-European ancestry, suggesting a need for further defining the genetics of diverse populations. Additionally, few heterozygous female carriers of known pathogenic variants have sufficiently confident phenotyping data, leaving researchers unable to determine subtle, less defined phenotypes. Using structure movement correlations to known pathogenic variants for the VUS, we determined seven clusters of likely pathogenic variants based on screening work. Conclusions: This work highlights the need to define missense variants, especially those for VUS and from subjects of non-European ancestry, as well as the roles of these variants in women’s physiology. Full article

The interaction of viral proteins with host factors represents a crucial aspect of the infection process in plants. In this work, we developed a strategy to identify host factors in Nicotiana tabacum that interact with movement proteins (MPs) of the 30K family, a group of viral proteins around 30 kDa related to the MP of tobacco mosaic virus, which enables virus movement between plant cells. Using the alfalfa mosaic virus (AMV) MP as a model, we incorporated tags into its coding sequence, without affecting its functionality, enabling the identification of 121 potential interactors through in vivo immunoprecipitation of the tagged MP. Further analysis of five selected candidates (histone 2B (H2B), actin, 14-3-3A protein, eukaryotic initiation factor 4A (elF4A), and a peroxidase-POX-) were conducted using bimolecular fluorescence complementation (BiFC). The interactions between these factors were also studied, revealing that some form part of protein complexes associated with AMV MP. Moreover, H2B, actin, 14-3-3, and eIF4A interacted with other MPs of the 30K family. This observation suggests that, beyond functional and structural features, 30K family MPs may share common interactors. Our results demonstrate that tagging 30K family MPs is an effective strategy to identify host factors associated with these proteins during viral infection. Full article

American plum line pattern virus (APLPV) is the most infrequently reported Ilarvirus infecting stone fruit trees and is of sufficient severity to be classified as an EPPO quarantine A1 pathogen. In late spring, yellow line pattern symptoms were observed on leaves in a few flowering cherries (Prunus serrulata Lindl.) grown in a public garden in Northwest Italy. RNA extracts from twenty flowering cherries were submitted to Ilarvirus multiplex and APLPV-specific RT-PCR assays already reported or developed in this study. One flowering cherry (T22) with mixed prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) infection also showed infection with APLPV. Blastn analysis of PCR products of the full coat protein (CP) and movement protein (MP) genes obtained from flowering cherry T22 showed 98.23% and 98.34% nucleotide identity with reference APLPV isolate NC_003453.1 from the USA. Then, a LAMP-specific assay was designed to facilitate the fast and low-cost identification of this virus either in the laboratory or directly in the field. The developed assay allowed not only the confirmation of APLPV (PSer22IT isolate) infection in the T22 flowering cherry but also the identification of APLPV in an asymptomatic flowering cherry tree (TL1). The LAMP assay successfully worked with crude flowering cherry extracts, obtained after manually shaking a single plant extract in the ELISA extraction buffer for 3–5 min. The developed rapid, specific and economic LAMP assay was able to detect APLPV using crude plant extracts rather that RNA preparation in less than 20 min, making it suitable for in-field detection. Moreover, the LAMP assay proved to be more sensitive in APLPV detection in flowering cherry compared to the specific one-step RT-PCR assay. The new LAMP assay will permit the estimation of APLPV geographic spread in the territory, paying particular attention to surrounding gardens and propagated flowering cherries in ornamental nurseries. Full article

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. Full article

Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae) is one of the causal agents of wheat viral disease, which severely impacts wheat production in most wheat-growing regions in the world. Currently, there is little information about natural resistance against WDV in common wheat germplasms. CRISPR/Cas9 technology is being utilized to manufacture transgenic plants resistant to different diseases. In the present study, we used the CRISPR/Cas9 system targeting overlapping regions of coat protein (CP) and movement protein (MP) (referred to as CP/MP) or large intergenic region (LIR) in the wheat variety ‘Fielder’ to develop resistance against WDV. WDV-inoculated T₁ progenies expressing Cas9 and sgRNA for CP/MP and LIR showed complete resistance against WDV and no accumulation of viral DNA compared with control plants. Mutation analysis revealed that the CP/MP and LIR targeting sites have small indels in the corresponding Cas9-positive plants. Additionally, virus inhibition and indel mutations occurred in T₂ homozygous lines. Together, our work gives efficient results of the engineering of CRISPR/Cas9-mediated WDV resistance in common wheat plants, and the specific sgRNAs identified in this study can be extended to utilize the CRISPR/Cas9 system to confer resistance to WDV in other cereal crops such as barley, oats, and rye. Full article

Loquat leaves exhibiting obvious yellowing, blistering, mosaic, leaf upward cupping, crinkle, and leaf narrowing were identified in Panzhihua City, Sichuan Province, China. High-throughput sequencing (HTS) with the ribo-depleted cDNA library was employed to identify the virome in the loquat samples; only tomato mosaic virus (ToMV) and citrus exocortis viroid (CEVd) were identified in the transcriptome data. The complete genome sequence of ToMV and CEVd were obtained from the loquat leaves. The full-length genome of the ToMV-loquat is 6376 nt and comprises four open reading frames (ORFs) encoding 183 kDa protein, RNA-dependent RNA polymerase (RdRp), movement protein (MP), and coat protein (CP), respectively. A pairwise identity analysis showed that the complete sequence of the ToMV-loquat had a nucleotide identity between 98.5 and 99.3% with other ToMV isolates. A phylogenetic analysis indicated that ToMV-loquat was more closely related to ToMV-IFA9 (GenBank No. ON156781). A CEVd sequence with 361 nt in length was amplified based on the HTS contigs, sequence alignment indicated CEVd-loquat had the highest identity with the strain of CEVd-Balad (GenBank No. PP869624), phylogenetic analysis showed that CEVd-loquat was more closely related to CEVd-lettuce (GenBank No. ON993891). This significant discovery marks the first documentation and characterization of ToMV and CEVd infecting loquat plants, shedding light on potential threats to loquat cultivation and providing insights for disease management strategies. Full article

Plant virus genomes encode proteins that are involved in replication, encapsidation, cell-to-cell, and long-distance movement, avoidance of host detection, counter-defense, and transmission from host to host, among other functions. Even though the multifunctionality of plant viral proteins is well documented, contemporary functional repertoires of individual proteins are incomplete. However, these can be enhanced by modeling tools. Here, predictive modeling of proteins encoded by the two genomic RNAs, i.e., RNA1 and RNA2, of grapevine fanleaf virus (GFLV) and their satellite RNAs by a suite of protein prediction software confirmed not only previously validated functions (suppressor of RNA silencing [VSR], viral genome-linked protein [VPg], protease [Pro], symptom determinant [Sd], homing protein [HP], movement protein [MP], coat protein [CP], and transmission determinant [Td]) and previously identified putative functions (helicase [Hel] and RNA-dependent RNA polymerase [Pol]), but also predicted novel functions with varying levels of confidence. These include a T3/T7-like RNA polymerase domain for protein 1A^VSR, a short-chain reductase for protein 1B^Hel/VSR, a parathyroid hormone family domain for protein 1E^Pol/Sd, overlapping domains of unknown function and an ABC transporter domain for protein 2B^MP, and DNA topoisomerase domains, transcription factor FBXO25 domain, or DNA Pol subunit cdc27 domain for the satellite RNA protein. Structural predictions for proteins 2A^HP/Sd, 2B^MP, and 3A^? had low confidence, while predictions for proteins 1A^VSR, 1B^Hel*/VSR, 1C^VPg, 1D^Pro, 1E^Pol*/Sd, and 2C^CP/Td retained higher confidence in at least one prediction. This research provided new insights into the structure and functions of GFLV proteins and their satellite protein. Future work is needed to validate these findings. Full article

Cell-to-cell transport of plant viruses through plasmodesmata (PD) requires viral movement proteins (MPs) often associated with cell membranes. The genome of the Hibiscus green spot virus encodes two MPs, BMB1 and BMB2, which enable virus cell-to-cell transport. BMB2 is known to localize to PD-associated membrane bodies (PAMBs), which are derived from the endoplasmic reticulum (ER) structures, and to direct BMB1 to PAMBs. This paper reports the fine structure of PAMBs. Immunogold labeling confirms the previously observed localization of BMB1 and BMB2 to PAMBs. EM tomography data show that the ER-derived structures in PAMBs are mostly cisterns interconnected by numerous intermembrane contacts that likely stabilize PAMBs. These contacts predominantly involve the rims of the cisterns rather than their flat surfaces. Using FRET-FLIM (Förster resonance energy transfer between fluorophores detected by fluorescence-lifetime imaging microscopy) and chemical cross-linking, BMB2 is shown to self-interact and form high-molecular-weight complexes. As BMB2 has been shown to have an affinity for highly curved membranes at cisternal rims, the interaction of BMB2 molecules located at rims of adjacent cisterns is suggested to be involved in the formation of intermembrane contacts in PAMBs. Full article

The emulsions prepared by three non-meat proteins, sodium caseinate (SC), soy protein isolate (SPI) and egg white protein (EPI), were individually added to the continuous phase of myofibrillar protein (MP) sol to form MP composite gels to simulate meat products. The research aimed to investigate the effects of Transglutaminase (TGase) on the physicochemical properties, microstructure and water phase distribution of non-meat protein emulsion MP composite gels. The results of this study revealed that TGase played a crucial role in forming a tight gel network structure in the composite gels. This enhanced their ability to retain water and improved their overall gel strength. Additionally, TGase increased the gel formation temperature of myofibrillar proteins. Electrophoresis analysis showed that when catalyzed by TGase, there was a lighter band compared to those not catalyzed by TGase. This indicated that the addition of TGase facilitated cross-linking interactions between meat proteins and non-meat proteins in the composite gels. Furthermore, microscopy observations demonstrated that composite gels treated with TGase exhibited a more uniform microstructure. This could be attributed to an acceleration in relaxation time T2. The uniform network structure restricted the movement of water molecules in the gel matrix, thereby improving its water-holding capacity. Overall, these findings highlight how incorporating non-meat proteins into myofibrillar systems can be effectively achieved through enzymatic treatment with TGase. Such modifications not only enhanced important functional properties but also contributed towards developing alternative meat products with improved texture and moisture retention abilities. Full article

An analysis of the complete genome sequence of a novel isolate of tomato brown rugose fruit virus (ToBRFV) obtained from tomatoes in Iran and named ToBRFV-Ir is presented in this study. Comprehensive phylogenetic analysis utilizing key viral proteins, including 126 KDa, 183 KDa, movement protein (MP), and coat protein (CP), as well as the complete genome sequence, classified ToBRFV-Ir and 65 isolates from GenBank into three distinct clades. Notably, genetic diversity assessment revealed relatively low variability among the isolates, irrespective of their geographical or clade affiliation. Natural selection analysis based on the complete genome sequence showed that dN/dS values were consistently <1, indicating the prevailing role of negative selection across all populations. Analyses using the Recombination Detection Program and SplitsTree found no evidence of recombination events or signals in the complete genome sequence of the tested isolates. Thus, these results suggest that the genetic composition of ToBRFV remains stable without significant genetic exchange or recombination events occurring. A simple arithmetic comparison of the patristic distances and dates suggested that the time to the most recent common ancestor (TMRCA) of the ToBRFV populations is approximately 0.8 up to 2.7 with the closest tobamoviruses. An evolutionary study of the tested isolates from various countries based on the complete genome suggests Peruvian ancestry. The ToBRF-Ir isolate was successfully transmitted through mechanical inoculations to Solanum lycopersicum and Nicotiana rustica. These findings shed light on the genetic dynamics and transmission mechanisms of ToBRFV, providing valuable insights into its molecular characteristics and potential spread among susceptible plant species. Full article

To move from cell to cell through plasmodesmata, many plant viruses require the concerted action of two or more movement proteins (MPs) encoded by transport gene modules of virus genomes. A tetra-cistron movement block (TCMB) is a newly discovered transport module comprising four genes. TCMB encodes three proteins, which are similar to MPs of the transport module known as the “triple gene block”, and a protein unrelated to known viral MPs and containing a double-stranded RNA (dsRNA)-binding domain similar to that found in a family of cell proteins, including AtDRB4 and AtHYL1. Here, the latter TCMB protein, named vDRB for virus dsRNA-binding protein, is shown to bind both dsRNA and single-stranded RNA in vitro. In a turnip crinkle virus-based assay, vDRB exhibits the properties of a viral suppressor of RNA silencing (VSR). In the context of potato virus X infection, vDRB significantly decreases the number and size of “dark green islands”, regions of local antiviral silencing, supporting the VSR function of vDRB. Nevertheless, vDRB does not exhibit the VSR properties in non-viral transient expression assays. Taken together, the data presented here indicate that vDRB is an RNA-binding protein exhibiting VSR functions in the context of viral infection. Full article