The 6-kilodalton peptide 1 in plant viruses of the family Potyviridae is a viroporin.

Potyviridae, the largest family of plant RNA viruses, includes many important pathogens that significantly reduce the yields of many crops worldwide. In this study, we report that the 6-kilodalton peptide 1 (6K1), one of the least characterized potyviral proteins, is an endoplasmic reticulum-localized protein. AI-assisted structure modeling and biochemical assays suggest that 6K1 forms pentamers with a central hydrophobic tunnel, can increase the cell membrane permeability of Escherichia coli and Nicotiana benthamiana, and can conduct potassium in Saccharomyces cerevisiae. An infectivity assay showed that viral proliferation is inhibited by mutations that affect 6K1 multimerization. Moreover, the 6K1 or its homologous 7K proteins from other viruses of the Potyviridae family also have the ability to increase cell membrane permeability and transmembrane potassium conductance. Taken together, these data reveal that 6K1 and its homologous 7K proteins function as viroporins in viral infected cells.

A Zinc Finger Motif in the P1 N Terminus, Highly Conserved in a Subset of Potyviruses, Is Associated with the Host Range and Fitness of Telosma Mosaic Virus.

P1 is the first protein translated from the genomes of most viruses in the family Potyviridae, and it contains a C-terminal serine-protease domain that cis-cleaves the junction between P1 and HCPro in most cases. Intriguingly, P1 is the most divergent among all mature viral factors, and its roles during viral infection are still far from understood. In this study, we found that telosma mosaic virus (TelMV, genus Potyvirus) in passion fruit, unlike TelMV isolates present in other hosts, has two stretches at the P1 N terminus, named N1 and N2, with N1 harboring a Zn finger motif. Further analysis revealed that at least 14 different potyviruses, mostly belonging to the bean common mosaic virus subgroup, encode a domain equivalent to N1. Using the newly developed TelMV infectious cDNA clones from passion fruit, we demonstrated that N1, but not N2, is crucial for viral infection in both Nicotiana benthamiana and passion fruit. The regulatory effects of N1 domain on P1 cis cleavage, as well as the accumulation and RNA silencing suppression (RSS) activity of its cognate HCPro, were comprehensively investigated. We found that N1 deletion decreases HCPro abundance at the posttranslational level, likely by impairing P1 cis cleavage, thus reducing HCPro-mediated RSS activity. Remarkably, disruption of the Zn finger motif in N1 did not impair P1 cis cleavage and HCPro accumulation but severely debilitated TelMV fitness. Therefore, our results suggest that the Zn finger motif in P1s plays a critical role in viral infection that is independent of P1 protease activity and self-release, as well as HCPro accumulation and silencing suppression. IMPORTANCE Viruses belonging to the family Potyviridae represent the largest group of plant-infecting RNA viruses, including a variety of agriculturally and economically important viral pathogens. Like all picorna-like viruses, potyvirids employ polyprotein processing as the gene expression strategy. P1, the first protein translated from most potyvirid genomes, is the most variable viral factor and has attracted great scientific interest. Here, we defined a Zn finger motif-encompassing domain (N1) at the N terminus of P1 among diverse potyviruses phylogenetically related to bean common mosaic virus. Using TelMV as a model virus, we demonstrated that the N1 domain is key for viral infection, as it is involved both in regulating the abundance of its cognate HCPro and in an as-yet-undefined key function unrelated to protease processing and RNA silencing suppression. These results advance our knowledge of the hypervariable potyvirid P1s and highlight the importance for infection of a previously unstudied Zn finger domain at the P1 N terminus.

Mixed infection of ITPase-encoding potyvirid and secovirid in Mercurialis perennis: evidences for a convergent euphorbia-specific viral counterstrike.

BACKGROUND: In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have also been detected in the genomes of several plant RNA viruses infecting two euphorbia species. In particular, two ipomoviruses produce replicase-associated ITPases to cope with high concentration of non-canonical nucleotides found in cassava tissues. METHOD: Using high-throughput RNA sequencing on the wild euphorbia species Mercurialis perennis, two new members of the families Potyviridae and Secoviridae were identified. Both viruses encode for a putative ITPase, and were found in mixed infection with a new partitivirid. Following biological and genomic characterization of these viruses, the origin and function of the phytoviral ITPases were investigated. RESULTS: While the potyvirid was shown to be pathogenic, the secovirid and partitivirid could not be transmitted. The secovirid was found belonging to a proposed new Comovirinae genus tentatively named "Mercomovirus", which also accommodates other viruses identified through transcriptome mining, and for which an asymptomatic pollen-associated lifestyle is suspected. Homology and phylogenetic analyses inferred that the ITPases encoded by the potyvirid and secovirid were likely acquired through independent horizontal gene transfer events, forming lineages distinct from the enzymes found in cassava ipomoviruses. Possible origins from cellular organisms are discussed for these proteins. In parallel, the endogenous ITPase of M. perennis was predicted to encode for a C-terminal nuclear localization signal, which appears to be conserved among the ITPases of euphorbias but absent in other plant families. This subcellular localization is in line with the idea that nucleic acids remain protected in the nucleus, while deaminated nucleotides accumulate in the cytoplasm where they act as antiviral molecules. CONCLUSION: Three new RNA viruses infecting M. perennis are described, two of which encoding for ITPases. These enzymes have distinct origins, and are likely required by viruses to circumvent high level of cytoplasmic non-canonical nucleotides. This putative plant defense mechanism has emerged early in the evolution of euphorbias, and seems to specifically target certain groups of RNA viruses infecting perennial hosts.

Discovery and Characterization of Two Highly Divergent Variants of a Novel Potyvirus Species Infecting Madagascar Periwinkle (Catharanthus roseus).

During the summer of 2022, a cluster of Madagascar periwinkle plants with white and mauve flowers were observed with foliar mild yellow mosaic symptoms on a private property in Harlingen, Cameron County, Texas. The symptoms were reproduced on mechanically inoculated periwinkle and Nicotiana benthamiana plants. Virions of 776 to 849 nm in length and 11.7 to 14.8 nm in width were observed in transmission electron microscopy of leaf dip preparations made from symptomatic periwinkle leaves. High-throughput sequencing (HTS) analysis of total RNA extracts from symptomatic leaves revealed the occurrence of two highly divergent variants of a novel Potyvirus species as the only virus-like sequences present in the sample. The complete genomes of both variants were independently amplified via reverse transcriptase PCR, cloned, and Sanger sequenced. The 5' and 3' of the genomes were acquired using random amplification of cDNA ends methodology. The assembled virus genomes were 9,936 and 9,944 nucleotides (nt) long, and they shared 99.9 to 100% identities with the respective HTS-derived genomes. Each genome encoded hypothetical polyprotein of 3,171 amino acids (aa) (362.6 kilodaltons [kDa]) and 3,173 aa (362.7 kDa), respectively, and they shared 77.3/84.4% nt/aa polyprotein identities, indicating that they represent highly divergent variants of the same Potyvirus species. Both genomes also shared below-species-threshold polyprotein identity levels with the most closely phylogenetically related known potyviruses, thus indicating that they belong to a novel species. The name periwinkle mild yellow mosaic virus (PwMYMV) is given to the potyvirus with complete genomes of 9,936 nt for variant 1 (PwMYMV-1) and 9,944 nt for variant 2 (PwMYMV-2). We propose that PwMYMV be assigned into the genus Potyvirus (family Potyviridae).

Alfalfa vein mottling virus, a novel potyvirid infecting Medicago sativa L.

BACKGROUND: We have recently identified a novel virus detected in alfalfa seed material. The virus was tentatively named alfalfa-associated potyvirus 1, as its genomic fragments bore similarities with potyvirids. In this study, we continued investigating this novel species, expanding information on its genomic features and biological characteristics. METHODS: This research used a wide range of methodology to achieve end results: high throughput sequencing, bioinformatics tools, reverse transcription-polymerase chain reactions, differential diagnostics using indicator plants, virus purification, transmission electron microscopy, and others. RESULTS: In this study, we obtained a complete genome sequence of the virus and classified it as a tentative species in the new genus, most closely related to the members of the genus Ipomovirus in the family Potyviridae. This assumption is based on the genome sequence and structure, phylogenetic relationships, and transmission electron microscopy investigations. We also demonstrated its mechanical transmission to the indicator plant Nicotiana benthamiana and to the natural host Medicago sativa, both of which developed characteristic symptoms therefore suggesting a pathogenic nature of the disease. CONCLUSIONS: Consistent with symptomatology, the virus was renamed to alfalfa vein mottling virus. A name Alvemovirus was proposed for the new genus in the family Potyviridae, of which alfalfa vein mottling virus is a tentative member.

Molecular characterization of a divergent genetic variant of wheat Eqlid mosaic virus from a Texas wheat field.

Investigations conducted during the spring 2020 season to diagnose the associated viral agent of a severe mosaic disease of wheat in a Texas Panhandle field revealed the presence of wheat Eqlid mosaic virus (WEqMV; genus Tritimovirus, family Potyviridae) in the analyzed samples. The complete genome sequences of two WEqMV isolates were determined, and each was found to be 9,634 nucleotides (nt) in length (excluding the polyA tail) and to contain 5' and 3' untranslated regions of 135 nt and 169 nt, respectively, based on rapid amplification of cDNA ends (RACE) assays. Both sequences contained an open reading frame (ORF) of 9,330 nt encoding a polyprotein of 3,109 amino acids (aa). The ORF sequences of the two isolates were 100% identical to each other, but only 74.7% identical to that of the exemplar WEqMV-Iran isolate, with 85.7% aa sequence identity in the encoded polyprotein. The Texas WEqMV isolates also diverged significantly from WEqMV-Iran in the individual proteins at the nt and aa levels. This is the first report of WEqMV in the United States and the first report of this virus outside of Iran, indicating an expansion of its geographical range.

The Single Distinct Leader Protease Encoded by Alpinia oxyphylla Mosaic Virus (Genus Macluravirus) Suppresses RNA Silencing Through Interfering with Double-Stranded RNA Synthesis.

The genomic 5'-terminal regions of viruses in the family Potyviridae (potyvirids) encode two types of leader proteases: serine-protease (P1) and cysteine-protease (HCPro), which differ greatly in the arrangement and sequence composition among inter-genus viruses. Most potyvirids have the same tandemly arranged P1 and HCPro, whereas viruses in the genus Macluravirus encode a single distinct leader protease, a truncated version of HCPro with yet-unknown functions. We investigated the RNA silencing suppression (RSS) activity and its underpinning mechanism of the distinct HCPro from alpinia oxyphylla mosaic macluravirus (aHCPro). Sequence analysis revealed that macluraviral HCPros have obvious truncations in the N-terminal and middle regions when aligned to their counterparts in potyviruses (well-characterized viral suppressors of RNA silencing). Nearly all defined elements essential for the RSS activity of potyviral counterparts are not distinguished in macluraviral HCPros. Here, we demonstrated that aHCPro exhibits a similar anti-silencing activity with the potyviral counterpart. However, aHCPro fails to block both the local and systemic spreading of RNA silencing. In line, aHCPro interferes with the dsRNA synthesis, an upstream step in the RNA silencing pathway. Affinity-purification and NanoLC-MS/MS analysis revealed that aHCPro has no association with core components or their potential interactors involving in dsRNA synthesis from the protein layer. Instead, the ectopic expression of aHCPro significantly reduces the transcript abundance of RDR2, RDR6, SGS3, and SDE5. This study represents the first report on the anti-silencing function of Macluravirus-encoded HCPro and the underlying molecular mechanism.

First report of Nerine yellow stripe virus infecting crinium lily (Crinium sp.) in Texas.

Crinum sp. (family Amaryllidaceae) is an ornamental flower bulb that is commonly called crinum lily, cape lily, cemetery plant, spider lily, and swamp lily. In April 2023, two plants of Crinum sp. var. Maiden's Blush with yellow stripe symptoms (Fig. S1) were submitted to the Texas Plant Virus Diagnostic Laboratory, Weslaco, TX for virus diagnosis. Due to the resemblance of the observed symptoms to those described for potyviruses infecting ornamental flower bulbs (Pearson et al. 2009), total RNA extracts were made from each sample using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, USA), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 2 µg total RNA per sample with Oligo(dT) primers using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, USA) as recommended by the manufacturer. A 2µL aliquot of each cDNA template was initially subjected to PCR using the generic primer pair CIFor/CIRev (Ha et al., 2008) that targets a fragment of the cylindrical inclusion (CI) body of potyviruses. The expected ~700 bp DNA band was amplified from both samples using the Taq DNA polymerase, dNTPack kit (Sigma-Aldrich). The amplicons were cloned and sequenced (three recombinant clones per sample) as described by Hernandez et al. (2021) and the BLASTX analyses of the consensus sequence (GenBank acc. no. OR137018) returned significant hits only to nerine yellow stripe virus (NeYSV; Potyvirus, Potyviridae) at 100% query coverage. To further confirm the results, another pair of universal primers (Jordan et al. 2011) was used to amplify the expected ∼1,600 bp product specific to the partial nuclear inclusion body (NIb), coat protein (CP) cistron, and 3' untranslated region of potyviruses from the same samples. The amplicons were similarly cloned, and a consensus sequence obtained (OR137019). In pairwise comparisons, the partial CI sequence of NeYSV from Texas (NeYSV-TX; OR137018) shared 83% nucleotide (nt)/93% amino acids (aa) identities with the corresponding sequences of NeYSV isolate 63 (MT396083) from the United Kingdom. The partial (649 nt) NIb sequences of NeYSV-TX (OR137019) and the complete CP (OR137019) of NeYSV-TX shared 77-94%/88-94% and 83-99%/89-98% nt/aa identities with the corresponding sequences of global NeYSV isolates that were retrieved from GenBank. Phylogenetic analysis revealed a closer relationship between NeYSV-TX and the isolates Stenomesson (EU042758) and DC (MG012805) from the Netherlands and USA, respectively based on the partial NIb and CP cistrons (Fig. S2), suggesting that NeYSV-TX may have been introduced from foreign and/or domestic sources. NeYSV has been documented previously from the United Kingdom, the Netherlands, Australia, New Zealand, and India; its first report from the United States was a decade ago from Amaryllis belladonna in California (Guaragna et al. 2013). To the best of our knowledge, this is the first report of NeYSV in Texas, thus expanding the geographical range of the virus in the USA. Anecdotal information from the sample submitter implicated infected crinum lily bulbs as the likely source of NeYSV introduction into the property, with subsequent vegetative propagation of plants resulting in 100% incidence of symptomatic lilies (n>100) over time. Thus, the results underscore the importance of ensuring that only virus-free vegetative plant materials are distributed and propagated by florists to curtail virus spread.

ICTV Virus Taxonomy Profile: Potyviridae 2022.

The family Potyviridae includes plant viruses with single-stranded, positive-sense RNA genomes of 8-11 kb and flexuous filamentous particles 650-950 nm long and 11-20 nm wide. Genera in the family are distinguished by the host range, genomic features and phylogeny of the member viruses. Most genomes are monopartite, but those of members of the genus Bymovirus are bipartite. Some members cause serious disease epidemics in cultivated plants. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Potyviridae, which is available at ictv.global/report/potyviridae.

P1 of Sweet Potato Feathery Mottle Virus Shows Strong Adaptation Capacity, Replacing P1-HCPro in a Chimeric Plum Pox Virus.

Potyviridae is the largest family of plant RNA viruses. Their genomes are expressed through long polyproteins that are usually headed by the leader endopeptidase P1. This protein can be classified as type A or type B based on host proteolytic requirements and RNA silencing suppression (RSS) capacity. The main Potyviridae genus is Potyvirus, and a group of potyviruses infecting sweet potato presents an enlarged P1 protein with a polymerase slippage motif that produces an extra product termed P1N-PISPO. These two proteins display some RSS activity and are expressed followed by HCPro, which appears to be the main RNA silencing suppressor in these viruses. Here, we studied the behavior of the P1 protein of Sweet potato feathery mottle virus (SPFMV) using a viral system based on a canonical potyvirus, Plum pox virus (PPV), and discovered that this protein is able to replace both PPV P1 and HCPro. We also found that P1N-PISPO, produced after polymerase slippage, provides extra RNA silencing suppression capacity to SPFMV P1 in this viral context. In addition, the results showed that presence of two type A P1 proteins was detrimental for viral viability. The ample recombination spectrum that we found in the recovered viruses supports the strong adaptation capacity of P1 proteins and signals the N-terminal part of SPFMV P1 as essential for RSS activity. Further analyses provided data to add extra layers to the evolutionary history of sweet potato-infecting potyvirids. IMPORTANCE Plant viruses represent a major challenge for agriculture worldwide and Potyviridae, being the largest family of plant RNA viruses, is one of the primary players. P1, the leader endopeptidase, is a multifunctional protein that contributes to the successful spread of these viruses over a wide host range. Understanding how P1 proteins work, their dynamic interplay during viral infection, and their evolutionary path is critical for the development of strategic tools to fight the multiple diseases these viruses cause. We focused our efforts on the P1 protein of Sweet potato feathery mottle virus, which is coresponsible for the most devastating disease in sweet potato. The significance of our research is in understanding the capacity of this protein to perform several independent functions, using this knowledge to learn more about P1 proteins in general and the potyvirids infecting this host.

A Newly Identified Virus in the Family Potyviridae Encodes Two Leader Cysteine Proteases in Tandem That Evolved Contrasting RNA Silencing Suppression Functions.

Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis-cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability.IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

Novel approaches on identification of conserved miRNAs for broad-spectrum Potyvirus control measures.

Potyviridae comprises more than 200 ssRNA viruses, many of which have a broad host range and geographical distributions. Potyvirids (members of Potyviridae) infect several economically important plants such as saffron, cardamom, cucumber, pepper, potato, tomato, yam, etc. Cumulatively, potyvirids cause a substantial economic loss. The major bottleneck in developing an efficient antiviral strategy is that viruses quickly evade host immunity owing to their higher mutation and recombination rates. Due to this reason, the emergence of newer and improved broad-spectrum approaches to combat viral infections is essential. The use of microRNA's (miRNA) to circumvent viral infection against animal viruses has been successfully employed. Fewer studies reported the development of efficient miRNA-based antivirus resistant strategies against plant viruses and none focused on multiple virus resistance. We focused on potyviruses since studies are limited and identification of conserved miRNAs among various host plants would be an initiative to design broad-spectrum antivirus strategies. In this study, we predicted evolutionarily conserved miRNAs by BLAST searching of reported miRNAs from 15 plants against the GSS and EST sequences of banana. A total of nine miRNAs were predicted and screened in nine diverse potyvirids' hosts (Banana, Tomato, Green gram, Jasmine, Chilli, Coriander, Onion, Rose and Colocasia) belonging to eight different orders (Zingiberales, Solanales, Fabales, Lamiales, Apiales, Asperagales, Rosales and Alismatales). Results suggested that miR168 and miR162 are conserved among all the selected plants. This comprehensive study laid the foundations to design broad-spectrum antivirus resistance using miRNAs. To conclude miR168 and miR162 are conserved among many plants and play a crucial role in evading virus infection which could be used as a potential candidate for developing antiviral strategies against potyvirid infections.

First report of squash vein yellowing virus naturally infecting butternut squash (Cucurbita moschata) in Texas.

Virus diseases are major constraints to the production of cucurbits in the Texas Lower Rio Grande Valley. In September 2020, a ~8.1 ha butternut squash (Cucurbita moschata) field in Hidalgo County, Texas, was observed with virus-like symptoms of vein yellowing, leaf curl, mosaic, and foliar chlorosis. The proportion of plants with virus-like symptoms in this field was estimated at 30% and seven samples (symptomatic = 5; non-symptomatic = 2) were collected randomly for virus diagnosis. Initially, equimolar mixtures of total nucleic acid extracts (Dellaporta et. al. 1983) from two symptomatic samples from this field and extracts from 12 additional symptomatic samples from six other fields across south and central Texas was used to generate one composite sample for diagnosis by high throughput sequencing (HTS). The TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina) was used to construct cDNA library from the composite sample, which was then sequenced on the Illumina NextSeq 500 platform. More than 26 million single-end HTS reads (75 nt each) were obtained and their bioinformatic analyses (Al Rwahnih et al. 2018) revealed several virus-like contigs belonging to different species (data not shown). Among them, 6 contigs that ranged in length from 429 to 3,834 nt shared 96 to 100% identities with isolates of squash vein yellowing virus (SqVYV), genus Ipomovirus, family Potyviridae. To confirm the HTS results, total nucleic acid extracts from the cucurbit samples from all seven fields (n = 46) were used for cDNA synthesis with random hexamers and the PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio). A 1-µL aliquot of cDNA was used in 12.5-µL PCR reaction volumes with PrimeSTAR GXL DNA Polymerase (Takara Bio) and two pairs of SqVYV-specific primers designed based on the HTS derived contigs. The primer pairs SqYVV-v4762: 5'-CTGGATTCTGCTGGAAGATCA & SqYVV-c5512: 5'-CCACCATTAAGGCCATCAAAC and SqYVV-v8478: 5'-TTTCTGGGCAAACAAACATGG & SqYVV-c9715: 5'-TTCAGCGACGTCAAGTGAG targeted ~0.75 kb and ~1.2 kb fragments of the cylindrical inclusion (CI) and the complete coat protein (CP) gene sequences of SqVYV, respectively. The expected DNA band sizes were obtained only from the five symptomatic butternut squash samples from the Hidalgo Co. field. Two amplicons per primer pair from two samples were cloned into pJET1.2/Blunt vector (Life Technologies) and bidirectionally Sanger sequenced, generating 753 nt partial CI specific sequences (MW584341-342) and 1,238 nt that encompassed the complete CP (MW584343-344) of SqVYV. In pairwise comparisons, the partial CI sequences shared 100% nt/aa identity with each other and 98-99% nt/aa identity with corresponding sequences of SqVYV isolate IL (KT721735). The CP cistron of TX isolates shared 100% nt/aa identity with each other and 90-98% nt (97-100% aa) identities with corresponding sequences of several SqVYV isolates in GenBank, with isolates IL (KT721735) and Florida (EU259611) being at the high and low spectrum of nt/aa identity values, respectively. This is the first report of SqVYV in Texas, naturally occurring in butternut squash. SqVYV was first discovered in Florida (Adkins et al. 2007) and subsequently reported from few other states in the U.S. (Adkins et al. 2013; Egel and Adkins 2007; Batuman et al. 2015), Puerto Rico (Acevedo et al. 2013), and locations around the world. The finding shows an expansion of the geographical range of SqVYV and adds to the repertoire of cucurbit-infecting viruses in Texas. Further studies are needed to determine the prevalence of SqVYV in Texas cucurbit fields and an assessment of their genetic diversity.

Complete genome sequence and construction of an infectious full-length cDNA clone of celery latent virus - an unusual member of a putative new genus within the Potyviridae.

Celery latent virus (CeLV) is an incompletely described plant virus known to be sap and seed transmissible and to possess flexuous filamentous particles measuring about 900 nm in length, suggesting it as a possible member of the family Potyviridae. Here, an Italian isolate of CeLV was transmitted by sap to a number of host plants and shown to have a single-stranded and monopartite RNA genome being 11 519 nucleotides (nts) in size and possessing some unusual features. The RNA contains a large open reading frame (ORF) that is flanked by a short 5' untranslated region (UTR) of 13 nt and a 3' UTR consisting of 586 nt that is not polyadenylated. CeLV RNA shares nt sequence identity of only about 40 % with other members of the Potyviridae (potyvirids). The CeLV polyprotein is notable in that it starts with a signal peptide, has a putative P3N-PIPO ORF and shares low aa sequence identity (about 18 %) with other potyvirids. Although potential cleavage sites were not identified for the N-terminal two-thirds of the polyprotein, the latter possesses a number of sequence motifs, the identity and position of which are characteristic of other potyvirids. Attempts at constructing an infectious full-length cDNA clone of CeLV were successful following Rhizobium radiobacter infiltration of Nicotiana benthamiana and Apium graveolens. CeLV appears to have the largest genome of all known potyvirids and some unique genome features that may warrant the creation of a new genus, for which we propose the name 'celavirus'.

A Functional Link between RNA Replication and Virion Assembly in the Potyvirus Plum Pox Virus.

Accurate assembly of viral particles in the potyvirus Plum pox virus (PPV) has been shown to depend on the contribution of the multifunctional viral protein HCPro. In this study, we show that other viral factors, in addition to the capsid protein (CP) and HCPro, are necessary for the formation of stable PPV virions. The CP produced in Nicotiana benthamiana leaves from a subviral RNA termed LONG, which expresses a truncated polyprotein that lacks P1 and HCPro, together with HCPro supplied in trans, was assembled into virus-like particles and remained stable after in vitro incubation. In contrast, deletions in multiple regions of the LONG coding sequence prevented the CP stabilization mediated by HCPro. In particular, we demonstrated that the first 178 amino acids of P3, but not a specific nucleotide sequence coding for them, are required for CP stability and proper assembly of PPV particles. Using a sequential coagroinfiltration assay, we observed that the subviral LONG RNA replicates and locally spreads in N. benthamiana leaves expressing an RNA silencing suppressor. The analysis of the effect of both point and deletion mutations affecting RNA replication in LONG and full-length PPV demonstrated that this process is essential for the assembly of stable viral particles. Interestingly, in spite of this requirement, the CP produced by a nonreplicating viral RNA can be stably assembled into virions as long as it is coexpressed with a replication-proficient RNA. Altogether, these results highlight the importance of coupling encapsidation to other viral processes to secure a successful infection.IMPORTANCE Viruses of the family Potyviridae are among the most dangerous threats for basically every important crop, and such socioeconomical relevance has made them a subject of many research studies. In spite of this, very little is currently known about proteins and processes controlling viral genome encapsidation by the coat protein. In the case of Plum pox virus (genus Potyvirus), for instance, we have previously shown that the multitasking viral factor HCPro plays a role in the production of stable virions. Here, by using this potyvirus as a model, we move further to show that additional factors are also necessary for the efficient production of potyviral particles. More importantly, a comprehensive screening for such factors led us to the identification of a functional link between virus replication and packaging, unraveling a previously unknown connection of these two key events of the potyviral infection cycle.

Sterol isomerase HYDRA1 interacts with RNA silencing suppressor P1b and restricts potyviral infection.

Plants use RNA silencing as a strong defensive barrier against virus challenges, and viruses counteract this defence by using RNA silencing suppressors (RSSs). With the objective of identifying host factors helping either the plant or the virus in this interaction, we have performed a yeast two-hybrid screen using P1b, the RSS protein of the ipomovirus Cucumber vein yellowing virus (CVYV, family Potyviridae), as a bait. The C-8 sterol isomerase HYDRA1 (HYD1), an enzyme involved in isoprenoid biosynthesis and cell membrane biology, and required for RNA silencing, was isolated in this screen. The interaction between CVYV P1b and HYD1 was confirmed in planta by Bimolecular Fluorescence Complementation assays. We demonstrated that HYD1 negatively impacts the accumulation of CVYV P1b in an agroinfiltration assay. Moreover, expression of HYD1 inhibited the infection of the potyvirus Plum pox virus, especially when antiviral RNA silencing was boosted by high temperature or by coexpression of homologous sequences. Our results reinforce previous evidence highlighting the relevance of particular composition and structure of cellular membranes for RNA silencing and viral infection. We report a new interaction of an RSS protein from the Potyviridae family with a member of the isoprenoid biosynthetic pathway.

Areca Palm Necrotic Ringspot Virus, Classified Within a Recently Proposed Genus Arepavirus of the Family Potyviridae, Is Associated With Necrotic Ringspot Disease in Areca Palm.

Areca palm (Areca catechu), one of the two most important commercial crops in Hainan, China, has been severely damaged by a variety of pathogens and insects. Here, we report a new disease, tentatively referred to as areca palm necrotic ringspot disease (ANRSD), which is highly epidemic in the main growing regions in Hainan. Transmission electron microscopy observation and small RNA deep sequencing revealed the existence of a viral agent of the family Potyviridae in a diseased areca palm plant (XC1). The virus was tentatively named areca palm necrotic ringspot virus (ANRSV). Subsequently, the positive-sense single-stranded genome of ANRSV isolate XC1 was completely determined. The genome annotation revealed the existence of two cysteine proteinases in tandem (HC-Pro1 and HC-Pro2) in the genomic 5' terminus of ANRSV. Sequence comparison and phylogenetic analysis suggested the taxonomic classification of ANRSV into the recently proposed genus Arepavirus in the family Potyviridae. Given the close relationship of ANRSV with another newly reported arepavirus (areca palm necrotic spindle-spot virus), the exact taxonomic status of ANRSV needs to be further investigated. In this study, a reverse transcription polymerase chain reaction assay for ANRSV-specific detection was developed and a close association between ANRSV and ANRSD was found.

Salicylic acid is an indispensable component of the Ny-1 resistance-gene-mediated response against Potato virus Y infection in potato.

The purpose of the study was to investigate the role of salicylic acid (SA) signalling in Ny-1-mediated hypersensitive resistance (HR) of potato (Solanum tuberosum L.) to Potato virus Y (PVY). The responses of the Ny-1 allele in the Rywal potato cultivar and transgenic NahG-Rywal potato plants that do not accumulate SA were characterized at the cytological, biochemical, transcriptome, and proteome levels. Analysis of noninoculated and inoculated leaves revealed that HR lesions started to develop from 3 d post inoculation and completely restricted the virus spread. At the cytological level, features of programmed cell death in combination with reactive oxygen species burst were observed. In response to PVY infection, SA was synthesized de novo. The lack of SA accumulation in the NahG plants led to the disease phenotype due to unrestricted viral spreading. Grafting experiments show that SA has a critical role in the inhibition of PVY spreading in parenchymal tissue, but not in vascular veins. The whole transcriptome analysis confirmed the central role of SA in orchestrating Ny-1-mediated responses and showed that the absence of SA leads to significant changes at the transcriptome level, including a delay in activation of expression of genes known to participate in defence responses. Moreover, perturbations in the expression of hormonal signalling genes were detected, shown as a switch from SA to jasmonic acid/ethylene signalling. Viral multiplication in the NahG plants was accompanied by downregulation of photosynthesis genes and activation of multiple energy-producing pathways.

Old and New Aphid-Borne Viruses in Coriander in Chile: An Epidemiological Approach.

In Chile, edible herbs are mainly grown by small farmers. This type of horticultural crop typically requires intensive management because it is highly susceptible to insects, some of which transmit viruses that severely affect crop yield and quality. In 2019, in coriander plants tested negative for all previously reported viruses, RNA-Seq analysis of one symptomatic plant revealed a plethora of viruses, including one virus known to infect coriander, five viruses never reported in coriander, and a new cytorhabdovirus with a 14,180 nucleotide RNA genome for which the species name Cytorhabdovirus coriandrum was proposed. Since all the detected viruses were aphid-borne, aphids and weeds commonly growing around the coriander field were screened for viruses. The results showed the occurrence of the same seven viruses and the alfalfa mosaic virus, another aphid-borne virus, in aphids and weeds. Together, our findings document the presence of multiple viruses in coriander and the potential role of weeds as virus reservoirs for aphid acquisition.

Metatranscriptome and small RNA sequencing revealed a mixed infection of newly identified bymovirus and bean yellow mosaic virus on peas.

Peas (Pisum sativum L.) are widely cultivated in temperate regions and are susceptible hosts for various viruses across different families. The discovery and identification of new viruses in peas has significant implications for field disease management. Here, we identified a mixed infection of two viruses from field-collected peas exhibiting virus-like symptoms using metatranscriptome and small RNA sequencing techniques. Upon identification, one of the viruses was determined to be a newly isolated and discovered bymovirus from peas, named "pea bymovirus 1 (PBV1)". The other was identified as a novel variant of bean yellow mosaic virus (BYMV-HZ1). Subsequently, mechanical inoculation and RT-PCR assays confirmed that both viruses could be inoculated back onto peas and tobaccos, showing mixed infection by PBV1 and BYMV-HZ1. To our knowledge, this is the first isolation of a bymovirus from pea and the first documented case of mixed infection of peas by PBV1 and BYMV-HZ1 in China.