A single amino acid at position 31 in the N-terminus of the coat protein of cucumber mosaic virus determines its avirulence function for RCY1-conferred virus resistance.

The coat protein (CP) of the cucumber mosaic virus (CMV) yellow strain [CMV(Y)], but not the CMV B2 strain [CMV(B2)], serves as an avirulence determinant against the NB-LRR class RCY1 of Arabidopsis thaliana. To investigate the avirulence function, a series of binary vectors were constructed by partially exchanging the CP coding sequence between CMV(Y) and CMV(B2) or introducing nucleotide substitutions. These vectors were transiently expressed in Nicotiana benthamiana leaves transformed with modified RCY1 cDNA. Analysis of hypersensitive resistance-cell death (HCD), CP accumulation, and defense gene expression at leaf sites infiltrated with Agrobacterium indicated that a single amino acid at position 31 of the CP seems to determine the avirulence function.

Single-cell mutation rate of turnip crinkle virus (-)-strand replication intermediates.

Viruses with single-stranded, positive-sense (+) RNA genomes incur high numbers of errors during replication, thereby creating diversified genome populations from which new, better adapted viral variants can emerge. However, a definitive error rate is known for a relatively few (+) RNA plant viruses, due to challenges to account for perturbations caused by natural selection and/or experimental set-ups. To address these challenges, we developed a new approach that exclusively profiled errors in the (-)-strand replication intermediates of turnip crinkle virus (TCV), in singly infected cells. A series of controls and safeguards were devised to ensure errors inherent to the experimental process were accounted for. This approach permitted the estimation of a TCV error rate of 8.47 X 10-5 substitution per nucleotide site per cell infection. Importantly, the characteristic error distribution pattern among the 50 copies of 2,363-base-pair cDNA fragments predicted that nearly all TCV (-) strands were products of one replication cycle per cell. Furthermore, some of the errors probably elevated error frequencies by lowering the fidelity of TCV RNA-dependent RNA polymerase, and/or permitting occasional re-replication of progeny genomes. In summary, by profiling errors in TCV (-)-strand intermediates incurred during replication in single cells, this study provided strong support for a stamping machine mode of replication employed by a (+) RNA virus.

Intracellular bottlenecking permits no more than three tomato yellow leaf curl virus genomes to initiate replication in a single cell.

Viruses are constantly subject to natural selection to enrich beneficial mutations and weed out deleterious ones. However, it remains unresolved as to how the phenotypic gains or losses brought about by these mutations cause the viral genomes carrying the very mutations to become more or less numerous. Previous investigations by us and others suggest that viruses with plus strand (+) RNA genomes may compel such selection by bottlenecking the replicating genome copies in each cell to low single digits. Nevertheless, it is unclear if similarly stringent reproductive bottlenecks also occur in cells invaded by DNA viruses. Here we investigated whether tomato yellow leaf curl virus (TYLCV), a small virus with a single-stranded DNA genome, underwent population bottlenecking in cells of its host plants. We engineered a TYLCV genome to produce two replicons that express green fluorescent protein and mCherry, respectively, in a replication-dependent manner. We found that among the cells entered by both replicons, less than 65% replicated both, whereas at least 35% replicated either of them alone. Further probability computation concluded that replication in an average cell was unlikely to have been initiated with more than three replicon genome copies. Furthermore, sequential inoculations unveiled strong mutual exclusions of these two replicons at the intracellular level. In conclusion, the intracellular population of the small DNA virus TYLCV is actively bottlenecked, and such bottlenecking may be a virus-encoded, evolutionarily conserved trait that assures timely selection of new mutations emerging through error-prone replication.

Activation of plant immunity by exposure to dinitrogen pentoxide gas generated from air using plasma technology.

Reactive nitrogen species (RNS) play an important role in plant immunity as signaling factors. We previously developed a plasma technology to partially convert air molecules into dinitrogen pentoxide (N2O5), an RNS whose physiological action is poorly understood. To reveal the function of N2O5 gas in plant immunity, Arabidopsis thaliana was exposed to plasma-generated N2O5 gas once (20 s) per day for 3 days, and inoculated with Botrytis cinerea, Pseudomonas syringae pv. tomato DC3000 (Pst), or cucumber mosaic virus strain yellow (CMV(Y)) at 24 h after the final N2O5 gas exposure. Lesion size with B. cinerea infection was significantly (P < 0.05) reduced by exposure to N2O5 gas. Propagation of CMV(Y) was suppressed in plants exposed to N2O5 gas compared with plants exposed to the air control. However, proliferation of Pst in the N2O5-gas-exposed plants was almost the same as in the air control plants. These results suggested that N2O5 gas exposure could control plant disease depending on the type of pathogen. Furthermore, changes in gene expression at 24 h after the final N2O5 gas exposure were analyzed by RNA-Seq. Based on the gene ontology analysis, jasmonic acid and ethylene signaling pathways were activated by exposure of Arabidopsis plants to N2O5 gas. A time course experiment with qRT-PCR revealed that the mRNA expression of the transcription factor genes, WRKY25, WRKY26, WRKY33, and genes for tryptophan metabolic enzymes, CYP71A12, CYP71A13, PEN2, and PAD3, was transiently induced by exposure to N2O5 gas once for 20 s peaking at 1-3 h post-exposure. However, the expression of PDF1.2 was enhanced beginning from 6 h after exposure and its high expression was maintained until 24-48 h later. Thus, enhanced tryptophan metabolism leading to the synthesis of antimicrobial substances such as camalexin and antimicrobial peptides might have contributed to the N2O5-gas-induced disease resistance.

High-Resolution X-ray Phase-Contrast Imaging and Sensory and Rheometer Tests in Cooked Edamame.

Although several reports exist on the use of X-ray analysis in vegetables and fruits to examine internal disorders, cavities, and porosity, information on X-ray analysis of qualities, such as texture, is lacking as well as information on X-ray analysis of legumes. Therefore, this study aimed to perform X-ray analysis with sensory and rheometer tests in cooked vegetable soybean (edamame). Edamame is popular worldwide due to its deliciousness and nutritional value. Vascular structures and cracks around them were clearly visualized using X-ray phase-contrast computed tomography (CT) imaging. In addition, we observed the fine structure of the seed coat, which could be important for seed development, germination, and processing. The density in the edamame beans declined as the boiling time increased, promoting a reduction in hardness described in sensory and rheometer tests. The reduction in density proceeded from the gap between cotyledons, the opposite side of the hypocotyl, and the crack. Collectively, the findings show that the high-resolution X-ray phase-contrast CT imaging conducted in a nondestructive manner may help in effectively evaluating the quality of vegetables and in observing the internal structures related to plant development.

Executive summary of the artificial intelligence in surgery series.

As opportunities for artificial intelligence to augment surgical care expand, the accompanying surge in published literature has generated both substantial enthusiasm and grave concern regarding the safety and efficacy of artificial intelligence in surgery. For surgeons and surgical data scientists, it is increasingly important to understand the state-of-the-art, recognize knowledge and technology gaps, and critically evaluate the deluge of literature accordingly. This article summarizes the experiences and perspectives of a global, multi-disciplinary group of experts who have faced development and implementation challenges, overcome them, and produced incipient evidence thereof. Collectively, evidence suggests that artificial intelligence has the potential to augment surgeons via decision-support, technical skill assessment, and the semi-autonomous performance of tasks ranging from resource allocation to patching foregut defects. Most applications remain in preclinical phases. As technologies and their implementations improve and positive evidence accumulates, surgeons will face professional imperatives to lead the safe, effective clinical implementation of artificial intelligence in surgery. Substantial challenges remain; recent progress in using artificial intelligence to achieve performance advantages in surgery suggests that remaining challenges can and will be overcome.

Plant death caused by inefficient induction of antiviral R-gene-mediated resistance may function as a suicidal population resistance mechanism.

Land plant genomes carry tens to hundreds of Resistance (R) genes to combat pathogens. The induction of antiviral R-gene-mediated resistance often results in a hypersensitive response (HR), which is characterized by virus containment in the initially infected tissues and programmed cell death (PCD) of the infected cells. Alternatively, systemic HR (SHR) is sometimes observed in certain R gene-virus combinations, such that the virus systemically infects the plant and PCD induction follows the spread of infection, resulting in systemic plant death. SHR has been suggested to be the result of inefficient resistance induction; however, no quantitative comparison has been performed to support this hypothesis. In this study, we report that the average number of viral genomes that establish cell infection decreased by 28.7% and 12.7% upon HR induction by wild-type cucumber mosaic virus and SHR induction by a single-amino acid variant, respectively. These results suggest that a small decrease in the level of resistance induction can change an HR to an SHR. Although SHR appears to be a failure of resistance at the individual level, our simulations imply that suicidal individual death in SHR may function as an antiviral mechanism at the population level, by protecting neighboring uninfected kin plants.

Isolation and Characterization of a Novel Jumbo Phage from Leaf Litter Compost and Its Suppressive Effect on Rice Seedling Rot Diseases.

Jumbo phages have DNA genomes larger than 200 kbp in large virions composed of an icosahedral head, tail, and other adsorption structures, and they are known to be abundant biological substances in nature. In this study, phages in leaf litter compost were screened for their potential to suppress rice seedling rot disease caused by the bacterium Burkholderia glumae, and a novel phage was identified in a filtrate-enriched suspension of leaf litter compost. The phage particles consisted of a rigid tailed icosahedral head and contained a DNA genome of 227,105 bp. The phage could lyse five strains of B. glumae and six strains of Burkholderia plantarii. The phage was named jumbo Burkholderia phage FLC6. Proteomic tree analysis revealed that phage FLC6 belongs to the same clade as two jumbo Ralstonia phages, namely RSF1 and RSL2, which are members of the genus Chiangmaivirus (family: Myoviridae; order: Caudovirales). Interestingly, FLC6 could also lyse two strains of Ralstonia pseudosolanacearum, the causal agent of bacterial wilt, suggesting that FLC6 has a broad host range that may make it especially advantageous as a bio-control agent for several bacterial diseases in economically important crops. The novel jumbo phage FLC6 may enable leaf litter compost to suppress several bacterial diseases and may itself be useful for controlling plant diseases in crop cultivation.

Cucumber Mosaic Virus Infection in Arabidopsis: A Conditional Mutualistic Symbiont?

A cucumber mosaic virus isolate, named Ho [CMV(Ho)], was isolated from a symptomless Arabidopsis halleri field sample containing low virus titers. An analysis of CMV(Ho) RNA molecules indicated that the virus isolate, besides the usual cucumovirus tripartite RNA genome, additionally contained defective RNA3 molecules and a satellite RNA. To study the underlying mechanism of the persistent CMV(Ho) infection in perennial A. halleri, infectious cDNA clones were generated for all its genetic elements. CMV, which consists of synthetic transcripts from the infectious tripartite RNA genomes, and designated CMV(Ho)tr, multiplied in A. halleri and annual Arabidopsis thaliana Col-0 to a similar level as the virulent strain CMV(Y), but did not induce any symptoms in them. The response of Col-0 to a series of reassortant CMVs between CMV(Ho)tr and CMV(Y) suggested that the establishment of an asymptomatic phenotype of CMV(Ho) infection was due to the 2b gene of CMV RNA2, but not due to the presence of the defective RNA3 and satellite RNA. The accumulation of CMV(Ho) 2b protein tagged with the FLAG epitope (2b.Ho-FLAG) in 2b.Ho-FLAG-transformed Col-0 did not induce any symptoms, suggesting a 2b-dependent persistency of CMV(Ho)tr infection in Arabidopsis. The 2b protein interacted with Argonaute 4, which is known to regulate the cytosine methylation levels of host genomic DNA. Whole genomic bisulfite sequencing analysis of CMV(Ho)tr- and mock-inoculated Col-0 revealed that cytosine hypomethylation in the promoter regions of 82 genes, including two genes encoding transcriptional regulators (DOF1.7 and CBP1), was induced in response to CMV(Ho)tr infection. Moreover, the increased levels of hypomethylation in the promoter region of both genes, during CMV(Ho)tr infection, were correlated with the up- or down-regulation of their expression. Taken altogether, the results indicate that during persistent CMV(Ho) infection in Arabidopsis, host gene expression may be epigenetically modulated resulting from a 2b-mediated cytosine hypomethylation of host genomic DNA.

Priming for enhanced ARGONAUTE2 activation accompanies induced resistance to cucumber mosaic virus in Arabidopsis thaliana.

Systemic acquired resistance (SAR) is a broad-spectrum disease resistance response that can be induced upon infection from pathogens or by chemical treatment, such as with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH). SAR involves priming for more robust activation of defence genes upon pathogen attack. Whether priming for SAR would involve components of RNA silencing remained unknown. Here, we show that upon leaf infiltration of water, BTH-primed Arabidopsis thaliana plants accumulate higher amounts of mRNA of ARGONAUTE (AGO)2 and AGO3, key components of RNA silencing. The enhanced AGO2 expression is associated with prior-to-activation trimethylation of lysine 4 in histone H3 and acetylation of histone H3 in the AGO2 promoter and with induced resistance to the yellow strain of cucumber mosaic virus (CMV[Y]). The results suggest that priming A. thaliana for enhanced defence involves modification of histones in the AGO2 promoter that condition AGO2 for enhanced activation, associated with resistance to CMV(Y). Consistently, the fold-reduction in CMV(Y) coat protein accumulation by BTH pretreatment was lower in ago2 than in wild type, pointing to reduced capacity of ago2 to activate BTH-induced CMV(Y) resistance. A role of AGO2 in pathogen-induced SAR is suggested by the enhanced activation of AGO2 after infiltrating systemic leaves of plants expressing a localized hypersensitive response upon CMV(Y) infection. In addition, local inoculation of SAR-inducing Pseudomonas syringae pv. maculicola causes systemic priming for enhanced AGO2 expression. Together our results indicate that defence priming targets the AGO2 component of RNA silencing whose enhanced expression is likely to contribute to SAR.

Complete genomic sequence of a novel phytopathogenic Burkholderia phage isolated from fallen leaf compost.

In contrast to most Burkholderia species, which affect humans or animals, Burkholderia glumae is a bacterial pathogen of plants that causes panicle blight disease in rice seedlings, resulting in serious damage to rice cultivation. Attempts to combat this disease would benefit from research involving a phage known to attack this type of bacterium. Some Burkholderia phages have been isolated from soil or bacterial species in the order Burkholderiales, but so far there has been no report of a complete genome nucleotide sequence of a phage of B. glumae. In this study, a novel phage, FLC5, of the phytopathogen B. glumae was isolated from leaf compost, and its complete genome nucleotide sequence was determined. The genome consists of a 32,090-bp circular DNA element and exhibits a phylogenetic relationship to members of the genus Peduovirus, with closest similarity to B. multivorans phage KS14. In addition to B. glumae, FLC5 was also able to lyse B. plantarii, a pathogen causing rice bacterial damping-off disease. This is the first report of isolation of a P2-like phage from phytopathogenic Burkholderia, determination of its complete genomic sequence, and the finding of its potential to infect two Burkholderia species: B. glumae and B. plantarii.

Milestones for autonomous in vivo microrobots in medical applications.

In light of recent developments within both health care and robotics, the use of robots within the human body has become attainable. Here we discuss the milestones for the realization of autonomous microrobots in medical applications. The desired tasks were classified by identifying the difficulties and requirements faced by the robot. In addition, we classified the levels of autonomy seen in microrobots for these uses. The aim of this article is to provide readers with a good understanding of the current state and future possibilities in this field.

Bottleneck, Isolate, Amplify, Select (BIAS) as a mechanistic framework for intracellular population dynamics of positive-sense RNA viruses.

Many positive-sense RNA viruses, especially those infecting plants, are known to experience stringent, stochastic population bottlenecks inside the cells they invade, but exactly how and why these populations become bottlenecked are unclear. A model proposed ten years ago advocates that such bottlenecks are evolutionarily favored because they cause the isolation of individual viral variants in separate cells. Such isolation in turn allows the viral variants to manifest the phenotypic differences they encode. Recently published observations lend mechanistic support to this model and prompt us to refine the model with novel molecular details. The refined model, designated Bottleneck, Isolate, Amplify, Select (BIAS), postulates that these viruses impose population bottlenecks on themselves by encoding bottleneck-enforcing proteins (BNEPs) that function in a concentration-dependent manner. In cells simultaneously invaded by numerous virions of the same virus, BNEPs reach the bottleneck-ready concentration sufficiently early to arrest nearly all internalized viral genomes. As a result, very few (as few as one) viral genomes stochastically escape to initiate reproduction. Repetition of this process in successively infected cells isolates viral genomes with different mutations in separate cells. This isolation prevents mutant viruses encoding defective viral proteins from hitchhiking on sister genome-encoded products, leading to the swift purging of such mutants. Importantly, genome isolation also ensures viral genomes harboring beneficial mutations accrue the cognate benefit exclusively to themselves, leading to the fixation of such beneficial mutations. Further interrogation of the BIAS hypothesis promises to deepen our understanding of virus evolution and inspire new solutions to virus disease mitigation.

Establishment of Neurospora crassa as a model organism for fungal virology.

The filamentous fungus Neurospora crassa is used as a model organism for genetics, developmental biology and molecular biology. Remarkably, it is not known to host or to be susceptible to infection with any viruses. Here, we identify diverse RNA viruses in N. crassa and other Neurospora species, and show that N. crassa supports the replication of these viruses as well as some viruses from other fungi. Several encapsidated double-stranded RNA viruses and capsid-less positive-sense single-stranded RNA viruses can be experimentally introduced into N. crassa protoplasts or spheroplasts. This allowed us to examine viral replication and RNAi-mediated antiviral responses in this organism. We show that viral infection upregulates the transcription of RNAi components, and that Dicer proteins (DCL-1, DCL-2) and an Argonaute (QDE-2) participate in suppression of viral replication. Our study thus establishes N. crassa as a model system for the study of host-virus interactions.

Single Amino Acid Substitutions in the Cucumber Mosaic Virus 1a Protein Induce Necrotic Cell Death in Virus-Inoculated Leaves without Affecting Virus Multiplication.

When Arabidopsis thaliana ecotype Col-0 was inoculated with a series of reassortant viruses created by exchanging viral genomic RNAs between two strains of cucumber mosaic virus (CMV), CMV(Y), and CMV(H), cell death developed in the leaves inoculated with reassortant CMV carrying CMV(H) RNA1 encoding 1a protein, but not in noninoculated upper leaves. In general, cell death in virus-infected plants is a critical event for virus survival because virus multiplication is completely dependent on host cell metabolism. However, interestingly, this observed cell death did not affect either virus multiplication in the inoculated leaves or systemic spread to noninoculated upper leaves. Furthermore, the global gene expression pattern of the reassortant CMV-inoculated leaves undergoing cell death was clearly different from that in hypersensitive response (HR) cell death, which is coupled with resistance to CMV. These results indicated that the observed cell death does not appear to be HR cell death but rather necrotic cell death unrelated to CMV resistance. Interestingly, induction of this necrotic cell death depended on single amino acid substitutions in the N-terminal region surrounding the methyltransferase domain of the 1a protein. Thus, development of necrotic cell death might not be induced by non-specific damage as a result of virus multiplication, but by a virus protein-associated mechanism. The finding of CMV 1a protein-mediated induction of necrotic cell death in A. thaliana, which is not associated with virus resistance and HR cell death, has the potential to provide a new pathosystem to study the role of cell death in virus-host plant interactions.

Catalytic self-folding of 2D structures through cascading magnet reactions.

While thousands of proteins involved in development of the human body are capable of self-assembling in a distributed manner from merely 20 types of amino acid, macroscopic products that can be assembled spontaneously from 'alive' components remains an aspiration in engineering. To attain such a mechanism, a major challenge lies in understanding which attributes from the bio-molecular realm must be leveraged at the macro-scale. Inspired by protein folding, we present a centimetre-size 1D tile chain whose self-folding processes are directed by structure-embedded magnetic interactions, which can theoretically self-assemble into convex 2D structures of any size or shape without the aid of a global 'controller'. Each tile holds two magnets contained in paths designed to control their interactions. Once initiated by a magnetic unit (termed Catalyst), the chain self-reconfigures by consuming magnetic potential energy stored between magnet pairs, until the final 2D structure is reached at an energetic minimum. Both simulation and experimental results are presented to illustrate the method's efficacy on chains of arbitrary length. Results demonstrate the promise of a physically implemented, bottom-up, and scalable self-assembly method for novel 2D structure manufacturing, bridging the bio-molecular and mechanical realms.

Atmospheric-pressure plasma irradiation can disrupt tobacco mosaic virus particles and RNAs to inactivate their infectivity.

Low-temperature atmospheric-pressure air plasma is a source of charged and neutral gas species. In this study, N-carrying tobacco plants were inoculated with plasma irradiated and non-irradiated tobacco mosaic virus (TMV) solution, resulting in necrotic local lesions on non-irradiated, but not on irradiated, TMV-inoculated leaves. Virus particles were disrupted by plasma irradiation in an exposure-dependent manner, but the viral coat protein subunit was not. TMV RNA was also fragmented in a time-dependent manner. These results indicate that plasma irradiation of TMV can collapse viral particles to the subunit level, degrading TMV RNA and thereby leading to a loss of infectivity.

Robotic metamorphosis by origami exoskeletons.

Changing the inherent physical capabilities of robots by metamorphosis has been a long-standing goal of engineers. However, this task is challenging because of physical constraints in the robot body, each component of which has a defined functionality. To date, self-reconfiguring robots have limitations in their on-site extensibility because of the large scale of today's unit modules and the complex administration of their coordination, which relies heavily on on-board electronic components. We present an approach to extending and changing the capabilities of a robot by enabling metamorphosis using self-folding origami "exoskeletons." We show how a cubical magnet "robot" can be remotely moved using a controllable magnetic field and hierarchically develop different morphologies by interfacing with different origami exoskeletons. Activated by heat, each exoskeleton is self-folded from a rectangular sheet, extending the capabilities of the initial robot, such as enabling the manipulation of objects or locomotion on the ground, water, or air. Activated by water, the exoskeletons can be removed and are interchangeable. Thus, the system represents an end-to-end (re)cycle. We also present several robot and exoskeleton designs, devices, and experiments with robot metamorphosis using exoskeletons.

Multi-crease Self-folding by Global Heating.

This study demonstrates a new approach to autonomous folding for the body of a 3D robot from a 2D sheet, using heat. We approach this challenge by folding a 0.27-mm sheetlike material into a structure. We utilize the thermal deformation of a contractive sheet sandwiched by rigid structural layers. During this baking process, the heat applied on the entire sheet induces contraction of the contracting layer and thus forms an instructed bend in the sheet. To attain the targeted folding angles, the V-fold spans method is used. The targeted angle θout can be kinematically encoded into crease geometry. The realization of this angle in the folded structure can be approximately controlled by a contraction angle θin. The process is non-reversible, is reliable, and is relatively fast. Our method can be applied simultaneously to all the folds in multi-crease origami structures. We demonstrate the use of this method to create a lightweight mobile robot.

Viruses roll the dice: the stochastic behavior of viral genome molecules accelerates viral adaptation at the cell and tissue levels.

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1-20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence-tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell.