A phytoplasma effector acts as a ubiquitin-like mediator between floral MADS-box proteins and proteasome shuttle proteins.

Plant pathogenic bacteria have developed effectors to manipulate host cell functions to facilitate infection. A certain number of effectors use the conserved ubiquitin-proteasome system in eukaryotic to proteolyze targets. The proteasome utilization mechanism is mainly mediated by ubiquitin interaction with target proteins destined for degradation. Phyllogens are a family of protein effectors produced by pathogenic phytoplasmas that transform flowers into leaves in diverse plants. Here, we present a noncanonical mechanism for phyllogen action that involves the proteasome and is ubiquitin-independent. Phyllogens induce proteasomal degradation of floral MADS-box transcription factors (MTFs) in the presence of RADIATION-SENSITIVE23 (RAD23) shuttle proteins, which recruit ubiquitinated proteins to the proteasome. Intracellular localization analysis revealed that phyllogen induced colocalization of MTF with RAD23. The MTF/phyllogen/RAD23 ternary protein complex was detected not only in planta but also in vitro in the absence of ubiquitin, showing that phyllogen directly mediates interaction between MTF and RAD23. A Lys-less nonubiquitinated phyllogen mutant induced degradation of MTF or a Lys-less mutant of MTF. Furthermore, the method of sequential formation of the MTF/phyllogen/RAD23 protein complex was elucidated, first by MTF/phyllogen interaction and then RAD23 recruitment. Phyllogen recognized both the evolutionarily conserved tetramerization region of MTF and the ubiquitin-associated domain of RAD23. Our findings indicate that phyllogen functionally mimics ubiquitin as a mediator between MTF and RAD23.

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.

Complete genome sequence of a novel polerovirus infecting Cynanchum rostellatum.

We detected a virus-like sequence in Cynanchum rostellatum leaves showing yellow mottle symptoms, found in Tokyo, Japan. RNA-Seq analysis revealed that the complete nucleotide sequence of the virus genome was 5,878 nucleotides in length and that it contained seven open reading frames (ORFs) specific to members of the genus Polerovirus. Accordingly, phylogenetic analysis revealed that the virus clustered with poleroviruses in the family Solemoviridae. The amino acid sequence identity values obtained by comparison of the deduced proteins of this virus and those of known members of the genus Polerovirus were lower than 90%, which is the species demarcation criterion of the taxon. The results indicate that this virus is a novel member of the genus Polerovirus, for which the name "cynanchum yellow mottle-associated virus" is proposed.

Complete genome sequence of a new orthotospovirus associated with ringspot in Fatsia japonica.

Fatsia japonica is an evergreen shrub native to Japan. For decades, virus-like ringspot symptoms have been observed on leaves of F. japonica in Japan; however, previous attempts to identify the causal agents have been unsuccessful. In this study, we detected an orthotospovirus-like sequence in symptomatic F. japonica plants using RNA sequencing analysis. The complete nucleotide sequences of the L, M, and S segments of the virus were determined using conventional sequencing strategies. The virus had a typical orthotospovirus genome structure, and the putative nucleocapsid protein showed the highest sequence identity to that of groundnut chlorotic fan-spot virus, with 83.7% identity at the amino acid level (which is below the 90% species demarcation cutoff for the genus Orthotospovirus). Although we could not confirm the pathogenicity of the virus in F. japonica due to difficulties associated with mechanical inoculation, its association with the observed symptoms was suggested by the fact that the virus was detected only in symptomatic leaf areas. Based on these results, we consider this virus, which we have named "Fatsia japonica ringspot-associated virus" (FjRSaV), to be the first representative of a new orthotospovirus species, for which we propose the binomial "Orthotospovirus fatsiae".

Complete genome sequence of pleioblastus mosaic virus, a distinct member of the genus Potyvirus.

Pleioblastus mosaic virus (PleMV) is a tentative member of the genus Potyvirus in the family Potyviridae and was discovered in bamboo with mosaic symptoms in Tokyo, Japan. Since no information on the genome sequence of PleMV has been reported, its taxonomic position has long been uncertain. Here, we report the first complete genome sequences of two distinct PleMV isolates. Excluding the 3'-terminal poly(A) tail, their genomic RNA sequences consist of 9,634 and 9,643 nucleotides (nt); both contain a large open reading frame (ORF) encoding a polyprotein and a small ORF termed PIPO. The large ORFs of the two isolates share 79.2% and 87.6% sequence identity at the nucleotide (nt) and amino acid (aa) level, respectively, and were found to have the highest nt and aa sequence identity (69.0% and 69.9%) to the potyvirus johnsongrass mosaic virus (JGMV). Phylogenetic analysis showed that PleMV is most closely related to JGMV but forms its own clade. These results suggest that PleMV is a distinct member of the genus Potyvirus.

Complete genome sequence of viola mottle virus, revealing its synonymous relationship to tulip virus X.

Viola mottle virus (VMoV) was discovered in Viola odorata showing symptoms of reduced growth, leaf mottling, and whitish stripes on flowers in northern Italy in 1977. This virus has been provisionally classified as a member of the genus Potexvirus based on its morphological, serological, and biological characteristics. However, since genetic information of VMoV has never been reported, the taxonomic status of this virus is unclear. Here, we report the first complete genome sequence of VMoV to clarify its taxonomic position. Its genomic RNA is 6,052 nucleotides long, excluding the 3'-terminal poly(A) tail, and has five open reading frames (ORFs) typical of potexviruses. Among potexviruses, VMoV showed the most similarity to tulip virus X (TVX) with 81.1-81.2% nucleotide and 90.4-90.7% amino acid sequence identity in ORF1 and 82.9-83.5% nucleotide and 93.2-95.2% amino acid sequence identity in ORF5. These values are much higher than the species demarcation threshold for the genus. Phylogenetic analysis also indicated that VMoV is nested within the clade of TVX isolates. These data demonstrate that VMoV and TVX are members of the same species.

The Plant Noncanonical Antiviral Resistance Protein JAX1 Inhibits Potexviral Replication by Targeting the Viral RNA-Dependent RNA Polymerase.

Understanding the innate immune mechanisms of plants is necessary for the breeding of disease-resistant lines. Previously, we identified the antiviral resistance gene JAX1 from Arabidopsis thaliana, which inhibits infection by potexviruses. JAX1 encodes a unique jacalin-type lectin protein. In this study, we analyzed the molecular mechanisms of JAX1-mediated resistance. JAX1 restricted the multiplication of a potexviral replicon lacking movement-associated proteins, suggesting inhibition of viral replication. Therefore, we developed an in vitro potato virus X (PVX) translation/replication system using vacuole- and nucleus-free lysates from tobacco protoplasts, and we revealed that JAX1 inhibits viral RNA synthesis but not the translation of the viral RNA-dependent RNA polymerase (RdRp). JAX1 did not affect the replication of a resistance-breaking mutant of PVX. Blue native polyacrylamide gel electrophoresis of fractions separated by sucrose gradient sedimentation showed that PVX RdRp constituted the high-molecular-weight complex that seems to be crucial for viral replication. JAX1 was detected in this complex of the wild-type PVX replicon but not in that of the resistance-breaking mutant. In addition, JAX1 interacted with the RdRp of the wild-type virus but not with that of a virus with a point mutation at the resistance-breaking residue. These results suggest that JAX1 targets RdRp to inhibit potexviral replication.IMPORTANCE Resistance genes play a crucial role in plant antiviral innate immunity. The roles of conventional nucleotide-binding leucine-rich repeat (NLR) proteins and the associated defense pathways have long been studied. In contrast, recently discovered resistance genes that do not encode NLR proteins (non-NLR resistance genes) have not been investigated extensively. Here we report that the non-NLR resistance factor JAX1, a unique jacalin-type lectin protein, inhibits de novo potexviral RNA synthesis by targeting the huge complex of viral replicase. This is unlike other known antiviral resistance mechanisms. Molecular elucidation of the target in lectin-type protein-mediated antiviral immunity will enhance our understanding of the non-NLR-mediated plant resistance system.

Crystal structure of phyllogen, a phyllody-inducing effector protein of phytoplasma.

Phytoplasmas are plant pathogenic bacteria that often induce unique phyllody symptoms in which the floral organs are transformed into leaf-like structures. Recently, a novel family of bacterial effector genes, called phyllody-inducing genes (phyllogens), was identified as being involved in the induction of phyllody by degrading floral MADS-domain transcription factors (MTFs). However, the structural characteristics of phyllogens are unknown. In this study, we elucidated the crystal structure of PHYL1OY, a phyllogen of 'Candidatus Phytoplasma asteris' onion yellows strain, at a resolution of 2.4 Å. The structure of PHYL1 consisted of two α-helices connected by a random loop in a coiled-coil manner. In both α-helices, the distributions of hydrophobic residues were conserved among phyllogens. Amino acid insertion mutations into either α-helix resulted in the loss of phyllody-inducing activity and the ability of the phyllogen to degrade floral MTF. In contrast, the same insertion in the loop region did not affect either activity, indicating that both conserved α-helices are important for the function of phyllogens. This is the first report on the crystal structure of an effector protein of phytoplasmas.

Comprehensive screening of antimicrobials to control phytoplasma diseases using an in vitro plant-phytoplasma co-culture system.

Phytoplasmas are plant-pathogenic bacteria that infect many important crops and cause serious economic losses worldwide. However, owing to an inability to culture phytoplasmas, screening of antimicrobials on media is difficult. The only antimicrobials being used to control phytoplasmas are tetracycline-class antibiotics. In this study, we developed an accurate and efficient screening method to evaluate the effects of antimicrobials using an in vitro plant-phytoplasma co-culture system. We tested 40 antimicrobials, in addition to tetracycline, and four of these (doxycycline, chloramphenicol, thiamphenicol and rifampicin) decreased the accumulation of 'Candidatus (Ca.) Phytoplasma asteris'. The phytoplasma was eliminated from infected plants by the application of both tetracycline and rifampicin. We also compared nucleotide sequences of rRNAs and amino acid sequences of proteins targeted by antimicrobials between phytoplasmas and other bacteria. Since antimicrobial target sequences were conserved among various phytoplasma species, the antimicrobials that decreased accumulation of 'Ca. P. asteris' may also have been effective against other phytoplasma species. These approaches will provide new strategies for phytoplasma disease management.

'Candidatus Phytoplasma noviguineense', a novel taxon associated with Bogia coconut syndrome and banana wilt disease on the island of New Guinea.

Bogia coconut syndrome (BCS) is one of the lethal yellowing (LY)-type diseases associated with phytoplasma presence that are seriously threatening coconut cultivation worldwide. It has recently emerged, and is rapidly spreading in northern parts of the island of New Guinea. BCS-associated phytoplasmas collected in different regions were compared in terms of 16S rRNA gene sequences, revealing high identity among them represented by strain BCS-BoR. Comparative analysis of the 16S rRNA gene sequences revealed that BCS-BoR shared less than a 97.5 % similarity with other species of 'Candidatus Phytoplasma', with a maximum value of 96.08 % (with strain LY; GenBank accession no. U18747). This result indicates the necessity and propriety of a novel taxon for BCS phytoplasmas according to the recommendations of the IRPCM. Phylogenetic analysis was also conducted on 16S rRNA gene sequences, resulting in a monophyletic cluster composed of BCS-BoR and other LY-associated phytoplasmas. Other phytoplasmas on the island of New Guinea associated with banana wilt and arecanut yellow leaf diseases showed high similarities to BCS-BoR and were closely related to BCS phytoplasmas. Based on the uniqueness of their 16S rRNA gene sequences, a novel taxon 'Ca.Phytoplasma noviguineense' is proposed for these phytoplasmas found on the island of New Guinea, with strain BCS-BoR (GenBank accession no. LC228755) as the reference strain. The novel taxon is described in detail, including information on the symptoms of associated diseases and additional genetic features of the secY gene and rp operon.

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.

Phytoplasma-conserved phyllogen proteins induce phyllody across the Plantae by degrading floral MADS domain proteins.

ABCE-class MADS domain transcription factors (MTFs) are key regulators of floral organ development in angiosperms. Aberrant expression of these genes can result in abnormal floral traits such as phyllody. Phyllogen is a virulence factor conserved in phytoplasmas, plant pathogenic bacteria of the class Mollicutes. It triggers phyllody in Arabidopsis thaliana by inducing degradation of A- and E-class MTFs. However, it is still unknown whether phyllogen can induce phyllody in plants other than A. thaliana, although phytoplasma-associated phyllody symptoms are observed in a broad range of angiosperms. In this study, phyllogen was shown to cause phyllody phenotypes in several eudicot species belonging to three different families. Moreover, phyllogen can interact with MTFs of not only angiosperm species including eudicots and monocots but also gymnosperms and a fern, and induce their degradation. These results suggest that phyllogen induces phyllody in angiosperms and inhibits MTF function in diverse plant species.

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.

Nucleocapsid protein from fig mosaic virus forms cytoplasmic agglomerates that are hauled by endoplasmic reticulum streaming.

UNLABELLED: Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE: Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.

Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector, phyllogen, induces phyllody.

Plant pathogens alter the course of plant developmental processes, resulting in abnormal morphology in infected host plants. Phytoplasmas are unique plant-pathogenic bacteria that transform plant floral organs into leaf-like structures and cause the emergence of secondary flowers. These distinctive symptoms have attracted considerable interest for many years. Here, we revealed the molecular mechanisms of the floral symptoms by focusing on a phytoplasma-secreted protein, PHYL1, which induces morphological changes in flowers that are similar to those seen in phytoplasma-infected plants. PHYL1 is a homolog of the phytoplasmal effector SAP54 that also alters floral development. Using yeast two-hybrid and in planta transient co-expression assays, we found that PHYL1 interacts with and degrades the floral homeotic MADS domain proteins SEPALLATA3 (SEP3), APETALA1 (AP1) and CAULIFLOWER (CAL). This degradation of MADS domain proteins was dependent on the ubiquitin-proteasome pathway. The expression of floral development genes downstream of SEP3 and AP1 was disrupted in 35S::PHYL1 transgenic plants. PHYL1 was genetically and functionally conserved among other phytoplasma strains and species. We designate PHYL1, SAP54 and their homologs as members of the phyllody-inducing gene family of 'phyllogens'.

Cell Death Triggered by a Putative Amphipathic Helix of Radish mosaic virus Helicase Protein Is Tightly Correlated With Host Membrane Modification.

Systemic necrosis is one of the most severe symptoms caused by plant RNA viruses. Recently, systemic necrosis has been suggested to have similar features to a defense response referred to as the hypersensitive response (HR), a form of programmed cell death. In virus-infected plant cells, host intracellular membrane structures are changed dramatically for more efficient viral replication. However, little is known about whether this replication-associated membrane modification is the cause of the symptoms. In this study, we identified an amino-terminal amphipathic helix of the helicase encoded by Radish mosaic virus (RaMV) (genus Comovirus) as an elicitor of cell death in RaMV-infected plants. Cell death caused by the amphipathic helix had features similar to HR, such as SGT1-dependence. Mutational analyses and inhibitor assays using cerulenin demonstrated that the amphipathic helix-induced cell death was tightly correlated with dramatic alterations in endoplasmic reticulum (ER) membrane structures. Furthermore, the cell death-inducing activity of the amphipathic helix was conserved in Cowpea mosaic virus (genus Comovirus) and Tobacco ringspot virus (genus Nepovirus), both of which are classified in the family Secoviridae. Together, these results indicate that ER membrane modification associated with viral intracellular replication may be recognized to prime defense responses against plant viruses.

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.

Efficient foreign gene expression in planta using a plantago asiatica mosaic virus-based vector achieved by the strong RNA-silencing suppressor activity of TGBp1.

Plant virus expression vectors provide a powerful tool for basic research as well as for practical applications. Here, we report the construction of an expression vector based on plantago asiatica mosaic virus (PlAMV), a member of the genus Potexvirus. Modification of a vector to enhance the expression of a foreign gene, combined with the use of the foot-and-mouth disease virus 2A peptide, allowed efficient expression of the foreign gene in two model plant species, Arabidopsis thaliana and Nicotiana benthamiana. Comparison with the widely used potato virus X (PVX) vector demonstrated that the PlAMV vector retains an inserted foreign gene for a longer period than PVX. Moreover, our results showed that the GFP expression construct PlAMV-GFP exhibits stronger RNA silencing suppression activity than PVX-GFP, which is likely to contribute to the stability of the PlAMV vector.

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.

Use of the 23S rRNA gene as a target template in the universal loop-mediated isothermal amplification (LAMP) of genomic DNA from phytoplasmas.

Plant-pathogenic bacteria cause numerous diseases in host plants and can result in serious damage. Timely and accurate diagnostic techniques are, therefore, crucial. While advances in molecular techniques have led to diagnostic systems able to distinguish known plant pathogens at the species or strain level, systems covering larger categories are mostly lacking. In this study, a specific and universal LAMP-based diagnostic system was developed for phytoplasmas, a large group of insect-borne plant-pathogenic bacteria that cause significant agricultural losses worldwide. Targeting the 23S rRNA gene of phytoplasma, the newly designed primer set CaPU23S-4 detected 31 'Candidatus Phytoplasma' tested within 30 min. This primer set also showed high specificity, without false-positive results for other bacteria (including close relatives of phytoplasmas) or healthy plants. The detection sensitivity was ~10,000 times higher than that of PCR methods for phytoplasma detection. A simple, rapid method of DNA extraction, by boiling phytoplasma-infected tissues, was developed as well. When used together with the universal LAMP assay, it enabled the prompt and accurate detection of phytoplasmas from plants and insects. The results demonstrate the potential of the 23S rRNA gene as a versatile target for the LAMP-based universal detection of bacteria at the genus level and provide a novel avenue for exploring this gene as molecular marker for phytoplasma presence detection.IMPORTANCEPhytoplasmas are associated with economically important diseases in crops worldwide, including lethal yellowing of coconut palm, "flavescence dorée" and "bois noir" of grapevine, X-disease in stone fruits, and white leaf and grassy shoot in sugarcane. Numerous LAMP-based diagnostic assays, mostly targeting the 16S rRNA gene, have been reported for phytoplasmas. However, these assays can only detect a limited number of 'Candidatus Phytoplasma' species, whereas the genus includes at least 50 of these species. In this study, a universal, specific, and rapid diagnostic system was developed that can detect all provisionally classified phytoplasmas within 1 h by combining the LAMP technique targeting the 23S rRNA gene with a simple method for DNA extraction. This diagnostic system will facilitate the on-site detection of phytoplasmas and may aid in the discovery of new phytoplasma-associated diseases and putative insect vectors, irrespective of the availability of infrastructure and experimental resources.