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.

A 1-aminocyclopropane-1-carboxylic-acid (ACC) dipeptide elicits ethylene responses through ACC-oxidase mediated substrate promiscuity.

Plants produce the volatile hormone ethylene to regulate many developmental processes and to deal with (a)biotic stressors. In seed plants, ethylene is synthesized from 1-aminocyclopropane-1-carboxylic acid (ACC) by the dedicated enzyme ACC oxidase (ACO). Ethylene biosynthesis is tightly regulated at the level of ACC through ACC synthesis, conjugation and transport. ACC is a non-proteinogenic amino acid, which also has signaling roles independent from ethylene. In this work, we investigated the biological function of an uncharacterized ACC dipeptide. The custom-synthesized di-ACC molecule can be taken up by Arabidopsis in a similar way as ACC, in part via Lysine Histidine Transporters (e.g., LHT1). Using Nano-Particle Assisted Laser Desoprtion/Ionization (Nano-PALDI) mass-spectrometry imaging, we revealed that externally fed di-ACC predominantly localizes to the vasculature tissue, despite it not being detectable in control hypocotyl segments. Once taken up, the ACC dimer can evoke a triple response phenotype in dark-grown seedlings, reminiscent of ethylene responses induced by ACC itself, albeit less efficiently compared to ACC. Di-ACC does not act via ACC-signaling, but operates via the known ethylene signaling pathway. In vitro ACO activity and molecular docking showed that di-ACC can be used as an alternative substrate by ACO to form ethylene. The promiscuous nature of ACO for the ACC dimer also explains the higher ethylene production rates observed in planta, although this reaction occurred less efficiently compared to ACC. Overall, the ACC dipeptide seems to be transported and converted into ethylene in a similar way as ACC, and is able to augment ethylene production levels and induce subsequent ethylene responses in Arabidopsis.

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.

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.

Painful myokymia after surgery in a patient with Isaacs' syndrome: a case report.

BACKGROUND: Isaacs' syndrome is a peripheral nerve hyperexcitability syndrome and rare acquired channel disease. The symptoms (myokymia, neuromyotonia, and muscle spasm) emerge even during sleep. This report describes the anesthetic management, especially neuromuscular blocking drugs and postoperative pain, in a case of Isaacs' syndrome. CASE PRESENTATION: A 63-year-old woman with Isaacs' syndrome underwent elective laparoscopic distal gastrectomy under general anesthesia without epidural anesthesia. She received double filtration plasmapheresis four times to alleviate symptoms before surgery. To avoid a prolonged neuromuscular blockade, we performed total intravenous anesthesia and titrated muscle relaxant with neuromuscular monitoring. Anesthetic management was performed without any problems. However, pain management after surgery proved difficult as she experienced severe pain due to myokymia. CONCLUSIONS: Despite attempts to minimize symptoms, severe postoperative pain associated with myokymia occurred. Continuous regional anesthesia should be considered to treat pain from abnormal discharge in the central nervous system in Isaacs' syndrome.

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.

Complete Genome Sequence of a Lily virus X Isolate from Japan.

The complete genome sequence of Lily virus X (LVX), which infects lilies, was determined for the first time from lilies in Japan. As with previous reports, the genome of the Japanese LVX isolate lacked an AUG start codon for the triple gene block protein 3-like region.

Genome-Wide Analysis of the Transcription Start Sites and Promoter Motifs of Phytoplasmas.

Phytoplasmas are obligate intracellular parasitic bacteria that infect both plants and insects. We previously identified the sigma factor RpoD-dependent consensus promoter sequence of phytoplasma. However, the genome-wide landscape of RNA transcripts, including non-coding RNAs (ncRNAs) and RpoD-independent promoter elements, was still unknown. In this study, we performed an improved RNA sequencing analysis for genome-wide identification of the transcription start sites (TSSs) and the consensus promoter sequences. We constructed cDNA libraries using a random adenine/thymine hexamer primer, in addition to a conventional random hexamer primer, for efficient sequencing of 5'-termini of AT-rich phytoplasma RNAs. We identified 231 TSSs, which were classified into four categories: mRNA TSSs, internal sense TSSs, antisense TSSs (asTSSs), and orphan TSSs (oTSSs). The presence of asTSSs and oTSSs indicated the genome-wide transcription of ncRNAs, which might act as regulatory ncRNAs in phytoplasmas. This is the first description of genome-wide phytoplasma ncRNAs. Using a de novo motif discovery program, we identified two consensus motif sequences located upstream of the TSSs. While one was almost identical to the RpoD-dependent consensus promoter sequence, the other was an unidentified novel motif, which might be recognized by another transcription initiation factor. These findings are valuable for understanding the regulatory mechanism of phytoplasma gene expression.

Previously Undiagnosed Spinal and Bulbar Muscular Atrophy as a Cause of Airway Obstruction after Robot-Assisted Laparoscopic Prostatectomy.

BACKGROUND: Preoperative vocal cord paralysis is a risk factor for postoperative respiratory distress following extubation after general anesthesia. We present an unusual case where a geriatric patient developed airway obstruction after robot-assisted laparoscopic prostatectomy. CASE PRESENTATION: A 67-year-old male, who had suffered from left vocal cord paralysis of unknown etiology, was scheduled for robot-assisted laparoscopic prostatectomy (RALP). General anesthesia was performed without any problems. The patient, however, developed airway obstruction one hour after extubation and was reintubated following commencement of mechanical ventilation for one day. At the age of 70 years, the patient received an emergency tracheostomy due to bilateral vocal cord paralysis and then was diagnosed with spinal and bulbar muscular atrophy (SBMA). Although no muscle weakness of either upper or lower extremities was observed, rocuronium showed hypersensitivity during total laryngectomy under general anesthesia. CONCLUSIONS: Vocal cord paralysis combined with postoperative laryngeal edema, the cause of which was presumed to be SBMA, likely caused airway obstruction after RALP. As neuromuscular symptoms progress gradually in patients with SBMA, muscle relaxants should be used carefully, even if patients with SBMA present no immobility of their extremities.

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.

Evaluation of In Vivo Volumetric Dosimetry for Prostate Cancer Using Electronic Portal Imaging Device.

PURPOSE: Volumetric modulated arc therapy (VMAT) is capable of acquiring projection images using electronic portal imaging device (EPID). Commercial EPID-based dosimetry software, dosimetry check (DC), allows in vivo dosimetry using projection images. The purpose of this study was to evaluate in vivo dosimetry for prostate cancer using VMAT. METHOD: VMAT plans were generated for eight patients with prostate cancer using treatment planning system (TPS), and patient quality assurances (QAs) were carried out with phantom. We analyzed five plans as phantom study and five plans as patient study. Projection images were acquired during VMAT delivery. DC converted acquired images into fluence images and used a pencil beam algorithm to calculate dose distributions delivered on the CT images of the phantom and the patients. We evaluated isocenter point doses and gamma analysis in both studies and dose indexes of planning target volume (PTV), bladder and rectum in patient study. RESULTS AND DISCUSSION: Dose differences at the isocenter were less than a criterion in both studies. Pass rates of the gamma analysis were less than a criterion by two plans in the phantom study. Dose indexes of reconstructed distribution were lower than original plans and standard deviations of PTV in reconstructed distribution were larger than original plans. The errors were caused by some issues, such as the commissioning of DC, variations in patient anatomy, and patient positioning. CONCLUSION: The method was feasible to non-invasively perform in vivo dose evaluation for prostate cancer using VMAT.

First Complete Genome Sequence of Cherry virus A.

The 5'-terminal genomic sequence of Cherry virus A (CVA) has long been unknown. We determined the first complete genome sequence of an apricot isolate of CVA (7,434 nucleotides [nt]). The 5'-untranslated region was 107 nt in length, which was 53 nt longer than those of known CVA sequences.

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.

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.

[Winston-lutz test and acquisition of flexmap using rotational irradiation].

PURPOSE: IGRT (image guided radiation therapy) is a useful technique for implementing precisely targeted radiation therapy. Quality assurance and quality control (QA/QC) medical linear accelerators with a portal imaging system (electronic portal imaging device: EPID) are the key to ensuring safe IGRT. The Winston-Lutz test (WLT) provides an evaluation of the MV isocenter, which is the intersection of radiation, collimator, and couch isocenters. A flexmap can indicate a displacement of EPID from the beam center axis as a function of gantry angles which can be removed from the images. The purpose of this study was to establish a novel method for simultaneously carrying out WLT and acquiring a flexmap using rotational irradiation. We also observed long-term changes in flexmaps over a period of five months. METHOD: We employed rotational irradiation with a rectangular field (30×30 mm). First, the displacement of EPID from the beam center axis, indicated by the ball bearing (BB) center, was evaluated using an in-house program. The location of the BB center was then modified according to WLT. Second, a second irradiation was used to acquire a flexmap. We performed this examination regularly and evaluated long-term changes in the flexmap. RESULTS AND DISCUSSION: It proved feasible to perform WLT and flexmap measurements using our proposed methods. The precision of WLT using rotational irradiation was 0.1 mm. In flexmap analysis, the maximum displacement from the mean value for each angle was 0.4 mm over five months. CONCLUSION: We have successfully established a novel method of simultaneously carrying out WLT and flexmap acquisition using rotational irradiation. Maximum displacement from the mean in each angle was 0.4 mm over five months.

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.

Construction of an infectious cDNA clone of radish mosaic virus, a crucifer-infecting comovirus.

Radish mosaic virus (RaMV) is a crucifer-infecting comovirus that has been detected worldwide. Here, we report the successful construction of a full-length infectious cDNA clone of RaMV. The full-length cDNA clones corresponding to RNA1 and RNA2 of a Japanese isolate of RaMV were cloned into the pBlueScript plasmid or the binary vector pCAMBIA1301 downstream of the cauliflower mosaic virus 35S promoter. Mechanical inoculation or agroinoculation of Nicotiana benthamiana with these vectors resulted in systemic RaMV infections causing symptoms similar to those caused by the wild-type parental virus. The presence of progeny virus was verified by western blot analysis and electron microscopy.

Dose verification of volumetric modulated arc therapy (VMAT) by use of in-treatment linac parameters.

Linac parameters such as the multi-leaf collimator (MLC) position and jaw position, cumulative monitor units (MUs), and the corresponding gantry angle were recorded during the clinical delivery of volumetric modulated arc therapy for prostate, lung, and head/neck cancer patients. Then, linac parameters were converted into the beam-data format used in the treatment planning system, and the dose distribution was reconstructed. The dose-volume histogram and the dose difference (DD) were compared with the corresponding values in the treatment plan. A reproducible error of in-treatment linac parameters was observed when a sudden change of beam intensity or MLC/jaw speed occurred. The maximum cumulative MU error was more than 4 MU for lung cancer cases, and the maximum MLC position exceeded 5 mm for prostate and head/neck cancer patients. However, these errors were quickly compensated for at the next control point. All treatments analyzed in the present study were delivered within 0.4% accuracy at the planning target volume. The cumulative dose agreed with that of the plan within 3% of the prescribed dose. The 1% DD was 93.9, 99.9, and 93.4% of the prescription dose for prostate, lung, and head/neck cancer patients, respectively.

Fig mosaic emaravirus p4 protein is involved in cell-to-cell movement.

Fig mosaic virus (FMV), a member of the newly formed genus Emaravirus, is a segmented negative-strand RNA virus. Each of the six genomic FMV segments contains a single ORF: that of RNA4 encodes the protein p4. FMV-p4 is presumed to be the movement protein (MP) of the virus; however, direct experimental evidence for this is lacking. We assessed the intercellular distribution of FMV-p4 in plant cells by confocal laser scanning microscopy and we found that FMV-p4 was localized to plasmodesmata and to the plasma membrane accompanied by tubule-like structures. A series of experiments designed to examine the movement functions revealed that FMV-p4 has the capacity to complement viral cell-to-cell movement, prompt GFP diffusion between cells, and spread by itself to neighbouring cells. Altogether, our findings demonstrated that FMV-p4 shares several properties with other viral MPs and plays an important role in cell-to-cell movement.

Identification of three MAPKKKs forming a linear signaling pathway leading to programmed cell death in Nicotiana benthamiana.

BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily ancient mechanism of signal transduction found in eukaryotic cells. In plants, MAPK cascades are associated with responses to various abiotic and biotic stresses such as plant pathogens. MAPK cascades function through sequential phosphorylation: MAPK kinase kinases (MAPKKKs) phosphorylate MAPK kinases (MAPKKs), and phosphorylated MAPKKs phosphorylate MAPKs. Of these three types of kinase, the MAPKKKs exhibit the most divergence in the plant genome. Their great diversity is assumed to allow MAPKKKs to regulate many specific signaling pathways in plants despite the relatively limited number of MAPKKs and MAPKs. Although some plant MAPKKKs, including the MAPKKKα of Nicotiana benthamiana (NbMAPKKKα), are known to play crucial roles in plant defense responses, the functional relationship among MAPKKK genes is poorly understood. Here, we performed a comparative functional analysis of MAPKKKs to investigate the signaling pathway leading to the defense response. RESULTS: We cloned three novel MAPKKK genes from N. benthamiana: NbMAPKKKß, NbMAPKKKγ, and NbMAPKKKε2. Transient overexpression of full-length NbMAPKKKß or NbMAPKKKγ or their kinase domains in N. benthamiana leaves induced hypersensitive response (HR)-like cell death associated with hydrogen peroxide production. This activity was dependent on the kinase activity of the overexpressed MAPKKK. In addition, virus-induced silencing of NbMAPKKKß or NbMAPKKKγ expression significantly suppressed the induction of programmed cell death (PCD) by viral infection. Furthermore, in epistasis analysis of the functional relationships among NbMAPKKKß, NbMAPKKKγ, and NbMAPKKKα (previously shown to be involved in plant defense responses) conducted by combining transient overexpression analysis and virus-induced gene silencing, silencing of NbMAPKKKα suppressed cell death induced by the overexpression of the NbMAPKKKß kinase domain or of NbMAPKKKγ, but silencing of NbMAPKKKß failed to suppress cell death induced by the overexpression of NbMAPKKKα or NbMAPKKKγ. Silencing of NbMAPKKKγ suppressed cell death induced by the NbMAPKKKß kinase domain but not that induced by NbMAPKKKα. CONCLUSIONS: These results demonstrate that in addition to NbMAPKKKα, NbMAPKKKß and NbMAPKKKγ also function as positive regulators of PCD. Furthermore, these three MAPKKKs form a linear signaling pathway leading to PCD; this pathway proceeds from NbMAPKKKß to NbMAPKKKγ to NbMAPKKKα.