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To study viral infection, the direct structural visualization of the viral life cycle consisting of virus attachment, entry, replication, assembly and transport is essential. Although conventional electron microscopy (EM) has been extremely helpful in the investigation of virus-host cell interactions, three-dimensional (3D) EM not only provides important information at the nanometer resolution, but can also create 3D maps of large volumes, even entire virus-infected cells. Here, we determined the ultrastructural details of tomato spotted wilt virus (TSWV)-infected plant cells using focused ion beam scanning EM (FIB-SEM). The viral morphogenesis and dynamic transformation of paired parallel membranes (PPMs) were analyzed. The endoplasmic reticulum (ER) membrane network consisting of tubules and sheets was related to viral intracellular trafficking and virion storage. Abundant lipid-like bodies, clustering mitochondria, cell membrane tubules, and myelin-like bodies were likely associated with viral infection. Additionally, connecting structures between neighboring cells were found only in infected plant tissues and showed the characteristics of tubular structure. These novel connections that formed continuously in the cell wall or were wrapped by the cell membranes of neighboring cells appeared frequently in the large-scale 3D model, suggesting additional strategies for viral trafficking that were difficult to distinguish using conventional EM.

Phytohormone signalling pathways have an important role in defence against pathogens mediated by cell-surface pattern recognition receptors and intracellular nucleotide-binding leucine-rich repeat class immune receptors

In recent years, the emergence of soybean stay-green syndrome (SGS), also referred to as “zhengqing”, in the Huang-Huai-Hai region of China has resulted in significant yield losses, with some areas experiencing a complete reduction in seed yield. SGS is a phenomenon characterized by the delayed senescence of soybean, resulting in stay-green leaves, flat pods, and stunted seed development at harvest. Our group was the first to identify a distinct geminivirus, named soybean stay-green associated geminivirus (SoSGV), as the causative agent of SGS by fulfilling Koch’s postulate. To further understand the epidemiology of SoSGV, in this study, we collected 368 stay-green samples from 17 regions in 8 provinces including the Huang-Huai-Hai region and surrounding areas of China. The results showed that 228 samples tested positive for SoSGV (61.96%), and 96.93% of these positive samples showed severe pod deflation. Our epidemiological assessment reveals SGS caused by the SoSGV is prevalent in the fields, and it is undergoing geographical expansion and genetic differentiation. Additionally, we determined the other natural hosts grown in the Huang-Huai-Hai region of China. By capturing insects in the field and conducting laboratory vector transmission tests, we confirmed that the common brown leafhopper (Orosius orientalis) is the transmitting vector of SoSGV. With a better understanding of the epidemiology of SoSGV and its transmission, we can develop more effective strategies for managing and mitigating its impact on soybean yields.

Orthotospoviruses, the plant-infecting bunyaviruses, cause serious diseases in agronomic crops and pose major threats to global food security. The family of Tospoviridae contains more than 30 members that are classified into two geographic groups, American-type and Euro/Asian-type orthotospovirus. However, the genetic interaction between different species and the possibility, during mixed infections, for transcomplementation of gene functions by orthotospoviruses from different geographic groups remains underexplored. In this study, minireplicon-based reverse genetics (RG) systems have been established for Impatiens necrotic spot virus (INSV) (an American-type orthotospovirus) and for Calla lily chlorotic spot virus and Tomato zonate spot virus (CCSV and TZSV) (two representative Euro/Asian orthotospoviruses). Together with the earlier established RG system for Tomato spotted wilt virus (TSWV), a type species of the Orthotospovirus American-clade, viral replicase/movement proteins were exchanged and analyzed on interspecies transcomplementation. Whereas the homologous RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) protein supported the replication of orthotospoviruses from both geographic groups, heterologous combinations of RdRp from one group and N from the other group were unable to support the replication of viruses from both groups. Furthermore, the NSm movement protein (MP), from both geographic groups of orthotospoviruses, was able to transcomplement heterologous orthotospoviruses or a positive-strand Cucumber mosaic virus (CMV) in their movement, albeit with varying efficiency. MP from Rice stripe tenuivirus (RSV), a plant-infecting bunyavirus that is distinct from orthotospoviruses, or MP from CMV also moves orthotospoviruses. Our findings gain insights into the genetic interaction/reassortant potentials for the segmented plant orthotospoviruses. IMPORTANCE Orthotospoviruses are agriculturally important negative-strand RNA viruses and cause severe yield-losses on many crops worldwide. Whereas the emergence of new animal-infecting bunyaviruses is frequently associated with genetic reassortants, this issue remains underexposed with the plant-infecting orthotospovirus. With the development of reverse genetics systems for orthotospoviruses from different geographic regions, the interspecies/intergroup replication/movement complementation between American- and Euro/Asian-type orthotospoviruses were investigated. Genomic RNAs from American orthotospoviruses can be replicated by the RdRp and N from those of Euro/Asia-group orthotospoviruses, and vice versa. However, their genomic RNAs cannot be replicated by a heterologous combination of RdRp from one geographic group and N from another geographic group. Cell-to-cell movement of viral entity is supported by NSm from both geographic groups, with highest efficiency by NSm from viruses belonging to the same group. Our findings provide important insights into the genetic interaction and exchange ability of viral gene functions between different species of orthotospovirus.

Rice stripe disease caused by Rice stripe virus (RSV) is one of the most devastating plant viruses of rice and causes enormous losses in production. RSV is transmitted from plant to plant by the small brown planthopper ( Laodelphax striatellus) in a circulative–propagative manner. The recent reemergence of this pathogen in East Asia since 2000 has made RSV one of the most studied plant viruses over the past two decades. Extensive studies of RSV have resulted in substantial advances regarding fundamental aspects of the virus infection. Here, we compile and analyze recent information on RSV with a special emphasis on the strategies that RSV has adopted to establish infections. These advances include RSV replication and movement in host plants and the small brown planthopper vector, innate immunity defenses against RSV infection, epidemiology, and recent advances in the management of rice stripe disease. Understanding these issues will facilitate the design of novel antiviral therapies for management and contribute to a more detailed understanding of negative-sense virus–host interactions at the molecular level.

Summary Plants use intracellular nucleotide‐binding leucine‐rich repeat immune receptors (NLRs) to recognize pathogen‐encoded effectors and initiate immune responses. Tomato spotted wilt virus (TSWV), which has been found to infect >1000 plant species, is among the most destructive plant viruses worldwide. The Sw‐5b is the most effective and widely used resistance gene in tomato breeding to control TSWV. However, broad application of tomato cultivars carrying Sw‐5b has resulted in an emergence of resistance‐breaking (RB) TSWV. Therefore, new effective genes are urgently needed to prevent further RB TSWV outbreaks. In this study, we conducted artificial evolution to select Sw‐5b mutants that could extend the resistance spectrum against TSWV RB isolates. Unlike regular NLRs, Sw‐5b detects viral elicitor NSm using both the N‐terminal Solanaceae‐specific domain (SD) and the C‐terminal LRR domain in a two‐step recognition process. Our attempts to select gain‐of‐function mutants by random mutagenesis involving either the SD or the LRR of Sw‐5b failed; therefore, we adopted a stepwise strategy, first introducing a NSmRB‐responsive mutation at the R927 residue in the LRR, followed by random mutagenesis involving the Sw‐5b SD domain. Using this strategy, we obtained Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q mutants, which are effective against TSWV RB carrying the NSmC118Y or NSmT120N mutation, and against other American‐type tospoviruses. Thus, we were able to extend the resistance spectrum of Sw‐5b; the selected Sw‐5b mutants will provide new gene resources to control RB TSWV.

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/Interleukin-1 receptor (TIR) domain detect pathogen effectors to produce TIR-catalyzed signaling molecules for activation of plant immunity. Plant immune signaling by TIR-containing NLR (TNL) proteins converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TNL signaling also require helper NLRs N requirement gene 1 (NRG1) and activated disease resistance 1 (ADR1). In two recent remarkable papers published in Science, the authors show that the TIR-containing proteins catalyze and produce two types of signaling molecules, ADPr-ATP/diADPR and pRib-AMP/ADP. Importantly, they demonstrate that EDS1-SAG101 and EDS1-PAD4 modules are the receptor complexes for ADPr-ATP/diADPRp and Rib-AMP/ADP, respectively, which allosterically promote EDS1-SAG101 interaction with NRG1 and EDS1-PAD4 interaction with ADR1. Thus, two different small molecules catalyzed by TIR-containing proteins selectively activate the downstream two distinct branches of EDS1-mediated immune signalings. These breakthrough studies significantly advance our understanding of TNL downstream signaling pathway.

This study aimed to investigate the efficacy of intraoperative motor evoked potential (MEP) and somatosensory evoked potential (SSEP) monitoring for predicting postoperative motor deficits (PMDs) in patients with internal carotid artery (ICA) aneurysms. The data for 138 patients with ICA aneurysms who underwent surgical clipping as well as their intraoperative neuromonitoring data were retrospectively reviewed. The efficacy of MEP/SSEP changes for predicting PMDs was assessed using binary logistic regression analysis. Subsequently, receiver operating characteristic curve analysis was used to obtain a supplementary critical value of the MEP/SSEP deterioration duration. The sensitivity and specificity of MEP changes for predicting PMDs were 0.824 and 0.843, respectively. For SSEP changes, the sensitivity and specificity were 0.529 and 0.959, respectively. MEP and SSEP changes were identified as independent predictors for short-term (p = 0.002 and 0.011, respectively) and long-term PMDs (p = 0.040 and 0.006, respectively). The supplementary critical value for MEP deterioration duration for predicting PMDs was 14 min (p = 0.007, AUC = 0.805). For SSEP, the value was 14.5 min (p = 0.042, AUC = 0.875). The MEP/SSEP changes adjusted by those optimal values were also identified as independent predictors for short-term (p

Cereal yellow dwarf virus (CYDV-RPV) encodes a P0 protein that functions as a viral suppressor of RNA silencing (VSR). The strength of silencing suppression is highly variable among CYDV-RPV isolates. In this study, comparison of the P0 sequences of CYDV-RPV isolates and mutational analysis identified a single C-terminal amino acid that influenced P0 RNA-silencing suppressor activity. A serine at position 247 was associated with strong suppressor activity, whereas a proline at position 247 was associated with weak suppressor activity. Amino acid changes at position 247 did not affect the interaction of P0 with SKP1 proteins from Hordeum vulgare (barley) or Nicotiana benthamiana. Subsequent studies found P0 proteins containing a P247 residue were less stable than the P0 proteins containing an S247 residue. Higher temperatures contributed to the lower stability and in planta and the P247 P0 proteins were subject to degradation via the autophagy-mediated pathway. A P247S amino acid residue substitution in P0 increased CYDV-RPV replication after expression in agroinfiltrated plant leaves and increased viral pathogenicity of P0 generated from the heterologous Potato virus X expression vector system. Moreover, an S247 CYDV-RPV could outcompete the P247 CYDV-RPV in a mixed infection in natural host at higher temperature. These traits contributed to increased transmission by aphid vectors and could play a significant role in virus competition in warming climates. Our findings underscore the capacity of a plant RNA virus to adapt to climate warming through minor genetic changes in gene-silencing suppressor, resulting in the potential for disease persistence and prevalence.

Plant nucleotide-binding domain and leucine-rich repeat receptors (NLRs) play crucial roles in recognizing pathogen effectors and activating plant immunity. The tomato NLR Sw-5b is a coiled-coil NLR (CC-NLR) immune receptor that confers resistance against tospoviruses, which cause serious economic losses in agronomic crops worldwide. Compared with other CC-NLRs, Sw-5b possesses an extended N-terminal Solanaceae domain (SD). The SD of Sw-5b is critical for recognition of the tospovirus viral movement protein NSm. An SD is also frequently detected in many NLRs from Solanaceae plants. However, no sequences homologous to the SD have been detected in animals or in plants other than Solanaceae. The properties of the SD protein are largely unknown, and thus 3D structural information is vital in order to better understand its role in pathogen perception and the activation of immune receptors. Here, the expression, purification and crystallization of Sw-5b SD (amino acids 1–245) are reported. Native and selenomethionine-substituted crystals of the SD protein belonged to space group P3112, with unit-cell parameters a = 81.53, b = 81.53, c = 98.44 Å and a = 81.63, b = 81.63, c = 98.80 Å, respectively. This is the first report of a structural study of the noncanonical SD domain of the NLR proteins from Solanaceae plants.

Most cytoplasmic-replicating negative-strand RNA viruses (NSVs) initiate genome transcription by cap snatching. The source of host mRNAs from which the cytoplasmic NSVs snatch capped-RNA leader sequences has remained elusive. Earlier reports have pointed towards cytoplasmic-RNA processing bodies (P body, PB), although several questions have remained unsolved. Here, the nucleocapsid (N) protein of plant- and animal-infecting members of the order Bunyavirales, in casu Tomato spotted wilt virus (TSWV), Rice stripe virus (RSV), Sin nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV) and Schmallenberg virus (SBV) have been expressed and localized in cells of their respective plant and animal hosts. All N proteins localized to PBs as well as stress granules (SGs), but extensively to docking stages of PB and SG. TSWV and RSV N proteins also co-localized with Ran GTPase-activating protein 2 (RanGAP2), a nucleo-cytoplasmic shuttling factor, in the perinuclear region, and partly in the nucleus when co-expressed with its WPP domain containing a nuclear-localization signal. Upon silencing of PB and SG components individually or concomitantly, replication levels of a TSWV minireplicon, as measured by the expression of a GFP reporter gene, ranged from a 30% reduction to a four-fold increase. Upon the silencing of RanGAP homologs in planta, replication of the TSWV minireplicon was reduced by 75%. During in vivo cap-donor competition experiments, TSWV used transcripts destined to PB and SG, but also functional transcripts engaged in translation. Altogether, the results implicate a more complex situation in which, besides PB, additional cytoplasmic sources are used during transcription/cap snatching of cytoplasmic-replicating and segmented NSVs.

2-Amino-3-methylhexanoic acid (AMHA) was synthetized as a non-natural amino acid more than 70 years ago; however, its possible function as an inducer of plant resistance has not been reported. Plant resistance inducers, also known as plant elicitors, are becoming a novel and important development direction in crop protection and pest management. We found that free AMHA accumulated in the mycelia but not in fermentation broths of four fungal species, Magnaporthe oryzae and three Alternaria spp. We unequivocally confirmed that AMHA is a naturally occurring endogenous (2S, 3S)-α-amino acid, based on isolation, purification and structural analyses. Further experiments demonstrated that AMHA has potent activity-enhancing resistance against extreme temperature stresses in several plant species. It is also highly active against fungal, bacterial and viral diseases by inducing plant resistance. AMHA pretreatment strongly protected wheat against powdery mildew, Arabidopsis against Pseudomonas syringae DC3000 and tobacco against Tomato spotted wilt virus. AMHA exhibits a great potential to become a unique natural elicitor protecting plants against biotic and abiotic stresses.

Negative-strand RNA (NSR) viruses contain not only medical important animal pathogens but also agricultural important plant pathogens. These NSR viruses cause deadliest diseases in humans, livestocks and agronomic crops and pose great threat to the health of human being and food security worldwide. The virus reverse genetics system is an important molecular genetic technology to study viral gene functions in virus infection cycle, disease pathology and virus-host interactions. For animal-infecting NSR viruses, the reverse genetics system has been established for more than twenty years. In contrast to the well-established reverse genetics systems for animal-infecting NSR viruses, establishment of such system for the plant-infecting NSR viruses turned out to be an extremely challenge work. Many research groups throughout the world have tried to use the similar strategies used for animal NSR viruses to establish reverse genetics systems for plant-infecting NSR viruses in the last twenty years but none of groups have succeeded. The absence of the reverse genetics system posed a major obstacle to molecular genetic manipulation and subsequent investigation of gene function and disease pathology for plant-infecting NSR viruses. Recently, big breakthroughs have been made in the establishment of the reverse genetics systems for plant NSR viruses by scientists from China and America, respectively. The systems were recently established for both nonsegmented and segmented plant NSR viruses. The reverse genetics system for a nonsegmented plant NSR virus was firstly developed for sonchus yellow net virus (SYNV), a non segmented nucleorhabdovirus NSR. Using the similar strategies as SYNV, barley yellow striate mosaic virus (BYSMV), a nonsegmented cytorhabdovirus NSR, was then succeeded in the establishment of reverse genetics system. The first reverse genetics system for a segmented plant NSR virus was developed using tomato spotted wilt virus (TSWV), a tospovirus with tripartite negative-stranded/ambisense RNA genomes. The reverse genetics system was also recently developed for rose rosette virus (RRV), an emaravirus with seven negative-sense mono-cistronic RNA genomes. The establishment of the reverse genetics system for plant NSR viruses typically involves two steps. The first step requires the construction of a mini-genome replication system. The viral genomic or antigenomic RNA was flanked by hammerhead and ribozyme to generate exact viral 5 ¢ and 3 ¢ end, and driven by 35S promoter. The mini-replicon construct was co-expressed with the constructs expressing nucleocapsid (N) protein, phosphoprotein (P; essential for rhabdovirus) and viral RNA-dependent RNA polymerase (RdRp). For the segmented plant NSR viruses such as TSWV, codon optimization and removal of intron-splicing sites of RdRp sequence are critical for the expression of a functional RdRp from 35S-driven constructs in planta . The second step involves the rescue of virus entirely from full-length infectious cDNA clones in plant. The recombinant virus can carry green fluorescence protein (GFP) gene reporter and infect Nicotiana benthamiana plant systemically. The establishment of reverse genetics for different plant-infecting NSR viruses now provided powerful systems to investigate all aspects of virus infection cycle and disease pathology. The recent breakthroughs of reverse genetics systems have opened a new era for the investigation of plant-infecting NSR viruses in the future.

Tomato spotted wilt virus (TSWV) is one of the most devastating plant viruses and often causes severe crop losses worldwide. Generally, mature plants become more resistant to pathogens, known as adult plant resistance. In this study, we demonstrated a new phenomenon involving developmentally regulated susceptibility of Arabidopsis thaliana to TSWV. We found that Arabidopsis plants become more susceptible to TSWV as plants mature. Most young 3‐week‐old Arabidopsis were not infected by TSWV. Infection of TSWV in 4‐, 5‐, and 6‐week‐old Arabidopsis increased from 9%, 21%, and 25%, respectively, to 100% in 7‐ to 8‐week‐old Arabidopsis plants. Different isolates of TSWV and different tospoviruses show a low rate of infection in young Arabidopsis but a high rate in mature plants. When Arabidopsis dcl2/3/4 or rdr1/2/6 mutant plants were inoculated with TSWV, similar results as observed for the wild‐type Arabidopsis plants were obtained. A cell‐to‐cell movement assay showed that the intercellular movement efficiency of TSWV NSm:GFP fusion was significantly higher in 8‐week‐old Arabidopsis leaves compared with 4‐week‐old Arabidopsis leaves. Moreover, the expression levels of pectin methylesterase and β‐1,3‐glucanase, which play critical roles in macromolecule cell‐to‐cell trafficking, were significantly up‐regulated in 8‐week‐old Arabidopsis leaves compared with 4‐week‐old Arabidopsis leaves during TSWV infection. To date, this mature plant susceptibility to pathogen infections has rarely been investigated. Thus, the findings presented here should advance our knowledge on the developmentally regulated mature host susceptibility to plant virus infection., Generally, mature plants become more resistance to pathogens. In this study we demonstrated a developmentally regulated mature host susceptibility to tomato spotted wilt virus infection in Arabidopsis.

To explore the relationship between postoperative motor deficits and the duration of reduced motor-evoked potentials (MEPs) in patients with middle cerebral artery (MCA) aneurysm. This study included 285 cases of MCA aneurysm treated with clipping surgery with MEP monitoring. The effects of MEP changes on postoperative motor function were assessed, and the key time point for minimizing the incidence of postoperative motor dysfunction was found through receiver operating characteristic (ROC) curve analysis. Motor dysfunction was significantly associated with the occurrence of MEP changes, and patients with irreversible changes were more likely to suffer motor dysfunction than were those with reversible changes. The critical duration of MEP changes that minimized the risk of postoperative motor dysfunction was 8.5 min. This study revealed that MEP monitoring is an effective method for preventing ischemic brain injury during surgical treatment of MCA aneurysm and proposes a critical cutoff for the duration of MEP deterioration of 8.5 min for predicting postoperative motor dysfunction.

Tomato spotted wilt virus (TSWV) is a negative-stranded RNA virus that infects hundreds of plant species, causing great economic loss. Infected Arabidopsis thaliana plants develop symptoms including chlorosis and wilt, which can lead to cell death. From 9 to 15 days after TSWV infection, symptoms progress through a three-stage process of appearance, severity, and death. In this study, deep sequencing technology was first used to explore gene expression in response to TSWV infection in model plant A. thaliana at different symptom development stages. We found that plant immune defense and protein degradation are induced by TSWV infection and that both inductions became stronger over time. The photosynthesis pathway was attenuated with TSWV infection. Cell wall metabolism had a large extent of downregulation while some genes were upregulated. These results illustrate the dynamic nature of TSWV infection in A. thaliana at the whole-transcriptome level. The link between biological processes and subpathway metabolism was further analyzed. Our study provides new insight into host regulatory networks and dynamic processes in response to TSWV infection.

Soybean is one of the most valuable legume crops in the world with high nutritional value. Recently, the outbreak of soybean stay-green syndrome has swept the soybean production in the Huang-Huai-Hai region of China, resulting in huge yield losses, which has become an epidemic and prominent problem in soybean production. However, the cause of the stay-green syndrome remains obscure. Here, we report a novel intergeneric recombinant geminivirus which causes soybean stay-green symptoms. Viral small RNA-based screening identified a new recombinant geminvirus from field soybean stay-green samples. The complete genome sequence of the virus contains 2762 nucleotide (nt) and appears to be an intergeneric recombinant virus in which protein coding for coat protein (V1) is similar to member of genus Mastrevirus, whereas proteins coding for V2, C2, C3 are most similar to those of viruses in the Maldovirus genus, and C1 and C4 are most similar to virus in genus Begomovirus. Inoculation of the infectious clone of the recombinant geminivirus through Agrobacterium rhizogenes causes typical soybean stay-green syndrome which resembles field symptoms including delayed leaf senescence, flat pods and abnormal seeds. The recombinant geminivirus can be detected in seed coat but not in cotyledon and embryo, thus failing to be transmitted by seeds. Moreover, the genome variation and epidemiological dynamic analysis were also carried out to help the continuous epidemiological surveillance of this emerging geminivirus. Collectively, this new geminivirus is tentatively named soybean stay-green associated virus (SoSGV). Our determination of the causal agent of soybean stay-green disease will bolster efforts to develop effective management strategies to control this prevalent disease in the field.

Sw-5b is an effective resistance gene used widely in tomato to control tomato spotted wilt virus (TSWV), which causes severe losses in crops worldwide. Sw-5b confers resistance by recognizing a 21-amino-acid peptide region of the viral movement protein NSm (NSm21, amino acids 115-135). However, C118Y or T120N mutation within this peptide region of NSm has given rise to field resistance-breaking (RB) TSWV isolates. To investigate the potential ability of TSWV to break Sw-5b-mediated resistance, we mutagenized each amino acid on NSm21 and determined which amino acid mutations would evade Sw-5b recognition. Among all alanine-scan mutants, NSm

Background: The current study aimed to investigate the predictive value of visual-evoked potential (VEP) latency for post-operative visual deterioration in patients undergoing craniopharyngioma resection via extended endoscopic endonasal approach (EEEA).Methods: Data from 90 patients who underwent craniopharyngioma resection via EEEA with intraoperative VEP monitoring were retrospectively reviewed. P100 latency was compared between patients with and without post-operative visual deterioration, and the threshold value of P100 latency for predicting post-operative visual deterioration was calculated by the receiver operating characteristic curve analysis. In addition, other potential prognostic factors regarding post-operative visual outcomes were also analyzed by multivariate analysis.Results: Patients with post-operative visual deterioration showed a significantly longer VEP latency than those without (p < 0.001). An extension over 8.61% in VEP latency was identified as a predictor of post-operative visual deterioration (p < 0.001). By contrast, longer preoperative visual impairment duration and larger tumor volume were not significant predictors for post-operative visual deterioration.Conclusions: The current study revealed that intraoperative VEP monitoring in EEEA is effective for predicting post-operative visual deterioration, and an extension over 8.61% in VEP latency can be used as a critical cut-off value to predict post-operative visual deterioration.

While G/U pairs are present in many RNAs, the lack of molecular studies to characterize the roles of multiple G/U pairs within a single RNA limits our understanding of their biological significance. From known RNA 3D structures, we observed that the probability a G/U will form a Watson–Crick (WC) base pair depends on sequence context. We analyzed 17 G/U pairs in the 359-nucleotide genome of Potato spindle tuber viroid (PSTVd), a circular non-coding RNA that replicates and spreads systemically in host plants. Most putative G/U base pairs were experimentally supported by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). Deep sequencing PSTVd genomes from plants inoculated with a cloned master sequence revealed naturally occurring variants, and showed that G/U pairs are maintained to the same extent as canonical WC base pairs. Comprehensive mutational analysis demonstrated that nearly all G/U pairs are critical for replication and/or systemic spread. Two selected G/U pairs were found to be required for PSTVd entry into, but not for exit from, the host vascular system. This study identifies critical roles for G/U pairs in the survival of an infectious RNA, and increases understanding of structure-based regulation of replication and trafficking of pathogen and cellular RNAs.

The tomato Sw-5b gene confers resistance to tomato spotted wilt virus (TSWV) and encodes a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal Solanaceae-specific domain (SD). Although our understanding of how Sw-5b recognizes the viral NSm elicitor has increased significantly, the process by which Sw-5b activates downstream defense signaling remains to be elucidated. In this study, we used a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate the roles of the SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 genes in the Sw-5b-mediated signaling pathway. We found that chaperone SGT1 was required for Sw-5b function, but co-chaperone RAR1 was not. Sw-5b-mediated immune signaling was independent of both EDS1 and NDR1. Silencing NPR1, which is a central component in SA signaling, did not result in TSWV systemic infection in Sw-5b-transgenic N. benthamiana plants. Helper NLR NRCs (NLRs required for cell death) were required for Sw-5b-mediated systemic resistance to TSWV infection. Suppression of NRC2/3/4 compromised the Sw-5b resistance. However, the helper NLRs ADR1 and NRG1 may not participate in the Sw-5b signaling pathway. Silencing ADR1, NRG1, or both genes did not affect Sw-5b-mediated resistance to TSWV. Our findings provide new insight into the requirement for conserved key components in Sw-5b-mediated signaling pathways.

The study aimed to investigate the predictive value of motor evoked potential (MEP) deterioration duration for postoperative motor deficits in patients undergoing intracranial aneurysm surgery.Data from 587 patients were reviewed and 92 patients with MEP deterioration were enrolled. MEP deterioration duration was compared between patients with and without postoperative motor deficits. Receiver operating characteristic (ROC) curve analysis was performed to define the threshold value for predicting postoperative motor deficit risk. Additionally, the association between MEP deterioration duration and postoperative CT findings was explored.Patients with postoperative motor deficits had a significantly longer MEP deterioration duration (p 0.01). An MEP deterioration duration greater than or equal to 13 min was identified as an independent predictor of immediate (p 0.01), short-term (p 0.01), and long-term postoperative motor deficits (p 0.05). There was no significant association between MEP deterioration duration and new CT abnormalities.MEP deterioration duration could be used for predicting intracranial aneurysm surgical outcome.The study first proposed a threshold value of MEP deterioration duration (13 min) for predicting the risk of postoperative motor deficits in patients undergoing intracranial aneurysm surgery.

Plant intracellular nucleotide binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen surveillance. Most plant NLRs characterized so far were found to use a single domain/sensor to recognize pathogen effectors. Here we report that the Sw-5b NLR immune receptor uses two distinct domains to detect the viral movement protein NSm encoded by tospovirus. In addition to its leucine-rich repeat (LRR) domain that has been previously reported, the N-terminal Solanaceae domain (SD) of Sw-5b also interacts with NSm and a conserved 21-amino-acid region of NSm (NSm21). The specific interaction between Sw-5b SD and NSm is required for releasing the inhibitory effect of coiled-coil domain on the NB-ARC-LRR region. Furthermore, we found that the binding of NSm affects the nucleotide binding activity of the NB-ARC-LRR in vitro, while Sw-5b NB-ARC-LRR is activated only when NSm and NSm21 levels are high. Interestingly, Sw-5b SD could significantly enhance the ability of the NB-ARC-LRR to detect low levels of NSm effector and facilitate its activation and induction of defense response. An Sw-5b SD mutant that is disrupted in NSm recognition failed to enhance the ability of the NB-ARC-LRR to sense low levels of NSm and NSm21. Taken together, our results suggest that Sw-5b SD functions as an extra sensor and the NB-ARC-LRR as an activator, and that Sw-5b NLR adopts a two-step recognition mechanism to enhance viral effector perception.

Although the application of somatosensory evoked potential (SSEP) in intracranial aneurysm surgery has been well demonstrated, the relationship between the duration of SSEP deterioration and postoperative neurological deficits (PNDs) is still not clear. The objectives of this study were (1) to detect the relationship between the SSEP deterioration duration and PND; and (2) detect the relationship between SSEP deterioration duration and postoperative computed tomography (CT) findings. Data from 587 patients were reviewed and 40 patients with SSEP deterioration were enrolled. Four patients presented irreversible disappearance and 36 patients presented reversible deterioration (including 9 [25%] patients with reversible reduction and 27 [75%] patients with reversible disappearance). In the patients with reversible SSEP deterioration, 17 patients had PNDs, and the SSEP deterioration duration was 42 ± 46 min, ranging from 5 to 180 min. Nineteen patients did not have PNDs, and their duration of SSEP deterioration was 11 ± 9 min (range 2-40 min). The SSEP deterioration duration significantly differed between patients with or without PND (P 0.01). Eleven minutes is the optimal cut-off value of motor evoked potential change duration avoiding PND (area under the curve = 0.84). Patients with a SSEP deteriorating duration 11 min had a significant higher incidence rate of abnormal CT finding postoperatively (p 0.05). According to these results, we conclude that the duration of SSEP deterioration is extremely important to postoperative neurological function, and in order to avoid PND, the SSEP deterioration duration must not exceed 10 min. The SSEP deterioration duration is also associated with postoperative CT findings.

The selection of resistant crops is an effective method for controlling geminivirus diseases. ty-5 encodes a messenger RNA surveillance factor Pelota with a single amino acid mutation (PelotaV16G), which confers effective resistance to tomato yellow leaf curl virus (TYLCV). No studies have investigated whether ty-5 confers resistance to other geminiviruses. Here, we demonstrate that the tomato ty-5 line exhibits effective resistance to various geminiviruses. It confers resistance to two representative begomoviruses, tomato yellow leaf curl China virus/tomato yellow leaf curl China betasatellite complex and tomato leaf curl Yunnan virus. The ty-5 line also exhibits partial resistance to a curtovirus beet curly top virus. Importantly, ty-5 confers resistance to TYLCV with a betasatellite. Southern blotting and quantitative polymerase chain reaction analyses showed that significantly less DNA of these geminiviruses accumulated in the ty-5 line than in the susceptible line. Moreover, knockdown of Pelota expression converted a Nicotiana benthamiana plant from a geminivirus-susceptible host to a geminivirus-resistant host. Overall, our findings suggest that ty-5 is an important resistance gene resource for crop breeding to control geminiviruses.

Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in human and livestock, and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana. We first developed a mini-replicon system expressing RSV genomic RNA3 eGFP reporter (MR3(-)eGFP), a nucleocapsid (NP), and a codon usage optimized RNA-dependent RNA polymerase (RdRpopt), respectively. Using this mini-replicon system we determined that RSV NP and RdRpopt are indispensable for the eGFP expression from MR3(-)eGFP. The expression of eGFP from MR3(-)eGFP can be significantly enhanced in the presence of NSs and P19-HcPro-γb. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana. However, we found that the RSV NS3 coding sequence acts as a cis-element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that this mini-replicon system described here will allow the studies of RSV replication, transcription, cell-to-cell movement and host machinery underpinning RSV infection in plants.IMPORTANCEPlant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into 3 genera: Orthotospovirus, Tenuivirus and Emaravirus. The reverse-genetics systems have been established for members in the genera Orthotospovirus and Emaravirus, respectively. However, there is still no reverse-genetics system available for Tenuivirus. Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damages to rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana. This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plant.

Plant and animal intracellular nucleotide-binding and leucine-rich repeat (NLR) receptors play important roles in sensing pathogens and activating defense signaling. However, the molecular mechanisms underlying the activation of host defense signaling by NLR proteins remain largely unknown. Many studies have determined that the coil–coil (CC) or Toll and interleukin-1 receptor/resistance protein (TIR) domain of NLR proteins and their dimerization/oligomerization are critical for activating downstream defense signaling. In this study, we demonstrated that, in tomato, the nucleotide-binding (NB) domain Sw-5b NLR alone can activate downstream defense signaling, leading to elicitor-independent cell death. Sw-5b NB domains can self-associate, and this self-association is crucial for activating cell death signaling. The self-association was strongly compromised after the introduction of a K568R mutation into the P-loop of the NB domain. Consequently, the NBK568R mutant induced cell death very weakly. The NBCΔ20 mutant lacking the C-terminal 20 amino acids can self-associate but cannot activate cell death signaling. The NBCΔ20 mutant also interfered with wild-type NB domain self-association, leading to compromised cell death induction. By contrast, the NBK568R mutant did not interfere with wild-type NB domain self-association and its ability to induce cell death. Structural modeling of Sw-5b suggests that NB domains associate with one another and likely participate in oligomerization. As Sw-5b-triggered cell death is dependent on helper NLR proteins, we propose that the Sw-5b NB domain acts as a nucleation point for the assembly of an oligomeric resistosome, probably by recruiting downstream helper partners, to trigger defense signaling.

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors play critical roles in mediating host immunity to pathogen attack. We use tomato Sw-5b::tospovirus as a model system to study the specific role of the compartmentalized plant NLR in dictating host defenses against the virus at different infection steps. We demonstrated here that tomato NLR Sw-5b distributes to the cytoplasm and nucleus, respectively, to play different roles in inducing host resistances against tomato spotted wilt orthotospovirus (TSWV) infection. The cytoplasmic-enriched Sw-5b induces a strong cell death response to inhibit TSWV replication. This host response is, however, insufficient to block viral intercellular and long-distance movement. The nuclear-enriched Sw-5b triggers a host defense that weakly inhibits viral replication but strongly impedes virus intercellular and systemic movement. Furthermore, the cytoplasmic and nuclear Sw-5b act synergistically to dictate a full host defense of TSWV infection. We further demonstrated that the extended N-terminal Solanaceae domain (SD) of Sw-5b plays critical roles in cytoplasm/nucleus partitioning. Sw-5b NLR controls its cytoplasm localization. Strikingly, the SD but not coil-coil domain is crucial for Sw-5b receptor to import into the nucleus to trigger the immunity. The SD was found to interact with importins. Silencing both importin α and β expression disrupted Sw-5b nucleus import and host immunity against TSWV systemic infection. Collectively, our findings suggest that Sw-5b bifurcates disease resistances by cytoplasm/nucleus partitioning to block different infection steps of TSWV. The findings also identified a new regulatory role of extra domain of a plant NLR in mediating host innate immunity.

Antiviral RNA silencing/interference (RNAi) of negative-strand (-) RNA plant viruses (NSVs) has been studied less than for single-stranded, positive-sense (+)RNA plant viruses. From the latter, genomic and subgenomic mRNA molecules are targeted by RNAi. However, genomic RNA strands from plant NSVs are generally wrapped tightly within viral nucleocapsid (N) protein to form ribonucleoproteins (RNPs), the core unit for viral replication, transcription and movement. In this study, the targeting of the NSV tospoviral genomic RNA and mRNA molecules by antiviral RNA-induced silencing complexes (RISC) was investigated, in vitro and in planta. RISC fractions isolated from tospovirus-infected N. benthamiana plants specifically cleaved naked, purified tospoviral genomic RNAs in vitro, but not genomic RNAs complexed with viral N protein. In planta RISC complexes, activated by a tobacco rattle virus (TRV) carrying tospovirus NSs or Gn gene fragments, mainly targeted the corresponding viral mRNAs and hardly genomic (viral and viral-complementary strands) RNA assembled into RNPs. In contrast, for the (+)ssRNA cucumber mosaic virus (CMV), RISC complexes, activated by TRV carrying CMV 2a or 2b gene fragments, targeted CMV genomic RNA. Altogether, the results indicated that antiviral RNAi primarily targets tospoviral mRNAs whilst their genomic RNA is well protected in RNPs against RISC-mediated cleavage. Considering the important role of RNPs in the replication cycle of all NSVs, the findings made in this study are likely applicable to all viruses belonging to this group., Author summary Plants have evolved RNA silencing as the first line of defense against virus infection. Antiviral RNA silencing as well as viral counter-defense strategies have been extensively studied for ss(+)RNA plant viruses, but less for plant negative-strand (-)/ambisense RNA plant viruses (NSVs). Upon virus infection, RNA-induced silencing complexes (RISCs) are activated to bind and cleave invasive viral RNAs. Unlike ss(+)RNA plant viruses, genomic RNA molecules of plant NSVs are not naked and tightly associate with viral nucleocapsid protein into ribonucleoproteins (RNPs), the core infectious unit for NSVs. However, their viral mRNAs are not assembled into RNPs. Whether the assembly of genomic RNA into RNPs provides another counter-defense strategy to protect against antiviral RISC activity has not been determined for any NSV. In this study, using tospovirus as a representative plant NSV model system, it is demonstrated that antiviral RISC mainly targets viral mRNAs but not genomic RNA embedded in RNPs. Our findings suggest that the RNA targets of RISC differ between plant NSVs and ss(+)RNA plant viruses and that RNPs provide another counter-defense way for NSVs to (partially) evade/escape from cytoplasmic RISC-mediated degradation.

Negative-strand (-) RNA viruses (NSVs) comprise a large and diverse group of viruses that are generally divided in those with non-segmented and those with segmented genomes. Whereas most NSVs infect animals and humans, the smaller group of the plant-infecting counterparts is expanding, with many causing devastating diseases worldwide, affecting a large number of major bulk and high-value food crops. In 2018, the taxonomy of segmented NSVs faced a major reorganization with the establishment of the order Bunyavirales. This article overviews the major plant viruses that are part of the order, i.e., orthospoviruses (Tospoviridae), tenuiviruses (Phenuiviridae), and emaraviruses (Fimoviridae), and provides updates on the more recent ongoing research. Features shared with the animal-infecting counterparts are mentioned, however, special attention is given to their adaptation to plant hosts and vector transmission, including intra/intercellular trafficking and viral counter defense to antiviral RNAi.

SummaryPlant intracellular nucleotide binding-leucine-rich repeat (NLR) receptors play critical roles in mediating host immunity to pathogen attack. We use tomato Sw-5b::tospovirus as a model system to study the specific role of the compartmentalized plant NLR in dictating host defense against virus at different infection steps.We demonstrated here that tomato NLR Sw-5b translocates to cytoplasm and nucleus, respectively, to play different roles in inducing host resistances against Tomato spotted wilt tospovirus (TSWV) infection. The cytoplasmic Sw-5b functions to induce a strong cell death response to inhibit TSWV replication. This host response is, however, insufficient to block viral intercellular and long-distance movement. The nucleus-localized Sw-5b triggers a host defense that weakly inhibits viral replication but strongly impedes virus intercellular and systemic movement. Furthermore, the cytoplasmic and nuclear Sw-5b act synergistically to dictate full host defense to TSWV infection.We further demonstrated that the extended N-terminal Solanaceae domain (SD) of Sw-5b plays critical roles in cytoplasm/nucleus partitioning. Sw-5b nucleotide-binding leucine-rich repeat (NB-LRR) controls its cytoplasm localization. Strikingly, the SD but not coil-coil (CC) domain is crucial for Sw-5b receptor to translocate from cytoplasm to nucleus to trigger the immunity. The SD was found to interact with importins. Silencing both importin α and β expression disrupted Sw-5b nucleus translocation and host immunity against TSWV systemic infection.Collectively, our findings suggest that Sw-5b bifurcates disease resistances by cytoplasm/nucleus partitioning to block different infection steps of TSWV. The findings also identified a new regulatory role of extra domain of a plant NLR in mediating host innate immunity.

Background High-frequency oscillation (HFO) represents a promising biomarker of epileptogenicity. However, the significant interindividual differences among patients limit its application in clinical practice. Here, we applied and evaluated an individualized, frequency-based approach of HFO analysis in stereoelectroencephalography (SEEG) data for localizing the epileptogenic zones (EZs). Methods Clinical and SEEG data of 19 patients with drug-resistant focal epilepsy were retrospectively analyzed. The individualized spectral power of all signals recorded by electrode array, i.e., the relative strength of HFO, was computed with a wavelet method for each patient. Subsequently, the clinical value of the relative strength of HFO for identifying the EZ was evaluated. Results Focal increase in the relative strength of HFO in SEEG recordings were identified in all 19 patients. HFOs identified inside the clinically identified seizure onset zone had more spectral power than those identified outside (p < 0.001), and HFOs in 250-500 Hz band (fast ripples) seemed to be more specific identifying the EZ than in those in 80-250 Hz band (ripples) (p < 0.01). The resection of brain regions generating HFOs resulted in a favorable seizure outcome in 17 patients (17/19; 89.5%), while in the cases of other patients with poor outcomes, the brain regions generating HFOs were not removed completely. Conclusion The relative strength of HFO, especially fast ripples, is a promising effective biomarker for identifying the EZ and can lead to a favorable seizure outcome if used to guide epilepsy surgery.

RNA granules are dynamic cellular foci that are widely spread in eukaryotic cells and play essential roles in cell growth and development, and immune and stress responses. Different types of granules can be distinguished, each with a specific function and playing a role in, for example, RNA transcription, modification, processing, decay, translation, and arrest. By means of communication and exchange of (shared) components, they form a large regulatory network in cells. Viruses have been reported to interact with one or more of these either cytoplasmic or nuclear granules, and act either proviral, to enable and support viral infection and facilitate viral movement, or antiviral, protecting or clearing hosts from viral infection. This review describes an overview and recent progress on cytoplasmic and nuclear RNA granules and their interplay with virus infection, first in animal systems and as a prelude to the status and current developments on plant viruses, which have been less well studied on this thus far.

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S((−))-genomic (g) or S((+))-antigenomic (ag) RNA template, flanked by the 5′ hammerhead and 3′ ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M((−))-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S((+))-agRNA, M((−))-gRNA, and L((+))-agRNA in which the glycoprotein gene sequence of M((−))-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.

Although many spherical and rod-shaped plant virus purification protocols are now available, only a few protocols on filamentous plant virus purification have been published. Here, we report a protocol for large-scale purification of Rice stripe virus (RSV) from RSV-infected rice tissues. RSV virions with high infectivity were first precipitated with polyethylene glycol (PEG) followed by pelleting through primary ultracentrifugation, ultracentrifugation in a glycerol cushion and ultracentrifugation in density gradient. The purified RSV virions can not only be viewed as filamentous particles under an electron microscope, but can also be acquired by insect vector through direct injection into insect body or through membrane feeding prior to transmission to rice plants.

Objective To evaluate a new technique in brainstem surgery, neuronavigation (NN)-guided corticospinal tract (CST) mapping, in a retrospective study of patients undergoing brainstem tumor surgery. Methods We studied 40 patients with a brainstem tumor who were enrolled in this study. Patients whose worst preoperative muscle strength of the 4 limbs was greater than 3 levels from normal on the Lovett scale were divided into 2 groups: a treatment group of 21 patients who underwent NN-guided CST mapping and routine intraoperative neurophysiology monitoring (IONM) and a control group of 19 patients who underwent routine NN and IONM. Preoperative muscle strength and postoperative (day 90 postsurgery) muscle strength were assessed and compared between the 2 groups. Results In the NN-guided CST mapping group, 3 patients (14.3%) had a decrease in muscle strength by 1 level postoperatively, and no patient experienced a decrease of >1 level. In the control group, 4 patients (21.1%) had a 1-level decrease in muscle strength, and 5 (26.3%) had a decrease of >1 level. Patients in the NN-guided CST mapping group had significantly better surgical outcomes compared with those in the control group (P = 0.018, Fisher exact test). Conclusions Brainstem tumor resection using NN-guided CST mapping achieved better preservation of motor function compared with routine NN and IONM. NN-guided CST mapping not only decreased the difficulty of the surgery, but also significantly improved the efficiency of surgery.

We determined the complete genome sequence of a lettuce chlorosis crinivirus (LCV) from China (LCV-NJ). The bipartite genome of LCV-NJ consists of RNA1 and RNA2 which are 8165 and 8454 nucleotides (nt) in length, respectively. The genomic structure of LCV-NJ RNA1 resembles that of LCV-California, an isolate with four open reading frames (ORFs) in RNA1. Although the amino acid sequences of ORF 1a and 1b have 92 and 99% identity between LCV-NJ and LCV-California, ORF 2 and ORF3 of LCV-NJ share only 63 and 71% identity with those of LCV-California, respectively. In addition LCV-NJ RNA2 contains 9 ORFs, compared to 10 ORFs in LCV-California. ORF10 was missing due to the deletion of a 173-nt sequence within the 3'-terminal region of LCV-NJ RNA2. Insertion or deletion of sequences of varying lengths was also observed in RNA1 and other regions of RNA2. Based on these findings, we propose that LCV-NJ/LCV-California may have undergone genome recombination and/or rearrangement in RNA1 and RNA2.

The complete genome sequence of a Capsicum chlorosis virus from China (CaCV-Hainan) was determined. The tripartite genome of CaCV-Hainan consists of small (S), medium (M), and large (L) RNAs of 3629, 4859, and 8912 nucleotides (nt), respectively. The S and M RNAs contain intergenic regions (IGRs) of 1348 and 462 nt, respectively. Strikingly, sequence comparisons among CaCV isolates revealed that the S RNA IGR of CaCV-Hainan derived from the CaCV-Qld-3432 Australia isolate through deletion of two stretches of 25- and 325-nt sequences within the S RNA IGR of CaCV-Qld-3432. Moreover, the S RNA IGR of CaCV-Hainan was inserted with two stretches of 10- and 20-nt sequences of an unknown origin. The S RNA IGR of CaCV-Ph from Taiwan and CaCV-NRA from Thailand also derived from the CaCV-Qld-3432 through deletion of 218-nt sequences. Our findings provide valuable new insight into the structural variations and evolutionary origin of CaCV IGRs.

Rice stripe tenuivirus (RSV) is a filamentous, negative-strand RNA virus causing severe diseases on rice in Asian countries. The viral particle is composed predominantly of a nucleocapsid protein (NP) and genomic RNA. However, the molecular details of how the RSV NP interacts with genomic RNA during particle assembly remain largely unknown. Here, we modeled the NP-RNA complex and show that polar amino acids within a predicted groove of NP are critical for RNA binding and protecting the RNA from RNase digestion. RSV NP formed pentamers, hexamers, heptamers, and octamers. By modeling the higher-order structures, we found that oligomer formation was driven by the N-terminal amino arm of the NP. Deletion of this arm abolished oligomerization; the N-terminally truncated NP was less able to interact with RNA and protect RNA than was the wild type. These findings afford valuable new insights into molecular mechanism of RSV NPs interacting with genomic RNA.

The XIth International Symposium on Thysanoptera and Tospoviruses co-hosted by the Yunnan Academy of Agricultural Sciences, and Nanjing Agricultural University was held from September 21–25 in Kunming, China (Figure 1) [...]

The group of negative strand RNA viruses (NSVs) includes not only dangerous pathogens of medical importance but also serious plant pathogens of agronomical importance. Tomato spotted wilt tospovirus (TSWV) is one of those plant NSVs that cause severe diseases on agronomic crops and pose major threats to global food security. Its negative-strand segmented RNA genome has, however, always posed a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from cDNA clones, the first reverse genetics (RG) system for a segmented plant NSV. First, a replication and transcription competent mini-genome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by a 5’ Hammerhead and 3’ Ribozyme sequence of Hepatitis Delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA dependent RNA polymerase (RdRp) gene. Next, a movement competent mini-genome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. After further optimization, infectious TSWV and derivatives carrying eGFP reporters were successfully rescuedin plantavia simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA and L(+)-agRNA. Viral rescue occurred in the additional presence of various viral suppressors of RNAi, but TSWV NSs interfered with the rescue of genomic RNA. The establishment of a RG system for TSWV now allows detailed molecular genetic analysis of all aspects of tospovirus life cycle and their pathogenicity.SignificanceFor many different animal-infecting segmented negative-strand viruses (NSVs), a reverse genetics system has been established that allows the generation of mutant viruses to study disease pathology and the role ofcis- andtrans-acting elements in the virus life cycle. In contrast to the relative ease to establish RG systems for animal-infecting NSVs, establishment of such system for the plant-infecting NSVs with a segmented RNA genome so far has not been successful. Here we report the first reverse genetics system for a segmented plant NSV, the Tomato spotted wilt tospovirus, a virus with a tripartite RNA genome. The establishment of this RG system now provides us with a new and powerful platform to study their disease pathology during a natural infection.

Tospoviruses are among the most important plant pathogens and cause serious crop losses worldwide. Tospoviruses have evolved to smartly utilize the host cellular machinery to accomplish their life cycle. Plants mount two layers of defense to combat their invasion. The first one involves the activation of an antiviral RNA interference (RNAi) defense response. However, tospoviruses encode an RNA silencing suppressor that enables them to counteract antiviral RNAi. To further combat viral invasion, plants also employ intracellular innate immune receptors (e.g., Sw-5b and Tsw) to recognize different viral effectors (e.g., NSm and NSs). This leads to the triggering of a much more robust defense against tospoviruses called effector-triggered immunity (ETI). Tospoviruses have further evolved their effectors and can break Sw-5b-/Tsw-mediated resistance. The arms race between tospoviruses and both layers of innate immunity drives the coevolution of host defense and viral genes involved in counter defense. In this review, a state-of-the-art overview is presented on the tospoviral life cycle and the multilined interplays between tospoviruses and the distinct layers of defense.

The tomato resistance protein Sw-5b differs from the classical coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) resistance proteins by having an extra N-terminal domain (NTD). To understand how NTD, CC and NB-LRR regulate autoinhibition and activation of Sw-5b, we dissected the function(s) of each domain. When viral elicitor was absent, Sw-5b LRR suppressed the central NB-ARC to maintain autoinhibition of the NB-LRR segment. The CC and NTD domains independently and additively enhanced the autoinhibition of NB-LRR. When viral elicitor was present, the NB-LRR segment of Sw-5b was specifically activated to trigger a hypersensitive response. Surprisingly, Sw-5b CC suppressed the activation of NB-LRR, whereas the extra NTD of Sw-5b became a positive regulator and fully activated the resistance protein, probably by relieving the inhibitory effects of the CC. In infection assays of transgenic plants, the NB-LRR segment alone was insufficient to confer resistance against Tomato spotted wilt tospovirus; the layers of NTD and CC regulation on NB-LRR were required for Sw-5b to confer resistance. Based on these findings, we propose that, to counter the negative regulation of the CC on NB-LRR, Sw-5b evolved an extra NTD to coordinate with the CC, thus developing a multilayered regulatory mechanism to control autoinhibition and activation.

Tospovirus is a tripartite negative stranded RNA virus and is considered as one of the most devastating plant viruses. Successful virus infection in plant requires many host factors. To date, very few host factors have been identified as important in Tospovirus infection in plants. We reported earlier that NSm protein encoded by Tomato spotted wilt virus (TSWV), a type species of the genus Orthotospovirus, plays critical roles in viral cell-to-cell and long-distance movement. In this study, we determined that molecular co-chaperone NbSGT1 interacted with TSWV NSm in Nicotiana benthamiana. TSWV infection significantly upregulated the expression of NbSGT1 gene and transient overexpression of NbSGT1 in N. benthamiana leaves accelerated TSWV infection. In contrast, silencing the NbSGT1 gene expression using a virus-induced gene silencing (VIGS) approach strongly inhibited TSWV NSm cell-to-cell movement, as well as TSWV local and systemic infection in N. benthamiana plants. Furthermore, NbSGT1 was found to regulate the infection of both American and Euro/Asia type tospoviruses in N. benthamiana plant. Collectively, our findings presented in this paper and the results published previously indicated that molecular co-chaperone NbSGT1 plays important roles in modulating both positive stranded and tripartite negative stranded RNA virus infection in plants.

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

Tomato chlorosis virus (ToCV) is the causal agent of an emerging virus disease of tomatoes which causes significant economic losses worldwide. The rapid spread of ToCV is associated with the increasing whitefly population in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the rapid detection of ToCV in infected plant or whitefly. This RT-LAMP assay can detect ToCV either in total RNA or crude RNA extracted from infected plants using a water bath within 1 h. The presence of ToCV in RT-LAMP products could be evaluated as ladder-like bands in an agarose gel or visualized in-tube with inclusion of a SYBR Green I dye under UV or daylight. RT-LAMP was 100–1000 times more sensitive compared to conventional RT-PCR for the detection of ToCV. RT-LAMP amplification was specific to ToCV, and no false reactions were detected when other viruses were tested. The developed RT-LAMP assay was also able to detect ToCV from purified RNA extracted from its vector whitefly. This is the first report of the application of the RT-LAMP to detect ToCV. The RT-LAMP assay developed in this study provides a rapid, sensitive and practical approach to facilitate the surveillance and management of ToCV.