A Novel Attenuated Enterovirus A71 Mutant with VP1-V238A,K244R Exhibits Reduced Efficiency of Cell Entry/Exit and Augmented Binding Affinity to Sulfated Glycans.

Enterovirus A71 (EV-A71) is one of the major etiological agents of hand, foot, and mouth disease (HFMD), and infection occasionally leads to fatal neurological complications in children. However, only inactivated whole-virus vaccines against EV-A71 are commercially available in Mainland China. Furthermore, the mechanisms underlying the infectivity and pathogenesis of EV-A71 remain to be better understood. By adaptation of an EV-A71 B5 strain in monkey Vero cells in the presence of brilliant black BN (E151), an anti-EV-A71 agent, a double mutant with VP1-V238A,K244R emerged whose infection was enhanced by E151. The growth of the reverse genetics (RG) mutant RG/B5-VP1-V238A,K244R (RG/B5-AR) was promoted by E151 in Vero cells but inhibited in other human and murine cells, while its parental wild type, RG/B5-wt, was strongly prevented by E151 from infection in all tested cells. In the absence of E151, RG/B5-AR exhibited defective cell entry/exit, resulting in reduced viral transmission and growth in vitro. It had augmented binding affinity to sulfated glycans, cells, and tissue/organs, which probably functioned as decoys to restrict viral dissemination and infection. RG/B5-AR was also attenuated, with a 355 times higher 50% lethal dose (LD50) and a shorter timing of virus clearance than those of RG/B5-wt in suckling AG129 mice. However, it remained highly immunogenic in adult AG129 mice and protected their suckling mice from lethal EV-A71 challenges through maternal neutralizing antibodies. Overall, discovery of the attenuated mutant RG/B5-AR contributes to better understanding of virulence determinants of EV-A71 and to further development of novel vaccines against EV-A71. IMPORTANCE Enterovirus A71 (EV-A71) is highly contagious in children and has been responsible for thousands of deaths in Asia-Pacific region since the 1990s. Unfortunately, the virulence determinants and pathogenesis of EV-A71 are not fully clear. We discovered that a novel EV-A71 mutant, VP1-V238A,K244R, showed growth attenuation with reduced efficiency of cell entry/exit. In the Vero cell line, which has been approved for manufacturing EV-A71 vaccines, the growth defects of the mutant were compensated by a food dye, brilliant black BN. The mutant also showed augmented binding affinity to sulfated glycans and other cellular components, which probably restricted viral infection and dissemination. Therefore, it was virulence attenuated in a mouse model but still retained its immunogenicity. Our findings suggest the mutant as a promising vaccine candidate against EV-A71 infection.

In Vitro and In Vivo Inhibition of the Infectivity of Human Enterovirus 71 by a Sulfonated Food Azo Dye, Brilliant Black BN.

Hand, foot, and mouth disease (HFMD), a highly contagious disease in children, is caused by human enteroviruses, including enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus A6 (CVA6). Although HFMD is usually mild and self-limiting, EV71 infection occasionally leads to fatal neurological disorders. Currently, no commercial antiviral drugs for HFMD treatment are available. Here, numerous sulfonated azo dyes, widely used as food additives, were identified as having potent antiviral activities against human enteroviruses. Among them, brilliant black BN (E151) was able to inhibit all EV71, CVA16, and CVA6 strains tested. In rhabdomyosarcoma cells, the 50% inhibitory concentrations of the dye E151 for various strains of EV71 ranged from 2.39 µM to 28.12 µM, whereas its 50% cytotoxic concentration was 1,870 µM. Food azo dyes, including E151, interacted with the vertex of the 5-fold axis of EV71 and prevented viral entry. Their efficacy in viral inhibition was regulated by amino acids at VP1-98, VP1-145, and/or VP1-246. Dye E151 not only prevented EV71 attachment but also eluted attached viruses in a concentration-dependent manner. Moreover, E151 inhibited the interaction between EV71 and its cellular uncoating factor cyclophilin A. In vivo studies demonstrated that E151 at a dose of 200 mg/kg of body weight/day given on the initial 4 days of challenge protected AG129 mice challenged with 10× the 50% lethal dose of wild-type EV71 isolates. Taken together, these data highlight E151 as a promising antiviral agent against EV71 infection.IMPORTANCE Human enterovirus 71 (EV71) is one of the causative agents of hand, foot, and mouth disease in children and is responsible for thousands of deaths in the past 20 years. Food azo dyes have been widely used since the nineteenth century; however, their biological effects on humans and microbes residing in humans are poorly understood. Here, we discovered that one of these dyes, brilliant black BN (E151), was particularly effective in inhibiting the infectivity of EV71 in both cell culture and mouse model studies. Mechanistic studies demonstrated that these sulfonated dyes mainly competed with EV71 attachment factors for viral binding to block viral attachment/entry to host cells. As no commercial antiviral drugs against EV71 are currently available, our findings open an avenue to exploit the development of permitted food dye E151 as a potential anti-EV71 agent.

Characterization of GAB3 and its association with NNV resistance in the Asian seabass.

Understanding the functions of genes related to disease resistance and identifying polymorphisms in these genes are essential in molecular breeding for disease resistance. Viral nervous necrosis (VNN) is one of the major diseases in the Asian seabass, Lates calcarifer. Our previous works on QTL mapping, GWAS and cell-line transcriptome analysis of the Asian seabass after NNV challenge revealed that the gene GAB3 might be a candidate gene for VNN resistance. In this study, we cloned and characterized GAB3, and identified SNPs in the gene of the Asian seabass. The cDNA of the gene was 2165 bp, containing an ORF of 1674 bp encoding 557 amino acids. The gene consisted of 10 exons and nine introns. It was ubiquitously expressed in normal fish. An analysis of the association between two SNPs in the second intron and NNV resistance in 1035 fish descended from 43 families revealed that the two SNPs were significantly associated with VNN resistance. After NNV infection, the expression of GAB3 was significantly increased in the brain, spleen, muscle and gut, and was suppressed in the liver. The GAB3 protein was localized in the nucleus. Overexpression of GAB3 with specific GAB3-pcDNA was positively correlated to increased viral RNA and titer in NNV-infected Asian seabass cells. Our study provides new evidence to support that GAB3 may be an important gene related to NNV resistance. In addition, the SNPs provide DNA markers for the selection of candidate genes resistance to NNV at the juvenile stage of Asian seabass.

Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids.

Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for Cymbidium orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 107/g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of Cymbidium. In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in Cymbidium orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops.

Proximity-dependent biotinylation screening identifies NbHYPK as a novel interacting partner of ATG8 in plants.

BACKGROUND: Autophagy is a conserved, highly-regulated catabolic process that plays important roles in growth, development and innate immunity in plants. In this study, we compared the rate of autophagy induction in Nicotiana benthamiana plants infected with Tobacco mosaic virus or the TMV 24A + UPD mutant variant, which replicates at a faster rate and induces more severe symptoms. Using a BirA* tag and proximity-dependent biotin identification (BioID) analysis, we identified host proteins that interact with the core autophagy protein, ATG8 in TMV 24A + UPD infected plants. By combining the use of a fast replicating TMV mutant and an in vivo protein-protein screening technique, we were able to gain functional insight into the role of autophagy in a compatible virus-host interaction. RESULTS: Our study revealed an increased autophagic flux induced by TMV 24A + UPD, as compared to TMV in N. benthamiana. Analysis of the functional proteome associated with ATG8 revealed a total of 67 proteins, 16 of which are known to interact with ATG8 or its orthologs in mammalian and yeast systems. The interacting proteins were categorized into four functional groups: immune system process, response to ROS, sulphur amino acid metabolism and calcium signalling. Due to the presence of an ubiquitin-associated (UBA) domain, which is demonstrated to interact with ATG8, the Huntingtin-interacting protein K-like (HYPK) was selected for validation of the physical interaction and function. We used yeast two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and subcellular localization to validate the ATG8-HYPK interaction. Subsequent down-regulation of ATG8 by virus-induced gene silencing (VIGS) showed enhanced TMV symptoms, suggesting a protective role for autophagy during TMV 24A + UPD infection. CONCLUSION: This study presents the use of BioID as a suitable method for screening ATG8 interacting proteins in planta. We have identified many putative binding partners of ATG8 during TMV 24A + UPD infection in N. benthamiana plants. In addition, we have verified that NbHYPK is an interacting partner of ATG8. We infer that autophagy plays a protective role in TMV 24A + UPD infected plants.

ICTV Virus Taxonomy Profile: Virgaviridae.

The family Virgaviridae is a family of plant viruses with rod-shaped virions, a ssRNA genome with a 3'-terminal tRNA-like structure and a replication protein typical of alpha-like viruses. Differences in the number of genome components, genome organization and the mode of transmission provide the basis for genus demarcation. Tobacco mosaic virus (genus Tobamovirus) was the first virus to be discovered (in 1886); it is present in high concentrations in infected plants, is extremely stable and has been extensively studied. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Virgaviridae, which is available at www.ictv.global/report/virgaviridae.

Characterization of a novel disease resistance gene rtp3 and its association with VNN disease resistance in Asian seabass.

Asian seabass, an important food fish in Southeast Asia, has suffered from nervous necrosis virus (NNV) infection, resulting in massive mortality of Asian seabass larvae and enormous economic losses. Identification and characterization of disease resistance genes is important. Previous transcriptome analysis of Asians seabass epithelial cells after NNV infection revealed a highly inducible gene, receptor-transporting protein 3 (rtp3), indicating it could play an important role in Asian seabass - NNV interaction. To characterize this gene, we determined its expression pattern and subcellular localization. The rtp3 was highly induced in most examined tissues and organs of Asian seabass after NNV infection, and protein Rtp3 was localized in cytoplasm. Further association study in multiple families revealed that a microsatellite marker, (GT)ntt(GT)n, in the 3' UTR of rtp3 was significantly associated with VNN disease resistance in Asian seabass. Our results imply that rtp3 may be a novel disease resistance gene in Asian seabass. This data could improve our understanding of molecular interaction between Asian seabass and NNV, and has the potential to be applied in marker-assisted selection for disease resistance breeding in Asian seabass.

Transcriptome analysis of genes responding to NNV infection in Asian seabass epithelial cells.

Asian seabass is an important food fish in Southeast Asia. Viral nervous necrosis (VNN) disease, triggered by nervous necrosis virus (NNV) infection, has caused mass mortality of Asian seabass larvae, resulting in enormous economic losses in the Asian seabass industry. In order to better understand the complex molecular interaction between Asian seabass and NNV, we investigated the transcriptome profiles of Asian seabass epithelial cells, which play an essential role in immune regulation, after NNV infection. Using the next generation sequencing (NGS) technology, we sequenced mRNA from eight samples (6, 12, 24, 48 h post-inoculation) of mock and NNV-infected Asian seabass epithelial cell line, respectively. Clean reads were de novo assembled into a transcriptome consisting of 89026 transcripts with a N50 of 2617 bp. Furthermore, 251 differentially expressed genes (DEGs) in response to NNV infection were identified. Top DEGs include protein asteroid homolog 1-like (ASTE1), receptor-transporting protein 3 (RTP3), heat shock proteins 30 (HSP30) and 70 (HSP70), Viperin, interferon regulatory factor 3 (IRF3) and other genes related to innate immunity. Our data suggest that abundant and diverse genes corresponding to NNV infection. The results of this study could also offer vital information not only for identification of novel genes involved in Asian seabass-NNV interaction, but also for our understanding of the molecular mechanism of Asian seabass' response to viral infection. In addition, 24807 simple sequence repeats (SSRs) were detected in the assembled transcriptome, providing valuable resources for studying genetic variations and accelerating quantitative trait loci (QTL) mapping for disease resistance in Asian seabass in the future.

Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

Upregulation of LINC-AP2 is negatively correlated with AP2 gene expression with Turnip crinkle virus infection in Arabidopsis thaliana.

KEY MESSAGE: A long intergenic noncoding RNA LINC - AP2 is upregulated and negatively correlated with AP2 gene expression with Turnip crinkle virus infection in Arabidopsis. Plant vegetative growth and floral reproductive structure were severely retarded and distorted in Turnip crinkle virus (TCV)-infected Arabidopsis thaliana. Compared to mock-inoculated plants, the stamen filaments were shorter in flowers of TCV-infected plants. However, TCV-infected plants can still produce normal seeds through artificial pollination, indicating both its pollen and stigma were biologically functional. From our high-throughput RNA-Seq transcriptome analysis, a floral structure-related APETALA2 (AP2) gene was found to be downregulated and its neighboring long intergenic noncoding RNAs (lincRNA), At4NC069370 (named LINC-AP2 in this study), were upregulated significantly in TCV-infected plants. This LINC-AP2 was further confirmed for its existence using 5'RACE technology. LINC-AP2 overexpression (LINC-AP2 OE) transgenic Arabidopsis plants were generated to compare with TCV-infected WT plants. TCV-infected LINC-AP2 OE plants which contained lower AP2 gene expression displayed more severe symptoms (including floral structure distortion) and higher TCV-CP gene transcript and coat protein levels. Furthermore, compared to TCV-infected WT plants, TCV-infected ap2 mutant plants failed to open their flower buds and displayed more severe viral symptoms. In conclusion, upregulation of LINC-AP2 is negatively correlated with AP2 gene expression with TCV infection in Arabidopsis.

A plant virus hijacks phosphatidylinositol-3,5-bisphosphate to escape autophagic degradation in its insect vector.

Hosts can initiate macroautophagy/autophagy as an antiviral defense response, while viruses have developed multiple ways to evade the host autophagic degradation. However, little is known as to whether viruses can target lipids to subvert autophagic degradation. Here, we show that a low abundant signaling lipid, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), is required for rice black-streaked dwarf virus (RBSDV) to evade the autophagic degradation in the insect vector Laodelphax striatellus. RBSDV binds to PtdIns(3,5)P2 and elevates its level through its main capsid protein P10, leading to inhibited autophagy and promoted virus propagation. Furthermore, we show that PtdIns(3,5)P2 inhibits the autophagy pathway by preventing the fusion of autophagosomes and lysosomes through activation of Trpml (transient receptor potential cation channel, mucolipin), an effector of PtdIns(3,5)P2. These findings uncover a strategy whereby a plant virus hijacks PtdIns(3,5)P2 via its viral capsid protein to evade autophagic degradation and promote its survival in insects.

Hibiscus chlorotic ringspot virus coat protein upregulates sulfur metabolism genes for enhanced pathogen defense.

In both Hibiscus chlorotic ringspot virus (HCRSV)-infected and HCRSV coat protein (CP) agroinfiltrated plant leaves, we showed that sulfur metabolism pathway related genes-namely, sulfite oxidase (SO), sulfite reductase, and adenosine 5'-phosphosulfate kinase-were upregulated. It led us to examine a plausible relationship between sulfur-enhanced resistance (SED) and HCRSV infection. We broadened an established method to include different concentrations of sulfur (0S, 1S, 2S, and 3S) to correlate them to symptom development of HCRSV-infected plants. We treated plants with glutathione and its inhibitor to verify the SED effect. Disease resistance was induced through elevated glutathione contents during HCRSV infection. The upregulation of SO was related to suppression of symptom development induced by sulfur treatment. In this study, we established that HCRSV-CP interacts with SO which, in turn, triggers SED and leads to enhanced plant resistance. Thus, we have discovered a new function of SO in the SED pathway. This is the first report to demonstrate that the interaction of a viral protein and host protein trigger SED in plants. It will be interesting if such interaction applies generally to other host-pathogen interactions that will lead to enhanced pathogen defense.

Silencing Asian Seabass gab3 Inhibits Nervous Necrosis Virus Replication.

The nervous necrosis virus (NNV) causes the viral nervous necrosis (VNN) disease in aquatic animals and has been a major threat in aquaculture. Thus, it is essential for the development of a prevention method to minimize economic losses caused by NNV such as the identification of NNV resistance genes and application of these genes in molecular breeding to increase disease resistance. gab3 is an important NNV resistance gene in Asian seabass. However, the mechanism of gab3 in NNV resistance has not been elucidated. In this study, knockdown of gab3 in NNV-infected Asian seabass cells resulted in a significant decrease in viral RNA and virus titers. Knockout of gab3 in zebrafish led to an increased survival rate and resistant time after NNV infection. Cellular localization of the GAB3 and NNV by immunofluorescence staining showed that the GAB3 was translocated from the nucleus to the cytoplasm, and finally reached the cell membrane of SB cells after 48 h post NNV infection. Our study suggests that gab3 plays an important role in NNV replication and silencing gab3 can inhibit virus replication.

Identification of putative binding interface of PI(3,5)P2 lipid on rice black-streaked dwarf virus (RBSDV) P10 protein.

Rice black-streaked dwarf virus (RBSDV) is an important reovirus that infects both plants and its transmission vector small brown planthopper, causing severe crop loss. High affinity binding between RBSDV P10 and PI(3,5)P2 lipid layer was measured using biolayer interferometry (BLI). Subcellular co-localization of PI(3,5)P2 and RBSDV P10 was observed on membranous structures in insect cells with stochastic optical reconstruction microscopy (STORM) imaging. Putative interacting sites of PI(3,5)P2 lipid on a computational predicted RBSDV P10 structure were mapped to its "C-domain" (250-470 aa), using HDXMS data. The BLI and STORM results showed binding and co-localization of RBSDV P10, and PI(3,5)P2 on vesicle-like membranous structures were corroborated with the prediction of the binding interface. Understanding the lipid binding sites on viral proteins will lead to developing strategies to block viral-lipid interaction and disrupt viral pathogenesis in insect vectors and to block virus transmission and achieve disease control of crops in the field.

Sulfonated azo dyes enhance the genome release of enterovirus A71 VP1-98K variants by preventing the virions from being trapped by sulfated glycosaminoglycans at acidic pH.

Enterovirus A71 (EV-A71) is a causative agent of hand, foot and mouth disease and occasionally causes death in children. Its infectivity and pathogenesis, however, remain to be better understood. Three sulfonated azo dyes, including acid red 88 (Ar88), were identified to enhance the infectivity of EV-A71, especially isolates with VP1-98K, 145E (-KE), by mainly promoting viral genome release in vitro. Enzymatic removal of sulfated glycosaminoglycans (GAGs) or knockout of xylosyltransferase II (XT2) responsible for biosynthesis of sulfated GAGs weakened the Ar88 enhanced EV-A71 infection. Ar88 is proposed to prevent the -KE variants from being trapped by sulfated GAGs at acidic pH and to facilitate the viral interaction with uncoating factors for genome release in endosomes. The results suggest dual roles of sulfated GAGs as attachment factors and as decoys during host interaction of EV-A71 and caution that these artificial dyes in our environment can enhance viral infection.

Effects of rrm1 on NNV Resistance Revealed by RNA-seq and Gene Editing.

Viral nervous necrosis (VNN) disease caused by the nervous necrosis virus (NNV) is a major disease, leading to a huge economic loss in aquaculture. Previous GWAS and QTL mapping have identified a major QTL for NNV resistance in linkage group 20 in Asian seabass. However, no causative gene for NNV resistance has been identified. In this study, RNA-seq from brains of Asian seabass fingerlings challenged with NNV at four time points (5, 10, 15 and 20 days post-challenge) identified 1228, 245, 189 and 134 DEGs, respectively. Eight DEGs, including rrm1, were located in the major QTL for NNV resistance. An association study in 445 survived and 608 dead fingerlings after NNV challenge revealed that the SNP in rrm1 were significantly associated with NNV resistance. Therefore, rrm1 was selected for functional analysis, as a candidate gene for NNV resistance. The expression of rrm1 was significantly increased in the gill, liver, spleen and muscle, and was suppressed in the brain, gut and skin after NNV challenge. The rrm1 protein was localized in the nuclear membrane. Over-expression of rrm1 significantly decreased viral RNA and titer in NNV-infected Asian seabass cells, whereas knock-down of rrm1 significantly increased viral RNA and titer in NNV-infected Asian seabass cells. The rrm1 knockout heterozygous zebrafish was more susceptible to NNV infection. Our study suggests that rrm1 is one of the causative genes for NNV resistance and the SNP in the gene may be applied for accelerating genetic improvement for NNV resistance.

iTRAQ-based quantitative proteomics suggests mitophagy involvement after Rice black-streaked dwarf virus acquisition in insect vector small brown planthopper Laodelphax striatellus Fallén.

Plant viruses trigger numerous responses in their insect vectors. Using iTRAQ-based quantitative proteomics analysis, early responses of the insect vector, the small brown planthopper (Laodelphax striatellus Fallén, SBPH), after acquiring Rice black-streaked dwarf virus (RBSDV) at 3 days and 5 days post first access to diseased plants (padp) were revealed. A total of 582 differentially abundant proteins (DAPs) in SBPH with a fold change >1.500 or <0.667 (p-value < 0.05) were identified. The proteomic analysis in SBPH at 3 days padp revealed 106 highly abundant proteins and 193 of low abundance, while 5 days padp revealed 214 highly abundant proteins and 182 of low abundance. Among them, 51 highly abundant proteins and 42 of low abundance were shown consistently at both 3 days and 5 days padp. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis mapping and Gene Ontology (GO) term classification suggested impairment of mitochondria in SBPH after RBSDV acquisition, and the 77 out of 582 differentially abundant SBPH proteins analyzed by the STRING program revealed the interaction network of the mitochondrial DAPs, showing an overall down-regulation of mitochondrial proteins including the electron transport chain proteins and mitochondrial ribosome proteins. The high abundance of Parkin at 5 days padp suggests that activation of mitophagy induced degradation of mitochondria occurred. Further verification of autophagy/mitophagy-related genes by reverse-transcription quantitative RT-PCR (RT-qPCR) in SBPH after RBSDV acquisition showed up-regulation of the autophagy receptors Optineurin (OPTN), Sequestosome-1 (SQSTM1, also known as p62) and Tax1-binding protein 1 (TAX1BP1) which targets ubiquitinated damaged mitochondria during mitophagy. The phosphorylation of the three autophagy receptors may be up-regulated through an increase of transcription level TRAF-associated NFκB activator (TANK)-binding kinase 1 (TBK1). As a result, an overall reduction in the abundance of mitochondrial proteins was observed and the selective autophagic degradation was up-regulated through increased transcription level of OPTN, p62/SQSTM1, TAX1BP1 and TBK1. Therefore, acquisition of RBSDV associated with up-regulated autophagy and selective mitochondrial degradation in SBPH suggest prevention of mitochondrial-mediated apoptosis and extension of the vector life span. BIOLOGICAL SIGNIFICANCE: RBSDV causes severe yield loss in rice plants. RBSDV is transmitted efficiently only through SBPH. It is important to understand how RBSDV infects SBPH in a persistent, circulative and propagative manner. However, there has been no study on the interaction between RBSDV and SBPH at the early acquisition stage using a proteomics approach. In this study, we combined iTRAQ technique and LC-MS/MS to analyze the vector proteomics at both the initial and latent infection stages after RBSDV acquisition and verified the results by RT-qPCR. Our results revealed that significantly low DAPs were involved in various pathways, including biosynthesis of secondary metabolites, ribosomes, carbon metabolism, biosynthesis of amino acids and TCA cycle. Further clustering of the DAPs revealed significant changes in SBPH mitochondria, including decreased proteins in mitochondrial ribosomes and electron transport chain complex I, II and V. On the other hand, there was a high abundance of Parkin, suggesting the occurrence of mitochondria damage and subsequent Parkin-mediated mitophagy for clearance of impaired mitochondria. Moreover, the decreased level of PMPCB in terms of gene expression and protein abundance suggested decreased PINK1 turnover, promoting Parkin/PINK1-mediated mitophagy. Further analysis on autophagy/mitophagy-related gene transcription level indicated up-regulation of OPTN, p62/SQSTM1, TAX1BP1 and TBK1, promoting selective autophagy in SBPH after RBSDV acquisition. These findings provided new insights into the effects of RBSDV on SBPH after early acquisition by selective degradation of mitochondria, especially on reprogramming of energy metabolism and decreased mitochondria biogenesis, to prevent apoptosis and prolong the life span of SBPH post virus acquisition.

Application of plant viruses as nano drug delivery systems.

Nano-sized drug delivery systems based on virus-derived platforms have promising delivery and targeting efficiencies. To date, much of our understanding of these systems is obtained from studies of animal viruses. Application of plant viruses for drug delivery is in the nascent stage, but it is becoming apparent that plant viral particles can be engineered to possess novel properties to meet the unique requirements of targeted drug delivery. Chemical functionalization of a plant viral particle surface can impart stealth properties to prolong in vivo circulation half-life and/or targeting capability to direct drug delivery to diseased tissues. The amino acid sequence of the viral coat protein can be genetically manipulated to yield protein cages of specific chemistry and morphology, while the conformation of the protein cage can be directed, via the external environment, to disassemble, then reassemble in vitro to exchange native viral genomic material with exogenous cargo. The purpose of this commentary is to evaluate current literature to assess the potential of nano-scale plant-virus-based drug delivery systems for the targeted delivery of chemotherapeutic agents.

A Conserved Carboxylesterase Inhibits Tobacco mosaic virus (TMV) Accumulation in Nicotiana benthamiana Plants.

A carboxylesterase (CXE) or carboxylic-ester hydrolase is an enzyme that catalyzes carboxylic ester and water into alcohol and carboxylate. In plants, CXEs have been implicated in defense, development, and secondary metabolism. We discovered a new CXE gene in Nicotiana benthamiana that is related to virus resistance. The transcriptional level of NbCXE expression was significantly increased after Tobacco mosaic virus (TMV) infection. Transient over-expression of NbCXE inhibited TMV accumulation in N. benthamiana plants. Conversely, when the NbCXE gene was silenced with a Tobacco rattle virus (TRV)-based gene silencing system, TMV RNA accumulation was increased in NbCXE-silenced plants after infection. NbCXE protein was shown to interact with TMV coat protein (CP) in vitro. Additionally, the expressions of host defense-related genes were increased in transient NbCXE-overexpressed plants but decreased in NbCXE silenced N. benthamiana plants. In summary, our study showed that NbCXE is a novel resistance-related gene involved in host defense responses against TMV infection.

Author Correction: Genome-wide transcriptomic analysis reveals correlation between higher WRKY61 expression and reduced symptom severity in Turnip crinkle virus infected Arabidopsis thaliana.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.