Search

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 Nicotiana benthamiana PHYTOENE DESATURASE. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8–13.2%) 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, the transgenerational gene editing frequency of which 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. [ABSTRACT FROM AUTHOR]

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 tryptophanglycine (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. [ABSTRACT FROM AUTHOR]

Introduction More than 80 viral diseases of grapevines have been reported worldwide. The infectious degeneration disease complex causes growth reduction, stunting, shortening of internodes, bushy growth and decline in susceptible vines. Grapevine fanleaf viruses (GFLV), arabis mosaic virus (ArMV), grapevine deformation virus (GDefV), tomato ring spot virus (ToRSV) and tobacco ring spot virus (TRSV), which belong to the genus Nepovirus, are known to cause infectious degeneration. The genus Nepovirus has been divided into three subgroups A, B and C based on genome length, genomic organization, serological relationships, proteinase cleavage sites and the phylogenetic relationship of their coat protein (CP) gene. GDefV with the new name Nepovirus deformationis belongs to subgroup A of the genus Nepovirus and is closely related to ArMV and GFLV. Prevalence of GFLV in the vineyards of Iran raises the possibility that a mixed infection of GFLV and GDefV is also present in these areas. In Khorasan-Razavi Province, Iran's third-largest grape production center, research has addressed grape-affecting viruses with potential economic consequences, including reduced yield and product quality. Studying the distribution and genetic diversity of GDefV and other grape viruses is crucial for developing effective management and prevention strategies in the region's vineyards. In this study, GDefV was identified in the vineyards of Khorasan-Razavi province in Northeastern Iran and its complete genome was sequenced. In addition, the phylogenetic relationship of these isolates to other GDefV isolates deposited in GenBank was analyzed. Materials and Methods Samples from three grapevines were collected from a vineyard in Kashmer, Khorasan-Razavi Province; total RNA was extracted from the petiole of young leaves using the CTAB-PVPP method., GDefV and mixed infections of GDefV/GFLV were detected by PCR using specific primers. The PCR products were electrophoresed in a 1% agarose gel and sequenced. The miRNAs were extracted from grapevine petiole tissue using the modified CTAB method, and small RNA libraries were prepared using the TruSeq Small RNA Sample Prep Kit and sequenced on the Illumina Novaseq 6000 platform. After trimming the reads, contigs were generated using k-mer 15 in Velvet assembler 0.7.31. Contigs were verified using BLASTn and BLASTx in NCBI, and reconstruction of the GDefV genome from the NGS reads was performed using CLC Genomics Workbench (CLC Bio) software. Phylogenetic trees were generated in MEGA7 using the maximum likelihood (ML) method with 1000 replicates in the bootstrap test. The occurrence of possible recombinations in the GDefV genome was analyzed using the RDP v.6 package. Results and Discussion The samples showed leaf deformation, shortening of internodes, bushy growth, stunting and decline, but these symptoms are probably related to GFLV, and GDefV only causes leaf deformation in the infected vine. The PCR amplification and sequencing of a segment of the coat protein gene revealed a co-infection of GDefV and GFLV in the samples. GFLV is widely distributed in Iranian vineyards, GDefV has also been previously reported in the vineyards of northwestern Iran, but this is the first report of GDefV in the vineyards of Khorasan-Razavi Province. After refining the reads, approximately 15 million reads (92-99.7% of the original reads) remained for further analysis. The read sequences of each library were deposited in the Sequence Read Archive (SRA) under the accession number SAMN33747579-81. Three Iranian GDefV isolates obtained from three miRNA libraries were deposited in GenBank (accession numbers **-**). RNA1 and RNA2 of the three Iranian GDefV isolates had a length of 7386 and 3753 nucleotides, respectively. The RNA1 in GDefV had an open reading frame (ORF) of 6852 nucleotides in length, which started with the start codon AUG at position 288 and ended with the stop codons UAA or UAG at position 7142. The 5' end of the genome had 287 nucleotides long and contained two repeating sequences of 15 nucleotides that formed stem-loop structures. The length of the non-coding region at the 3' end had 244 nucleotides. Translation of RNA1 of the GDefV genome produces a polyprotein (p1) of 2285 amino acids (approximately 252 kda). The polyprotein p1 comprises the cofactor proteins proteinase, helicase, VPg, proteinase and polymerase with approximate weights of 45, 88, 3, 25 and 91 kDa, respectively. RNA2 also has one ORF, located between nucleotides 237 and 3560. This open reading frame also produces a 122 kDa polyprotein (P2) with 1108 amino acids. The second fragment of the GDeFV genome had 236 nucleotides as a 5'-noncoding region with three repeating sequences of 15 nucleotides that produced stem-loop structures with a 3-nucleotide loop and a 6bp stem. The 3'-noncoding region was also 193 nucleotides long. Polyprotein P2 comprised protein 2A, movement protein (MP) and coat protein (CP). The GDefV polyproteins had cysteine/alanine (C/A) and arginine/glycine (R/G) cleavage sites similar to those of the GFLV polyprotein. Comparison of the RNA1 sequences from three Iranian GDefV isolates with other GDefV isolates available in GenBank showed that the Iranian isolates had 88.1-92.2 % nucleotide identity with each other and 90.3-93.9 % with GenBank isolates at the nucleotide level. At the amino acid level, the Iranian isolates were 86.6-91.6 % identity with each other and 88.9-92.7 % with GenBank isolates. For RNA2, the Iranian isolates showed 89.4-92 % similar to each other and 89.6-94.2 % similar to GenBank isolates at the nucleotide level. The amino acid similarity between the Iranian GDefV isolates was 85-88.6 %. In the phylogenetic tree based on the nucleotide and amino acid sequences of RNA1 and RNA2 of the GDefV genome, the Iranian isolates of this study were clustered in a distinct clade than other GDefV isolates from Turkey (HE613269 and NC017939). GDefV was reported in 2003 and no further information is available on its distribution in vineyards around the world. GDefV has already been reported from Turkey and Iran, but the complete genome sequence of the Iranian GDefV isolate is being reported for the first time. Further studies on the population diversity of GDefV isolates in different regions of Iran are required to gain more insight into the mechanisms affecting the dynamics of GDefV populations. [ABSTRACT FROM AUTHOR]

Hibiscus is not native to Colombia but well suited to its arid soil and dry climates. A single hibiscus plant from Risaralda, showing black spots on upper and lower sides of its leaves, was collected for virome analysis using meta-transcriptomic high-throughput sequencing technology. Bioinformatic analysis identified 12.5% of the total reads in the Ribo-Zero cDNA library which mapped to viral genomes. BLAST searches revealed the presence of carlavirus, potexvirus, and of known members of the genera Betacarmovirus, Cilevirus, Nepovirus, and Tobamovirus in the sample; confirmed by RT-PCR with virus-specific primers followed by amplicon sequencing. Furthermore, in silico analysis suggested the possibility of a novel soymovirus, and a new hibiscus strain of citrus leprosis virus C2 in the mixed infection. Both RNA dependent RNA polymerase and coat protein gene sequences of the potex and carla viruses shared less than 72% nucleotide and 80% amino acid identities with any alphaflexi- and betaflexi-virus sequences available in GenBank, identifying three novel carlavirus and one potexvirus species in the Hibiscus rosa-sinensis plant. The detection of physalis vein necrosis nepovirus and passion fruit green spot cilevirus in hibiscus are also new reports from Colombia. Overall, the meta-transcriptome analysis identified the complex virome associated with the black spot symptoms on hibiscus leaves and demonstrated the diversity of virus genera tolerated in the mixed infection of a single H. rosa-sinensis plant. [ABSTRACT FROM AUTHOR]

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 1AVSR, a short-chain reductase for protein 1BHel/VSR, a parathyroid hormone family domain for protein 1EPol/Sd, overlapping domains of unknown function and an ABC transporter domain for protein 2BMP, and DNA topoisomerase domains, transcription factor FBXO25 domain, or DNA Pol subunit cdc27 domain for the satellite RNA protein. Structural predictions for proteins 2AHP/Sd, 2BMP, and 3A? had low confidence, while predictions for proteins 1AVSR, 1BHel*/VSR, 1CVPg, 1DPro, 1EPol*/Sd, and 2CCP/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. [ABSTRACT FROM AUTHOR]

Pawpaw (Asimina triloba) trees exhibiting stunting and foliar mosaic, chlorosis, or distortions were observed in New York. In 2021, leaf samples from two symptomatic trees and a sapling, as well as two asymptomatic trees, were tested for the presence of viruses and viroids by high-throughput sequencing (HTS) using total RNA after ribosomal RNA depletion. HTS sequence information revealed tobacco ringspot virus (TRSV) and tomato ringspot virus (ToRSV) in symptomatic but not in asymptomatic leaves. HTS reads and de novo-assembled contigs covering the genomes of both viruses were obtained, with a higher average read depth for RNA2 than RNA1. The occurrence of TRSV and ToRSV was confirmed in the original leaf samples used for HTS and 12 additional trees and saplings from New York and Maryland in 2022 by RT-PCR combined with Sanger sequencing, and DAS-ELISA. Single infections by TRSV in 11 of 14 trees and dual infections by TRSV and ToRSV in 3 of 14 trees were identified. The nucleotide sequence identity of partial gene fragments of TRSV and ToRSV was high among pawpaw isolates (94.9-100% and 91.8-100%, respectively) and between pawpaw isolates and isolates from other horticultural crops (93.6-100% and 71.3-99.3%, respectively). This study is the first to determine the virome of pawpaw.

Grapevine fanleaf virus (GFLV) (genus Nepovirus, family Secoviridae) causes fanleaf degeneration, one of the most damaging viral diseases of grapevines. Despite substantial advances at deciphering GFLV-host interactions, how this virus overcomes the host antiviral pathways of RNA silencing is poorly understood. In this study, we identified viral suppressors of RNA silencing (VSRs) encoded by GFLV, using fluorescence assays, and tested their capacity at modifying host gene expression in transgenic Nicotiana benthamiana expressing the enhanced green fluorescent protein gene (EGFP). Results revealed that GFLV RNA1-encoded protein 1A, for which a function had yet to be assigned, and protein 1BHel, a putative helicase, reverse systemic RNA silencing either individually or as a fused form (1ABHel) predicted as an intermediary product of RNA1 polyprotein proteolytic processing. The GFLV VSRs differentially altered the expression of plant host genes involved in RNA silencing, as shown by reverse transcription-quantitative PCR. In a co-infiltration assay with an EGFP hairpin construct, protein 1A upregulated NbDCL2, NbDCL4, and NbRDR6, and proteins 1BHel and 1A+1BHel upregulated NbDCL2, NbDCL4, NbAGO1, NbAGO2, and NbRDR6, while protein 1ABHel upregulated NbAGO1 and NbRDR6. In a reversal of systemic silencing assay, protein 1A upregulated NbDCL2 and NbAGO2 and protein 1ABHel upregulated NbDCL2, NbDCL4, and NbAGO1. This is the first report of VSRs encoded by a nepovirus RNA1 and of two VSRs that act either individually or as a predicted fused form to counteract the systemic antiviral host defense, suggesting that GFLV might devise a unique counterdefense strategy to interfere with various steps of the plant antiviral RNA silencing pathways during infection. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Despite its first description in 1977 and numerous reports of its presence in various plant species in many countries, the molecular information available in GenBank for artichoke Italian latent virus (AILV) is still limited to a single complete genome sequence (RNA1 and 2) of a grapevine isolate (AILV-V) and a partial portion of the RNA2 sequence from an isolate of unknown origin and host. Here, we report the results of molecular analyses conducted on the RNA2 of some AILV isolates, sequenced for the first time in this study, together with the first-time identification of AILV in a new host plant species, namely chard (Beta vulgaris subsp. vulgaris), associated with vein clearing and mottling symptoms on leaves. The different AILV isolates sequenced were from artichoke (AILV-C), gladiolus (AILV-G), Sonchus (AILV-S), and chard (AILV-B). At the molecular level, the sequencing results of the RNA2 segments showed that AILV-C, AILV-G, AILV-S, and AILV-B had a length of 4629 nt (excluding the 3′ terminal polyA tail), which is one nt shorter than that of the AILV-V reported in GenBank. A comparison of the RNA2 coding region sequences of all the isolates showed that AILV-V was the most divergent isolate, with the lowest sequence identities of 83.2% at the nucleotide level and 84.7% at the amino acid level. Putative intra-species sequence recombination sites were predicted among the AILV isolates, mainly involving the genomes of AILV-V, AILV-C, and AILV-B. This study adds insights into the variability of AILV and the occurrence of recombination that may condition plant infection. [ABSTRACT FROM AUTHOR]

Hibiscus is not native to Colombia but well suited to its arid soil and dry climates. A single hibiscus plant from Risaralda, showing black spots on upper and lower sides of its leaves, was collected for virome analysis using meta-transcriptomic high-throughput sequencing technology. Bioinformatic analysis identified 12.5% of the total reads in the Ribo-Zero cDNA library which mapped to viral genomes. BLAST searches revealed the presence of carlavirus, potexvirus, and of known members of the genera Betacarmovirus, Cilevirus, Nepovirus, and Tobamovirus in the sample; confirmed by RT-PCR with virus-specific primers followed by amplicon sequencing. Furthermore, in silico analysis suggested the possibility of a novel soymovirus, and a new hibiscus strain of citrus leprosis virus C2 in the mixed infection. Both RNA dependent RNA polymerase and coat protein gene sequences of the potex and carla viruses shared less than 72% nucleotide and 80% amino acid identities with any alphaflexi- and betaflexi-virus sequences available in GenBank, identifying three novel carlavirus and one potexvirus species in the Hibiscus rosa-sinensis plant. The detection of physalis vein necrosis nepovirus and passion fruit green spot cilevirus in hibiscus are also new reports from Colombia. Overall, the meta-transcriptome analysis identified the complex virome associated with the black spot symptoms on hibiscus leaves and demonstrated the diversity of virus genera tolerated in the mixed infection of a single H. rosa-sinensis plant.

Introduction: Soybean is a plant belonging to the Fabaceae family and is one of the most important oil seed in the world. The northern provinces of Iran are the main soybean cultivation regions in the country, but over the years, soybean podding disorder has reduced yield in this regions and caused up to 100% damage. The disorder is more severe in late planting farms, and symptoms can be observed as plant greening, accumulation of flowers and pods, abnormal pods, bud blight, and lack of seed in pod. The results of previous studies have shown the possible role of viruses in occurrence of soybean disorder. This research was conducted to investigate the Nepovirus effect on soybean yield and cultivars, and possible role on soybean podding disorder. Materials and Methods: 770 soybean sample from Golestan provinces were collected befor and after disorder appeared, and tested by DAS-ELISA and RT-PCR test for investigate the possible role of Nepovirus in disorder. The response of soybean cultivars to Nepovirus and soybean podding disorder, was evaluated in greenhouse and natural conditions. In greenhouse, 2 soybean cultivars (Katol, Saman) were inoculated mechanically at the 4-6 leaf stage. For comparison of soybean cultivars response to studied viruses and evaluation of possible role of Nepovirus on soybean podding disorder in natural condition, the experiment was performed in Golestan Agricuitural Research center as a split plot design with three factors: using net, virus inoculation and soybean cultivars with 3 replications in two years. Nepoviruses inoculated on specific plots on soybean plants at 4-6 leaf stages. ELISA and RT-PCR tests were used to ensure the plants infection with inoculated viruses, and growth indices were measured at physiological growth stage. Data were analyzed by two-way ANOVA, and the means were compared with LSD test at 5% confidence level, using SPSS software version 16.0. Results and Discussion: Comparing of viruse frequency in not disorder plants with podding disorder plants, did not show significant relationship between viruses and disorder incidence. In greenhouse, virus inoculation on soybean plants, caused chlorosis, stunt and systemic necrosis, but the typical symptoms of disorder such as severe falling of flowers, re-flowering, and plant greening did not show. In natural conditions, disorder was observed in all soybean cultivars, but disorder incidence was different among cultivars, and Williams variety showed less disorder in four treatments. The highest and lowest plant growth indices were observed in non-inoculation virus with use of net treatment and, virus inoculation with no use of net, respectively. Mechanical virus inoculation on different soybean cultivars, although reduced growth indices and soybeans yield, but had no significant effect on the podding disorder. In treatments with not controlling of sucker pests, the incidence of disorder was significantly higher than other treatments. Conclusion: Virus inoculation on soybean plants in greenhouse and natural conditions did not cause podding disorder syndrome. Viruses in soybean plants, reduce growth indices and aggravate disorder due to the stress they inflict on the soybean plant, but are not probably the cause of the disorder alone. In treatments with not controlling of sucker pests, disorder incidence was significantly higher, so the role of sucker pests was estimated effective and important. [ABSTRACT FROM AUTHOR]

Despite its first description in 1977 and numerous reports of its presence in various plant species in many countries, the molecular information available in GenBank for artichoke Italian latent virus (AILV) is still limited to a single complete genome sequence (RNA1 and 2) of a grapevine isolate (AILV-V) and a partial portion of the RNA2 sequence from an isolate of unknown origin and host. Here, we report the results of molecular analyses conducted on the RNA2 of some AILV isolates, sequenced for the first time in this study, together with the first-time identification of AILV in a new host plant species, namely chard (Beta vulgaris subsp. vulgaris), associated with vein clearing and mottling symptoms on leaves. The different AILV isolates sequenced were from artichoke (AILV-C), gladiolus (AILV-G), Sonchus (AILV-S), and chard (AILV-B). At the molecular level, the sequencing results of the RNA2 segments showed that AILV-C, AILV-G, AILV-S, and AILV-B had a length of 4629 nt (excluding the 3′ terminal polyA tail), which is one nt shorter than that of the AILV-V reported in GenBank. A comparison of the RNA2 coding region sequences of all the isolates showed that AILV-V was the most divergent isolate, with the lowest sequence identities of 83.2% at the nucleotide level and 84.7% at the amino acid level. Putative intra-species sequence recombination sites were predicted among the AILV isolates, mainly involving the genomes of AILV-V, AILV-C, and AILV-B. This study adds insights into the variability of AILV and the occurrence of recombination that may condition plant infection.

Plant viruses of the families Potyviridae and Secoviridae encode 3C-like proteases (3CL pro ) that are related to picornavirus 3C proteases. This review discusses recent advances in deciphering the multifunctional activities of plant virus 3CL pro . These proteases regulate viral polyprotein processing and facilitate virus replication. They are also determinants of host range, virulence, symptomatology and super-infection exclusion in some plant-virus interactions and facilitate aphid transmission. Potyvirid NIa-Pro proteases interact with host factors to interfere with a variety of defense mechanisms: salicylic acid-dependent signaling, ethylene-dependent signaling, transcriptional gene silencing and RNA decay. Potyvirid NIa-Pro also cleave host proteins at signature cleavage sites, although the biological impact of these cleavage remains to be determined. Recently, a plant defense mechanism was uncovered that inhibits the proteolytic activity of a comovirus 3CL pro . Future perspectives are discussed including using proteomic and degradomic techniques to elucidate the network of interactions of plant virus 3CL pro with the host proteome., Competing Interests: Declaration of competing interest I have nothing to declare., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Viral diseases affecting the essential oil rose, which is a valuable object of agricultural production, may have a significant negative impact on the economic value of this crop. Hence, the study and control of potentially dangerous viruses is essential to improving the quality of cultivars of this raw plant material, to enable production of valuable derivatives. The diversity of viruses affecting Rosa L. plants manifests itself in their conditional division into those that are specific to this crop, and those that are hosted by other plants. Representatives of both groups are found in different countries, however, a low number of viruses identified have been thoroughly studied through the use of experimental methods. In particular, with regard to many viruses, the issue of their spread remains open. The viruses infecting Rosa L. plants along with other crops are described in the literature in detail, as the range of hosts they affect is rather wide and well-studied. It is also possible to single out the three most significant viruses affecting this host—Prunus necrotic ringspot virus, Apple mosaic virus and Arabis mosaic virus which individually, or collectively, cause viral diseases that manifest themselves in mosaic symptoms. The most likely mechanisms for the spread of the Rosa L. species viruses are vegetative propagation procedures and transmission by various pests. These presumptions underlie viral infection control methods, including a well-thought-out planting scheme and provision of accurate plant care, which considers plant disinfection, disease monitoring associated with diagnostics and obtaining virus-free material through biotechnology techniques. [ABSTRACT FROM AUTHOR]

Plant viruses of the families Potyviridae and Secoviridae encode 3C-like proteases (3CLpro) that are related to picornavirus 3C proteases. This review discusses recent advances in deciphering the multifunctional activities of plant virus 3CLpro. These proteases regulate viral polyprotein processing and facilitate virus replication. They are also determinants of host range, virulence, symptomatology and super-infection exclusion in some plant-virus interactions and facilitate aphid transmission. Potyvirid NIa-Pro proteases interact with host factors to interfere with a variety of defense mechanisms: salicylic acid-dependent signaling, ethylene-dependent signaling, transcriptional gene silencing and RNA decay. Potyvirid NIa-Pro also cleave host proteins at signature cleavage sites, although the biological impact of these cleavage remains to be determined. Recently, a plant defense mechanism was uncovered that inhibits the proteolytic activity of a comovirus 3CLpro. Future perspectives are discussed including using proteomic and degradomic techniques to elucidate the network of interactions of plant virus 3CLpro with the host proteome. • Potyvirids and secovirids encode proteases related to picornavirus 3C protease. • Potyvirid NIa-Pro counteract plant defenses targeted at the virus or their vector. • The NIa-Pro of three potyviruses cleave a variety of host proteins. • A plant antiviral mechanism inhibits the activity of a comovirus protease. [ABSTRACT FROM AUTHOR]

Papa criolla is one of the most important agricultural products in the Colombian Andean region. Viral diseases, mostly caused by PYVV, PVS, PLRV and PVV, are one of the most limiting factors that constraint the productivity of this crop. In order to gain new knowledge of the S. phureja virome in Colombia, in this study using high-throughput sequencing (HTS) and quantitative RT-PCR (RT-qPCR) we present the first report of a natural infection with Potato virus B (PVB) in this country. PVB was detected in leaf samples from different S. phureja plots in Antioquia. Using bioinformatics analysis, it was possible to get the genome assembly of PVB, which comprised two segments of 7.126 nt (RNA1) and 4.298 nt (RNA2) coding for polyproteins P1 and P2. Genome sequences were used to design PVB-specific primers for RT-PCR and RT-qPCR. PVB was found in 35 % of S. phureja samples but was not detected in 20 samples of S. tuberosum var. Diacol-Capiro. The methodology used in this study could be useful in a PVB epidemiology surveillance program in Colombia and other Andean countries. [ABSTRACT FROM AUTHOR]

Tomato black ring virus (TBRV) and beet ringspot virus (BRSV) are closely related but distinct members of subgroup B of the genus Nepovirus. Both viruses have broad host ranges and are transmitted by seed, pollen, and ectoparasitic nematodes. Although 13 TBRV and 3 BRSV genome sequences were already available, no attempt has been made to link sequence data from these recent sequences with those of historical isolates studied in the pre‐sequencing era. High‐throughput sequencing was used to generate eight new TBRV and BRSV genome sequences from three historical >60‐year‐old and two >30‐year‐old isolates, and three more recent isolates. These eight isolates were from the Czech Republic, Germany, and the UK. We compared these with all genomes sequenced previously. Intraspecies recombination (three of four TBRV and two of four BRSV isolates) was frequent amongst the eight new genomes. Interspecies recombination was also present within the RNA1 of TBRV isolates BRSV‐3393 SG GB and BRSV‐9888 ST GB. No satellite RNAs were associated with the eight new genomes. Two commercial enzyme‐linked immunosorbent assay (ELISA) kits used to detect TBRV during routine testing differed in that one detected only TBRV and the other only BRSV, so they are likely to provide incorrect but potentially complementary virus occurrence information. We suggest both ELISA kits, or appropriate molecular tests, be used by biosecurity authorities to avoid this problem. This study illustrates the value of sequencing historical isolates preserved from the pre‐sequencing era. [ABSTRACT FROM AUTHOR]

BackgroundGenome-wide association study (GWAS) is a useful tool for detecting and characterizing traits of interest including those associated with disease resistance in soybean. The availability of 50,000 single nucleotide polymorphism (SNP) markers (SoySNP50K iSelect BeadChip; www.soybase.org ) on 19,652 soybean and wild soybean plant introductions (PIs) in the USDA Soybean Germplasm Collection allows for fast and robust identification of loci associated with a desired phenotype. By using a genome-wide marker set to predict phenotypic values, genomic prediction for phenotype-unknown but genotype-determined PIs has become possible. The goal of this study was to describe the genetic architecture associated with sensitivity to Tobacco ringspot virus (TRSV) infection in the USDA Soybean Germplasm Collection.ResultsTRSV-induced disease sensitivities of the 697 soybean PIs were rated on a one to five scale with plants rated as one exhibiting mild symptoms and plants rated as five displaying terminal bud necrosis (i.e., bud blight). The GWAS identified a single locus on soybean chromosome 2 strongly associated with TRSV sensitivity. Cross-validation showed a correlation of 0.55 (P < 0.01) to TRSV sensitivity without including the most significant SNP marker from the GWAS as a covariate, which was a better estimation compared to the mean separation by using significant SNPs. The genomic estimated breeding values for the remaining 18,955 unscreened soybean PIs in the USDA Soybean Germplasm Collection were obtained using the GAPIT R package. To evaluate the prediction accuracy, an additional 55 soybean accessions were evaluated for sensitivity to TRSV, which resulted in a correlation of 0.67 (P < 0.01) between actual and predicted severities.ConclusionA single locus responsible for TRSV sensitivity in soybean was identified on chromosome 2. Two leucine-rich repeat receptor-like kinase genes were located near the locus and may control sensitivity of soybean to TRSV infection. Furthermore, a comprehensive genomic prediction for TRSV sensitivity for all accessions in the USDA Soybean Germplasm Collection was completed.

Aim. The aim of the current work is to analyse the epiphytotic situation in the south of the Russian Far East in connection with viral diseases of legumes (Fabaceae Lindl., 1836).Discussion contains a description of 18 viruses that infect legumes in this region: Alfalfa mosaic (Martellivirales: Bromoviridae, Alfamovirus); Vicia unijuga mosaic (Martellivirales: Bromoviridae, Bromovirus); Cucumber mosaic (Martellivirales: Bromoviridae, Cucumovirus); Vicia unijuga ringspot virus (Martellivirales: Closteroviridae, Unidentified); Trifolium hybridum yellow mosaic virus, Bean common mosaic virus, Bean yellow mosaic virus, Trifolium repens mottle virus, Mountain clover mosaic virus, Red clover mosaic virus, Soybean chlorotic deformation virus, Soybean chlorotic mottle virus, Soybean mosaic virus, Soybean weak mosaic virus (Patatavirales: Genus, Potyvirus); Tobacco ringspot virus (Picornavirales: Secoviridae, Nepovirus); Pea enation mosaic virus (Tolivirales: Luteoviridae, Enamovirus); White clover mosaic virus (Tymovirales: Alphaflexiviridae, Potexvirus); Vicia pseudorobus necrotic mosaic virus (Tymovirales: Betaflexiviridae, Carlavirus). The description of the established natural reservoirs and the main vectors of these viruses is given.Conclusion. A list of measures are recommended for the prevention of viral diseases of legumes and a thesis is provided on the need to continue the planned monitoring of the phytovirological situation in the Russian Far East.

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 1B Hel , as well as their fused form (1AB Hel ). 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 1B Hel 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 1AB Hel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1AB Hel . 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 1B Hel . 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Choi, Browning, Schmitt-Keichinger and Fuchs.)

Pawpaw (Asimina triloba) trees exhibiting stunting and foliar mosaic, chlorosis, or distortions were observed in New York. In 2021, leaf samples from two symptomatic trees and a sapling, as well as two asymptomatic trees, were tested for the presence of viruses and viroids by high-throughput sequencing (HTS) using total RNA after ribosomal RNA depletion. HTS sequence information revealed tobacco ringspot virus (TRSV) and tomato ringspot virus (ToRSV) in symptomatic but not in asymptomatic leaves. HTS reads and de novo-assembled contigs covering the genomes of both viruses were obtained, with a higher average read depth for RNA2 than RNA1. The occurrence of TRSV and ToRSV was confirmed in the original leaf samples used for HTS and 12 additional trees and saplings from New York and Maryland in 2022 by RT-PCR combined with Sanger sequencing, and DAS-ELISA. Single infections by TRSV in 11 of 14 trees and dual infections by TRSV and ToRSV in 3 of 14 trees were identified. The nucleotide sequence identity of partial gene fragments of TRSV and ToRSV was high among pawpaw isolates (94.9–100% and 91.8–100%, respectively) and between pawpaw isolates and isolates from other horticultural crops (93.6–100% and 71.3–99.3%, respectively). This study is the first to determine the virome of pawpaw.

Abstract Background Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. Results The epitope sequence was genetically inserted in the αB-αB” domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. Conclusions The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.

In 2015 and 2016, lettuce (Lactuca sativa) plants showing yellow ringspots and line patterns were observed in Argolida (Peloponnese, Greece). Symptoms resembled those caused by nepoviruses, especially artichoke yellow ringspot virus (AYRSV), an endemic virus in artichoke (Cynara scolymus) and onion (Allium cepa var. cepa) crops in this area. The aim of this work was to identify and characterize the viral agent associated with the observed disease. We carried out mechanical inoculations onto Nicotiana benthamiana and lettuce plants and also virus‐specific reverse transcription (RT)‐PCR. Results confirmed the presence of AYRSV in all lettuce plants with symptoms tested, and the natural and experimental host range of the virus was studied. The species Heliotropium europaeum, Solanum nigrum, Origanum vulgare, Lactuca seriola, and Sinapis arvensis, are recorded as hosts of AYRSV for the first time. Also, AYRSV was detected by RT‐PCR in seeds and in the produced seedlings of lettuce, N. benthamiana, N. glutinosa, N. tabacum, and S. nigrum. Sequencing of a partial fragment of the gene encoding the RNA‐dependent RNA polymerase was performed for 29 isolates of AYRSV originating from different hosts (lettuce, artichoke, onion, and arable weeds). Phylogenetic analysis of AYRSV isolates classified them in diverse clusters that exhibited no host grouping. All groups included isolates from cultivated and wild plant species. In this study, AYRSV was associated with a new lettuce disease for the first time, thus increasing the known natural host range of the virus. [ABSTRACT FROM AUTHOR]

We have obtained precatalytic (enzyme-substrate complex) and postcatalytic (enzyme-product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme-substrate and enzyme-product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures.

The majority of picornaviral 3C proteinases (3Cpro) cleavage sites possess glutamine at the P1 position. Plant nepovirus 3C-like proteinases (3CLpro) show however much broader specificity, cleaving not only after glutamine, but also after several basic and hydrophobic residues. To investigate this difference, we employed AlphaFold to generate structural models of twelve selected 3CLpro, representing six substrate specificities. Generally, we observed favorable correlations between the architecture and charge of nepovirus proteinase S1 subsites and their ability to accept or restrict larger residues. The models identified a conserved aspartate residue close to the P1 residue in the S1 subsites of all nepovirus proteinases examined, consistent with the observed strong bias against negatively-charged residues at the P1 position of nepovirus cleavage sites. Finally, a cramped S4 subsite along with the presence of two unique histidine and serine residues explains the strict requirement of the grapevine fanleaf virus proteinase for serine at the P4 position. • We used AlphaFold to generate models of twelve nepovirus proteinases. • All models show close relationships to picornavirus 3C proteinases. • The residues found in the S1 pockets of the nepovirus proteinases differ. • These differences can explain the varied specificities of the nepovirus proteinases. • Comparison of the models illuminates the evolution of the proteinases. [ABSTRACT FROM AUTHOR]

Abstract. The aim of the presented work consists in the analysis of results of long-term monitoring of the members of one of the most important and interesting phytovirus taxons – genus Nepovirus (Picornavirales, Secoviridae, Comovirinae) – which is carried out since 1962 on the planned basis in the south of the Far East (Primorsky krai, Khabarovsk krai, the Sakhalin region) by the Laboratory of Virology of Federal Scientific Center of the East Asia terrestrial biodiversity of Far Eastern Branch of Russian Academy of Sciences (Vladivostok, Russia). Discussion. Many (it is possible – all) nepoviruses are capable to cause epiphytoties with significant economic damage for world economy. In the current article the organization of nepovirus genome, the principles of their division into three genetic groups (A, B, and C), virion morphology, the principles of division into three beyond density fractions (T, M, and B), the characteristics of phytopathology connected with nepoviruses, their ecology in the south of the Far East (including sources of isolation and vectors) are discussed as well as physical-chemical properties of some the most interesting strains from this region which apparently could represent new species of Nepovirus genus: Phryma asiatica mosaic virus (PhAMV); dayflower mosaic virus (DFMV); foenugreek necrotic spot virus (FoNSV); Capsicum annuum necrotic spot virus (CaNSV). Conclusion. Nepovirus genus is one of the most adapted for circulation in the phytocenoses of midlatitudes having natural reservoir among wild plants and pose serious threat for crops. On the basis of ecological features of nepoviruses the scientifically based list of preventive and protective actions against epiphytoties with nepovirus nature is provided. One of the most important elements of such actions is their planned monitoring at the regional level with obligatory comparison of the received results to universal data.

Grapevine fanleaf virus (GFLV) is one of the most severe virus diseases of grapevines, causing fanleaf degeneration that is transmitted by Xiphinema index. This paper aims to isolate Xiphinema species from Tunisian vineyard soil samples and assess their ability to acquire and transmit GFLV under natural and controlled conditions. Based on morphological and morphometric analyses, Tunisian dagger nematodes were identified as X. index and Xiphinema italiae. These results were confirmed with molecular identification tools using species-specific polymerase chain reaction primers. The total RNA of GFLV was extracted from specimens of Xiphinema and amplified based on real-time polymerase chain reaction using virus-specific primers. Our results showed that X. index could acquire and transmit the viral particles of GFLV. This nepovirus was not detected in X. italiae , under natural conditions; however, under controlled conditions, this nematode was able to successfully acquire and transmit the viral particles of GFLV.

Tomato necrotic ringspot virus (TNRV) was first reported in Thailand in 2011, where it continues to reduce the yield and quality of pepper and tomato crops. Here, we report the complete genome sequence of TNRV isolate chilli-CR derived from next-generation sequencing. The TNRV genome comprises 16,595 nucleotides (nt) on three RNA segments. The L RNA is 8,858 nt, the M RNA is 4,724 nt, and the S RNA is 3,013 nt in length. The genome structure and organization are typical of orthotospoviruses, encoding five proteins, named L, NSm, G N G C , NSs, and N. Pairwise comparison of each genomic RNA segment and its deduced amino acid (aa) sequence showed that TNRV chilli-CR shares 73.6-82.3% nt sequence identity and 81.1-91.9% aa sequence identity with pepper chlorotic spot virus (PCSV). Similar phylogenetic groupings were observed based on each genomic RNA or deduced aa sequence, and with concatenated genomic RNA sequences. The clustering of TNRV and PCSV in all phylogenetic analyses, and the 78.9% overall nt sequence identity observed using the concatenated genomic RNAs suggest that TNRV is a distinct orthotospovirus and that analysis of concatenated orthotospovirus genome sequences will be of value in future phylogenetic studies of this virus group., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Cotton is one of the most salient cash crops globally and in the United States. Lately, several virus-like diseases have been reported from cotton in the United States such as the tobacco ringspot virus (TRSV) in Oklahoma. TRSV has been reported from various hosts worldwide with minimal phylogenetic examination. In this study, complete genome sequences of four TRSV isolates from cotton were isolated, and the genetic diversity was investigated along with additional available TRSV isolates retrieved from GenBank. Phylogenetic analysis based on the complete RNA1 and RNA2 sequences distributed all TRSV isolates into three major phylogenetic clades exhibiting a differential clade composition depending on the segment. The TRSV cotton isolates exhibited differential grouping between the RNA1 and RNA2 analyses. Additionally, monophyletic subclades of isolates appeared to be conserved between both segments. Thirty-five recombination events in RNA1 and 23 in RNA2 were identified with implications in the variation of the phylogenetic analyses. Furthermore, multiple hypotheses of TRSV evolution were generated based on the phylogenetic analyses, but to test them, more complete genomes of TRSV will be needed. This study provides the first complete genome analysis of TRSV isolates infecting cotton in the United States and a detailed analysis of global TRSV isolates., Competing Interests: The author(s) declare no conflict of interest.

Tomato black ring virus (TBRV) is a member of the Nepovirus genus in the Secoviridae family, which infects a wide range of important crop species worldwide. In this work, we constructed four cDNA infectious clones of the TBRV tagged with the green fluorescent protein (TBRV-GFP), which varied in (i) the length of the sequences flanking the GFP insert, (ii) the position of the GFP insert within the RNA2 polyprotein, and (iii) the addition of a self-cutting 2A protein. The presence of the GFP coding sequence in infected plants was verified by RT-PCR, while the infectivity and stability of the constructs were verified by mechanical inoculation of the host plants. The systemic spread of TBRV-GFP within plants was observed under UV light at a macroscopic level, monitoring GFP-derived fluorescence in leaves, and at a microscopic level using confocal microscopy. The obtained clones are a valuable tool for future studies of TBRV-host interactions, virus biology, and the long-term monitoring of its distribution in infected plants.

Complete genome sequences of two cycas necrotic stunt virus (CNSV) isolates from Paeonia suffruticosa and Daphne odora were determined. Phylogenetic trees and pairwise comparisons using complete RNA1- and RNA2-encoded polyproteins showed that the two CNSV isolates are divergent (83.19%–89.42% in polyprotein 1 and 73.61%–85.78% in polyprotein 2). A comparative analysis based on taxonomic criteria for the species demarcation of nepoviruses confirmed that they are not new species but distinct variants. This is the first report of the complete genome sequences of CNSV detected in P. suffruticosa and D. odora, and the first report of CNSV infecting P. suffruticosa. [ABSTRACT FROM AUTHOR]

Abstract ARGONAUTEs (notably AGO1 and AGO2) are effectors of plant antiviral RNA silencing. AGO1 was shown to be required for the temperature-dependent symptom recovery of Nicotiana benthamiana plants infected with tomato ringspot virus (isolate ToRSV-Rasp1) at 27 °C. In this study, we show that symptom recovery from isolate ToRSV-GYV shares similar hallmarks of antiviral RNA silencing but occurs at a wider range of temperatures (21–27 °C). At 21 °C, an early spike in AGO2 mRNAs accumulation was observed in plants infected with either ToRSV-Rasp1 or ToRSV-GYV but the AGO2 protein was only consistently detected in ToRSV-GYV infected plants. Symptom recovery from ToRSV-GYV at 21 °C was not prevented in an ago2 mutant or by silencing of AGO1 or AGO2. We conclude that other factors (possibly other AGOs) contribute to symptom recovery under these conditions. The results also highlight distinct expression patterns of AGO2 in response to ToRSV isolates and environmental conditions. [ABSTRACT FROM AUTHOR]

Peach rosette mosaic disease was first described in the 1940s affecting peach and plum. It was later determined that peach rosette mosaic virus (PRMV) is the causal agent of the disease. PRMV, a member of the genus Nepovirus, infects several perennial crops including stone fruit, grape and blueberry as well as several weed species found in orchards around the world. The molecular characterization of the virus is limited to partial genome sequences making it difficult to develop reliable and sensitive molecular detection tests; the reason that detection is routinely performed using ELISA with antibodies risen against a single virus isolate. Given the potential economic impact of the virus and the modes of transmission which, in addition to nematodes, include seed we studied PRMV in more depth using a modified dsRNA extraction protocol to obtain the virus genome. We determined the full nucleotide sequence and developed a protocol that detects conserved regions present in RNA 1 and RNA 2, making it an excellent alternative to the detection protocols used today. [ABSTRACT FROM AUTHOR]

Summary: Since their discovery, single‐domain antigen‐binding fragments of camelid‐derived heavy‐chain‐only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode‐transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell‐to‐cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs. [ABSTRACT FROM AUTHOR]

An emerging virus-like flower yellowing disease (FYD) of green Sichuan pepper (Zanthoxylum armatum v. novemfolius) has been recently reported. Four new RNA viruses were discovered in the FYD-affected plant by the virome analysis using high-throughput sequencing of transcriptome and small RNAs. The complete genomes were determined, and based on the sequence and phylogenetic analysis, they are considered to be new members of the genera Nepovirus (Secoviridae), Idaeovirus (unassigned), Enamovirus (Luteoviridae), and Nucleorhabdovirus (Rhabdoviridae), respectively. Therefore, the tentative names corresponding to these viruses are green Sichuan pepper-nepovirus (GSPNeV), -idaeovirus (GSPIV), -enamovirus (GSPEV), and -nucleorhabdovirus (GSPNuV). The viral population analysis showed that GSPNeV and GSPIV were dominant in the virome. The small RNA profiles of these viruses are in accordance with the typical virus-plant interaction model for Arabidopsis thaliana. Rapid and sensitive RT-PCR assays were developed for viral detection, and used to access the geographical distributions. The results revealed a correlation between GSPNeV and the FYD. The viruses pose potential threats to the normal production of green Sichuan pepper in the affected areas due to their natural transmission and wide spread in fields. Collectively, our results provide useful information regarding taxonomy, transmission and pathogenicity of the viruses as well as management of the FYD.

The stone fruit genus Prunus, within the family Rosaceae, comprises more than 230 species, some of which have great importance or value as ornamental or fruit crops. Prunus are affected by numerous viruses and viroids linked to the vegetative propagation practices in many of the cultivated species. To date, 44 viruses and three viroids have been described in the 9 main cultivated Prunus species. Seven of these viruses and one viroid have been identified in Prunus hosts within the last 5 years. This work addresses recent advances and prospects in the study of viruses and viroids affecting Prunus species, mostly concerning the detection and characterisation of the agents involved, pathogenesis analysis and the search for new control tools. New sequencing technologies are quickly reshaping the way we can identify and characterise new plant viruses and isolates. Specific efforts aimed at virus identification or data mining of high-throughput sequencing data generated for plant genomics-oriented purposes can efficiently reveal the presence of known or novel viruses. These technologies have also been used to gain a deeper knowledge of the pathogenesis mechanisms at the gene and miRNA expression level that underlie the interactions between Prunus spp. and their main viruses and viroids. New biotechnological control tools include the transfer of resistance by grafting, the use of new sources of resistance and the development of gene silencing strategies using genetic transformation. In addition, the application of next generation sequencing and genome editing techniques will contribute to improving our knowledge of virus-host interactions and the mechanisms of resistance. This should be of great interest in the search to obtain new Prunus cultivars capable of dealing both with known viruses and viroids and with those that are yet to be discovered in the uncertain scenario of climate change. [ABSTRACT FROM AUTHOR]

Competing Interests: The author(s) declare no conflict of interest.

Grapevine fanleaf virus (GFLV) (genus Nepovirus , family Secoviridae ) causes fanleaf degeneration, one of the most damaging viral diseases of grapevines. Despite substantial advances at deciphering GFLV-host interactions, how this virus overcomes the host antiviral pathways of RNA silencing is poorly understood. In this study, we identified viral suppressors of RNA silencing (VSRs) encoded by GFLV, using fluorescence assays, and tested their capacity at modifying host gene expression in transgenic Nicotiana benthamiana expressing the enhanced green fluorescent protein gene ( EGFP ). Results revealed that GFLV RNA1-encoded protein 1A, for which a function had yet to be assigned, and protein 1B Hel , a putative helicase, reverse systemic RNA silencing either individually or as a fused form (1AB Hel ) predicted as an intermediary product of RNA1 polyprotein proteolytic processing. The GFLV VSRs differentially altered the expression of plant host genes involved in RNA silencing, as shown by reverse transcription-quantitative PCR. In a co-infiltration assay with an EGFP hairpin construct, protein 1A upregulated NbDCL2 , NbDCL4 , and NbRDR6 , and proteins 1B Hel and 1A+1B Hel upregulated NbDCL2 , NbDCL4 , NbAGO1 , NbAGO2 , and NbRDR6 , while protein 1AB Hel upregulated NbAGO1 and NbRDR6 . In a reversal of systemic silencing assay, protein 1A upregulated NbDCL2 and NbAGO2 and protein 1AB Hel upregulated NbDCL2 , NbDCL4 , and NbAGO1 . This is the first report of VSRs encoded by a nepovirus RNA1 and of two VSRs that act either individually or as a predicted fused form to counteract the systemic antiviral host defense, suggesting that GFLV might devise a unique counterdefense strategy to interfere with various steps of the plant antiviral RNA silencing pathways during infection. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

For quarantine purpose, we developed the RT- and nested PCR module of Tomato black ring virus (TBRV), Arabis mosaic virus (ArMV), Cherry leafroll virus (CLRV) and Grapevine fanleaf virus (GFLV). The PCR modules, developed in this study make diagnosis more convenient and speedy because of same PCR condition. And also, the methods are more accurate because it can check whether the result is contamination or not using the mutation-positive control. We discard or return the 27 cases of Nepovirus infection seed by employing the module past 3 years. This study provides a rapid and useful method for detection of four quarantine plant viruses.