Interaction of EXA1 and eIF4E Family Members Facilitates Potexvirus Infection in Arabidopsis thaliana.

Plant viruses depend on a number of host factors for successful infection. Deficiency of critical host factors confers recessively inherited viral resistance in plants. For example, loss of Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana confers resistance to potexviruses. However, the molecular mechanism of how EXA1 assists potexvirus infection remains largely unknown. Previous studies reported that the salicylic acid (SA) pathway is upregulated in exa1 mutants, and EXA1 modulates hypersensitive response-related cell death during EDS1-dependent effector-triggered immunity. Here, we show that exa1-mediated viral resistance is mostly independent of SA and EDS1 pathways. We demonstrate that Arabidopsis EXA1 interacts with three members of the eukaryotic translation initiation factor 4E (eIF4E) family, eIF4E1, eIFiso4E, and novel cap-binding protein (nCBP), through the eIF4E-binding motif (4EBM). Expression of EXA1 in exa1 mutants restored infection by the potexvirus Plantago asiatica mosaic virus (PlAMV), but EXA1 with mutations in 4EBM only partially restored infection. In virus inoculation experiments using Arabidopsis knockout mutants, EXA1 promoted PlAMV infection in concert with nCBP, but the functions of eIFiso4E and nCBP in promoting PlAMV infection were redundant. By contrast, the promotion of PlAMV infection by eIF4E1 was, at least partially, EXA1 independent. Taken together, our results imply that the interaction of EXA1-eIF4E family members is essential for efficient PlAMV multiplication, although specific roles of three eIF4E family members in PlAMV infection differ. IMPORTANCE The genus Potexvirus comprises a group of plant RNA viruses, including viruses that cause serious damage to agricultural crops. We previously showed that loss of Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana confers resistance to potexviruses. EXA1 may thus play a critical role in the success of potexvirus infection; hence, elucidation of its mechanism of action is crucial for understanding the infection process of potexviruses and for effective viral control. Previous studies reported that loss of EXA1 enhances plant immune responses, but our results indicate that this is not the primary mechanism of exa1-mediated viral resistance. Here, we show that Arabidopsis EXA1 assists infection by the potexvirus Plantago asiatica mosaic virus (PlAMV) by interacting with the eukaryotic translation initiation factor 4E family. Our results imply that EXA1 contributes to PlAMV multiplication by regulating translation.

ICTV Virus Taxonomy Profile: Phenuiviridae 2023.

The family Phenuiviridae comprises viruses with 2-8 segments of negative-sense or ambisense RNA, comprising 8.1-25.1 kb in total. Virions are typically enveloped with spherical or pleomorphic morphology but can also be non-enveloped filaments. Phenuivirids infect animals including livestock and humans, birds, plants or fungi, as well as arthropods that serve as single hosts or act as biological vectors for transmission to animals or plants. Phenuivirids include important pathogens of humans, livestock, seafood and agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Phenuiviridae, which is available at ictv.global/report/phenuiviridae.

Complete genome sequence of a novel ophiovirus associated with chlorotic disease of pepper (Capsicum annuum L.) in Japan.

In July 2018, pepper plants (Capsicum annuum L.) with chlorotic leaves and fruits were observed in Kochi prefecture, Japan. High-throughput sequencing (HTS) identified the possible presence of an ophiovirus-like virus possessing three RNA segments in a chlorotic leaf. Using Sanger sequencing with primers designed based on the HTS results and a different source of RNA from the one used for HTS, the complete nucleotide sequences of three RNA segments encoding four proteins on their complementary strand were determined. The amino acid sequences of these four proteins showed similarity to those of the RNA-dependent RNA polymerase, RNA-silencing suppressor protein, movement protein, and coat protein, respectively, of ophioviruses, which are negative-sense single-stranded RNA viruses. However, the coat protein amino acid sequence of the virus found on pepper plants was no more than 61.9% identical to those of any known ophioviruses, which is lower than the species demarcation threshold of 85 %. Hence, we suggest that this virus, which we have named "pepper chlorosis associated virus" (PepCaV) should be considered a member of a new species in the genus Ophiovirus, for which we propose the name "Ophiovirus capsici". The results of phylogenetic analysis using coat protein amino acid sequences of PepCaV and other ophioviruses also supported this conclusion. PepCaV RNA was found to have conserved nucleotide sequences at both the 5' and 3' termini of the different RNA segments, and the conserved terminal nucleotide sequences were predicted to form a self-complementary double-stranded region, resulting in a panhandle structure in each of the genomic RNAs.

Characterization of a ToMV isolate overcoming Tm-22 resistance gene in tomato.

Tomato mosaic virus (ToMV) is easily transmitted in soil and by contact. By these reasons, it is relatively difficult to control ToMV disease in tomato. Incorporation of the Tm-22 gene has been widely used as a control method for ToMV, but ToMV isolates that overcome this resistance gene have been reported worldwide in recent years. In this study, we determined the entire nucleotide sequences of ToMV isolate [named ToMV-KMT (LC650928)], which was isolated from tomato plants showing symptoms of systemic necrosis in Kumamoto prefecture, Japan. We also analyzed the viral gene of ToMV-KMT that overcome the Tm-22 gene by constructing its infectious cDNA clone and by generating chimeric viruses with a non-breaking strain. According to previous research, Tm-22 recognizes the viral movement protein (MP) and exerts resistance by inducing hypersensitive reaction or hypersensitive cell death. We discovered that a mutation in the 240th amino acid (aspartic acid to tyrosine) of the MP of ToMV-KMT, which may stabilize the protein's structure, is responsible for the ability of this isolate to overcome the resistance of Tm-22.

Characterization of tulip streak virus, a novel virus associated with the family Phenuiviridae.

In Japan, tulip-growing areas have been plagued by viral diseases for decades, but the viruses causing the damage remain undescribed. In this study, Nicotiana benthamiana and Chenopodium quinoa plants mechanically inoculated with crude sap from a symptomatic tulip flower exhibited necrosis symptoms. Additionally, flexuous and filamentous virus particles were detected by electron microscopy analysis. Moreover, we determined the complete sequences of two genomic segments of the tulip streak virus (TuSV), which is a new virus associated with streaking symptoms, on the basis of a next-generation sequencing analysis. Homology analyses of the amino acid sequence of RNA-dependent RNA polymerase and the terminal sequence of the genomic RNA indicated that TuSV is associated with viruses in the family Phenuiviridae, but differs substantially from other reported viruses.

Construction of an infectious dahlia common mosaic virus clone.

Dahlia is a major ornamental plant that is cultivated worldwide. However, dahlia plants, which are mainly propagated through vegetative reproduction, are susceptible to widespread damage by viruses, and viral control requires that the nature of the infecting virus(es) be known. In this study, dahlia common mosaic virus (DCMV) was detected for the first time in Japan and sequenced. This is the first report of an infectious DCMV clone being constructed, and it will aid in the characterization of DCMV.

EXA1, a GYF domain protein, is responsible for loss-of-susceptibility to plantago asiatica mosaic virus in Arabidopsis thaliana.

One of the plant host resistance machineries to viruses is attributed to recessive alleles of genes encoding critical host factors for virus infection. This type of resistance, also referred to as recessive resistance, is useful for revealing plant-virus interactions and for breeding antivirus resistance in crop plants. Therefore, it is important to identify a novel host factor responsible for robust recessive resistance to plant viruses. Here, we identified a mutant from an ethylmethane sulfonate (EMS)-mutagenized Arabidopsis population which confers resistance to plantago asiatica mosaic virus (PlAMV, genus Potexvirus). Based on map-based cloning and single nucleotide polymorphism analysis, we identified a premature termination codon in a functionally unknown gene containing a GYF domain, which binds to proline-rich sequences in eukaryotes. Complementation analyses and robust resistance to PlAMV in a T-DNA mutant demonstrated that this gene, named Essential for poteXvirus Accumulation 1 (EXA1), is indispensable for PlAMV infection. EXA1 contains a GYF domain and a conserved motif for interaction with eukaryotic translation initiation factor 4E (eIF4E), and is highly conserved among monocot and dicot species. Analysis using qRT-PCR and immunoblotting revealed that EXA1 was expressed in all tissues, and was not transcriptionally responsive to PlAMV infection in Arabidopsis plants. Moreover, accumulation of PlAMV and a PlAMV-derived replicon was drastically diminished in the initially infected cells by the EXA1 deficiency. Accumulation of two other potexviruses also decreased in exa1-1 mutant plants. Our results provided a functional annotation to GYF domain-containing proteins by revealing the function of the highly conserved EXA1 gene in plant-virus interactions.

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor.

RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR, but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans-acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/RDR6-dependent double-stranded RNA synthesis in the trans-acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs.

Lectin-mediated resistance impairs plant virus infection at the cellular level.

Plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. Lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. This study analyzed the resistance of certain Arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (PlAMV). Map-based positional cloning revealed that the lectin gene JACALIN-TYPE LECTIN REQUIRED FOR POTEXVIRUS RESISTANCE1 (JAX1) is responsible for the resistance. JAX1-mediated resistance did not show the properties of conventional resistance (R) protein-mediated resistance and was independent of plant defense hormone signaling. Heterologous expression of JAX1 in Nicotiana benthamiana showed that JAX1 interferes with infection by other tested potexviruses but not with plant viruses from different genera, indicating the broad but specific resistance to potexviruses conferred by JAX1. In contrast with the lectin gene RESTRICTED TEV MOVEMENT1, which inhibits the systemic movement of potyviruses, which are distantly related to potexviruses, JAX1 impairs the accumulation of PlAMV RNA at the cellular level. The existence of lectin genes that show a variety of levels of virus resistance, their targets, and their properties, which are distinct from those of known R genes, suggests the generality of lectin-mediated resistance in plant innate immunity.

Complete nucleotide sequence and genome structure of a Japanese isolate of hibiscus latent Fort Pierce virus, a unique tobamovirus that contains an internal poly(A) region in its 3' end.

In this study, we detected a Japanese isolate of hibiscus latent Fort Pierce virus (HLFPV-J), a member of the genus Tobamovirus, in a hibiscus plant in Japan and determined the complete sequence and organization of its genome. HLFPV-J has four open reading frames (ORFs), each of which shares more than 98 % nucleotide sequence identity with those of other HLFPV isolates. Moreover, HLFPV-J contains a unique internal poly(A) region of variable length, ranging from 44 to 78 nucleotides, in its 3'-untranslated region (UTR), as is the case with hibiscus latent Singapore virus (HLSV), another hibiscus-infecting tobamovirus. The length of the HLFPV-J genome was 6431 nucleotides, including the shortest internal poly(A) region. The sequence identities of ORFs 1, 2, 3 and 4 of HLFPV-J to other tobamoviruses were 46.6-68.7, 49.9-70.8, 31.0-70.8 and 39.4-70.1 %, respectively, at the nucleotide level and 39.8-75.0, 43.6-77.8, 19.2-70.4 and 31.2-74.2 %, respectively, at the amino acid level. The 5'- and 3'-UTRs of HLFPV-J showed 24.3-58.6 and 13.0-79.8 % identity, respectively, to other tobamoviruses. In particular, when compared to other tobamoviruses, each ORF and UTR of HLFPV-J showed the highest sequence identity to those of HLSV. Phylogenetic analysis showed that HLFPV-J, other HLFPV isolates and HLSV constitute a malvaceous-plant-infecting tobamovirus cluster. These results indicate that the genomic structure of HLFPV-J has unique features similar to those of HLSV. To our knowledge, this is the first report of the complete genome sequence of HLFPV.

Complete genome sequence of bean common mosaic virus from black bean in Vietnam.

This is a report of a complete genome sequence of bean common mosaic virus in Vietnam. This virus shares around 99% nucleotide identity with a Nepal isolate.

Development of a RT-LAMP assay for real-time detection of criniviruses infecting tomato.

Yellowing symptoms caused by tomato chlorosis virus (ToCV) and tomato infectious chlorosis virus (TICV), both assigned to the genus Crinivirus, resemble nutrient deficiencies. Therefore, early diagnosis of infections will prevent crop damage and the spread of the viruses. In this study, we established a rapid detection method for ToCV and TICV by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). We first designed primer sets for RT-LAMP specific for ToCV and TICV. Next, by selecting the optimum primer set and determining the optimum conditions for the RT-LAMP reaction, each virus was detected within 50 min by piercing the diseased area of a tomato leaf with a toothpick, immersing the toothpick in the reaction solution, and conducting the RT-LAMP reaction. To verify the accuracy of the procedure, 61 tomato leaf samples showing disease symptoms were collected from five regions of Indonesia, and the RT-LAMP results for the samples were identical to those obtained with the commonly used reverse transcription-polymerase chain reaction.

Unique morphological changes in plant pathogenic phytoplasma-infected petunia flowers are related to transcriptional regulation of floral homeotic genes in an organ-specific manner.

Abnormal flowers are often induced by infection of certain plant pathogens, e.g. phytoplasma, but the molecular mechanisms underlying these malformations have remained poorly understood. Here, we show that infection with OY-W phytoplasma (Candidatus Phytoplasma asteris, onion yellows phytoplasma strain, line OY-W) affects the expression of the floral homeotic genes of petunia plants in an organ-specific manner. Upon infection with OY-W phytoplasma, floral morphological changes, including conversion to leaf-like structures, were observed in sepals, petals and pistils, but not in stamens. As the expression levels of homeotic genes differ greatly between floral organs, we examined the expression levels of homeotic genes in each floral organ infected by OY-W phytoplasma, compared with healthy plants. The expression levels of several homeotic genes required for organ development, such as PFG, PhGLO1 and FBP7, were significantly downregulated by the phytoplasma infection in floral organs, except the stamens, suggesting that the unique morphological changes caused by the phytoplasma infection might result from the significant decrease in expression of some crucial homeotic genes. Moreover, the expression levels of TER, ALF and DOT genes, which are known to participate in floral meristem identity, were significantly downregulated in the phytoplasma-infected petunia meristems, implying that phytoplasma would affect an upstream signaling pathway of floral meristem identity. Our results suggest that phytoplasma infection may have complex effects on floral development, resulting in the unique phenotypes that were clearly distinct from the mutant flower phenotypes produced by the knock-out or the overexpression of certain homeotic genes.

A dual strategy for the suppression of host antiviral silencing: two distinct suppressors for viral replication and viral movement encoded by potato virus M.

Viruses encode RNA silencing suppressors to counteract host antiviral silencing. In this study, we analyzed the suppressors encoded by potato virus M (PVM), a member of the genus Carlavirus. In the conventional green fluorescent protein transient coexpression assay, the cysteine-rich protein (CRP) of PVM inhibited both local and systemic silencing, whereas the triple gene block protein 1 (TGBp1) showed suppressor activity only on systemic silencing. Furthermore, to elucidate the roles of these two suppressors during an active viral infection, we performed PVX vector-based assays and viral movement complementation assays. CRP increased the accumulation of viral RNA at the single-cell level and also enhanced viral cell-to-cell movement by inhibiting RNA silencing. However, TGBp1 facilitated viral movement but did not affect viral accumulation in protoplasts. These data suggest that CRP inhibits RNA silencing primarily at the viral replication step, whereas TGBp1 is a suppressor that acts at the viral movement step. Thus, our findings demonstrate a sophisticated viral infection strategy that suppresses host antiviral silencing at two different steps via two mechanistically distinct suppressors. This study is also the first report of the RNA silencing suppressor in the genus Carlavirus.

A comprehensive list of the Bunyavirales replication promoters reveals a unique promoter structure in Nairoviridae differing from other virus families.

Members of the order Bunyavirales infect a wide variety of host species, including plants, animals and humans, and pose a threat to public health. Major families in this order have tri-segmented negative-sense RNA genomes, the 5' and 3' ends of which form complementary strands that serve as a replication promoter. Elucidation of the mechanisms by which viral polymerases recognize the promoter to initiate RNA synthesis is important for understanding viral replication and pathogenesis, and developing antivirals. A list of replication promoter configuration patterns may provide details on the differences in the replication mechanisms among bunyaviruses. By using public sequence data of all known bunyavirus species, we constructed a comprehensive list of the replication promoters comprising 40 nucleotides in both the 5' and 3' ends of the genome that form a specific complementary strand. Among tri-segmented bunyaviruses, members of the family Nairoviridae, including the highly pathogenic Crimean-Congo hemorrhagic fever virus, have evolved a GC-rich promoter structure differing from that of other families. The unique promoter structure might be related to the large genome size of the family Nairoviridae among bunyaviruses, and the large genome architecture might confer pathogenic advantages. The promoter list provided in this report is useful for predicting the virus family-specific replication mechanisms of bunyaviruses.

A necrosis-inducing elicitor domain encoded by both symptomatic and asymptomatic Plantago asiatica mosaic virus isolates, whose expression is modulated by virus replication.

Systemic necrosis is the most destructive symptom induced by plant pathogens. We previously identified amino acid 1154, in the polymerase domain (POL) of RNA-dependent RNA polymerase (RdRp) of Plantago asiatica mosaic virus (PlAMV), which affects PlAMV-induced systemic necrosis in Nicotiana benthamiana. By point-mutation analysis, we show that amino acid 1,154 alone is not sufficient for induction of necrotic symptoms. However, PlAMV replicons that can express only RdRp, derived from a necrosis-inducing PlAMV isolate, retain their ability to induce necrosis, and transient expression of PlAMV-encoded proteins indicated that the necrosis-eliciting activity resides in RdRp. Moreover, inducible-overexpression analysis demonstrated that the necrosis was induced in an RdRp dose-dependent manner. In addition, during PlAMV infection, necrotic symptoms are associated with high levels of RdRp accumulation. Surprisingly, necrosis-eliciting activity resides in the helicase domain (HEL), not in the amino acid 1,154-containing POL, of RdRp, and this activity was observed even in HELs of PlAMV isolates of which infection does not cause necrosis. Moreover, HEL-induced necrosis had characteristics similar to those induced by PlAMV infection. Overall, our data suggest that necrotic symptoms induced by PlAMV infection depend on the accumulation of a non-isolate specific elicitor HEL (even from nonnecrosis isolates), whose expression is indirectly regulated by amino acid 1,154 that controls replication.

Complete Genome Sequence of a Pepper Yellow Leaf Curl Indonesia Virus Isolated from Tomato in Bali, Indonesia.

We report a complete genome sequence of a pepper yellow leaf curl Indonesia virus (PepYLCIV) isolated in Bali, Indonesia. This virus shares around 90% identity with other PepYLCIV DNA-As and 86% identity with DNA-Bs, suggesting that it is a novel isolate of PepYLCIV.

Development of a LAMP assay with a portable device for real-time detection of begomoviruses under field conditions.

The emergence of begomovirus infection is one of the most important problems affecting production of a variety of vegetable crops worldwide. Infection by begomoviruses has been detected and spread rapidly on Cucurbitaceae and Solanaceae plants in Indonesia. A rapid and simple detection assay for begomoviruses under field conditions for routine sampling of plants is needed. Primers for a loop-mediated isothermal amplification (LAMP) assay were designed based on the sequences of three Indonesian begomoviruses, namely Tomato leaf curl New Delhi virus (ToLCNDV), Pepper yellow leaf curl Indonesia virus (PepYLCIV), and Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), infecting Cucurbitaceae and Solanaceae plants. LAMP assays using a Genelyzer™ III portable fluorometer with a toothpick method successfully detected these begomoviruses in infected melon, pepper, and eggplant samples. LAMP assays conducted during a field survey for detection of the three begomoviruses on 104 fresh leaves indicated that most of the samples were positive; the findings were confirmed by PCR using universal primers of begomovirus as a common detection method. These results demonstrate that this simple and rapid LAMP assay using a fluorometer portable device may be used to achieve real-time detection of begomoviruses under field conditions.

Complete Genome Sequence of Tomato Leaf Curl New Delhi Virus from Luffa in Indonesia.

This is the first report of a begomovirus infecting luffa in Indonesia. The genome of this virus shares a close identity with that of Tomato leaf curl New Delhi virus (ToLCNDV). There is a 36-nucleotide duplicated sequence in the DNA-B component, suggesting the occurrence of an intraviral recombination.

Functional conservation of EXA1 among diverse plant species for the infection by a family of plant viruses.

Since the propagation of plant viruses depends on various host susceptibility factors, deficiency in them can prevent viral infection in cultivated and model plants. Recently, we identified the susceptibility factor Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana, and revealed that EXA1-mediated resistance was effective against three potexviruses. Although EXA1 homolog genes are found in tomato and rice, little is known about which viruses depend on EXA1 for their infection capability and whether the function of EXA1 homologs in viral infection is conserved across multiple plant species, including crops. To address these questions, we generated knockdown mutants using virus-induced gene silencing in two Solanaceae species, Nicotiana benthamiana and tomato. In N. benthamiana, silencing of an EXA1 homolog significantly compromised the accumulation of potexviruses and a lolavirus, a close relative of potexviruses, whereas transient expression of EXA1 homologs from tomato and rice complemented viral infection. EXA1 dependency for potexviral infection was also conserved in tomato. These results indicate that EXA1 is necessary for effective accumulation of potexviruses and a lolavirus, and that the function of EXA1 in viral infection is conserved among diverse plant species.