The Oncolytic Avian Reovirus p17 Protein Inhibits Invadopodia Formation in Murine Melanoma Cancer Cells by Suppressing the FAK/Src Pathway and the Formation of theTKs5/NCK1 Complex.

To explore whether the p17 protein of oncolytic avian reovirus (ARV) mediates cell migration and invadopodia formation, we applied several molecular biological approaches for studying the involved cellular factors and signal pathways. We found that ARV p17 activates the p53/phosphatase and tensin homolog (PTEN) pathway to suppress the focal adhesion kinase (FAK)/Src signaling and downstream signal molecules, thus inhibiting cell migration and the formation of invadopodia in murine melanoma cancer cell line (B16-F10). Importantly, p17-induced formation of invadopodia could be reversed in cells transfected with the mutant PTENC124A. p17 protein was found to significantly reduce the expression levels of tyrosine kinase substrate 5 (TKs5), Rab40b, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and matrix metalloproteinases (MMP9), suggesting that TKs5 and Rab40b were transcriptionally downregulated by p17. Furthermore, we found that p17 suppresses the formation of the TKs5/NCK1 complex. Coexpression of TKs5 and Rab40b in B16-F10 cancer cells reversed p17-modulated suppression of the formation of invadopodia. This work provides new insights into p17-modulated suppression of invadopodia formation by activating the p53/PTEN pathway, suppressing the FAK/Src pathway, and inhibiting the formation of the TKs5/NCK1 complex.

Thriving under Salinity: Growth, Ecophysiology and Proteomic Insights into the Tolerance Mechanisms of Obligate Halophyte Suaeda fruticosa.

Studies on obligate halophytes combining eco-physiological techniques and proteomic analysis are crucial for understanding salinity tolerance mechanisms but are limited. We thus examined growth, water relations, ion homeostasis, photosynthesis, oxidative stress mitigation and proteomic responses of an obligate halophyte Suaeda fruticosa to increasing salinity under semi-hydroponic culture. Most biomass parameters increased under moderate (300 mmol L-1 of NaCl) salinity, while high (900 mmol L-1 of NaCl) salinity caused some reduction in biomass parameters. Under moderate salinity, plants showed effective osmotic adjustment with concomitant accumulation of Na+ in both roots and leaves. Accumulation of Na+ did not accompany nutrient deficiency, damage to photosynthetic machinery and oxidative damage in plants treated with 300 mmol L-1 of NaCl. Under high salinity, plants showed further decline in sap osmotic potential with higher Na+ accumulation that did not coincide with a decline in relative water content, Fv/Fm, and oxidative damage markers (H2O2 and MDA). There were 22, 54 and 7 proteins in optimal salinity and 29, 46 and 8 proteins in high salinity treatment that were up-regulated, down-regulated or exhibited no change, respectively, as compared to control plants. These data indicate that biomass reduction in S. fruticosa at high salinity might result primarily from increased energetic cost rather than ionic toxicity.

Nucleocytoplasmic shuttling of BEFV M protein-modulated by lamin A/C and chromosome maintenance region 1 through a transcription-, carrier- and energy-dependent pathway.

This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) 6KKGKSK11 and nuclear export signal (NES) 98LIITSYL TI106 of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.

Halophilic Plant-Associated Bacteria with Plant-Growth-Promoting Potential.

The salinization of soils is a growing agricultural concern worldwide. Irrigation practices, drought, and climate change are leading to elevated salinity levels in many regions, resulting in reduced crop yields. However, there is potential for a solution in the microbiome of halophytes, which are naturally salt-tolerant plants. These plants harbor a salt-tolerant microbiome in their rhizosphere (around roots) and endosphere (within plant tissue). These bacteria may play a significant role in conferring salt tolerance to the host plants. This leads to the possibility of transferring these beneficial bacteria, known as salt-tolerant plant-growth-promoting bacteria (ST-PGPB), to salt-sensitive plants, enabling them to grow in salt-affected areas to improve crop productivity. In this review, the background of salt-tolerant microbiomes is discussed and their potential use as ST-PGPB inocula is explored. We focus on two Gram-negative bacterial genera, Halomonas and Kushneria, which are commonly found in highly saline environments. These genera have been found to be associated with some halophytes, suggesting their potential for facilitating ST-PGPB activity. The study of salt-tolerant microbiomes and their use as PGPB holds promise for addressing the challenges posed by soil salinity in the context of efforts to improve crop growth in salt-affected areas.

Salt-Induced Modulation of Ion Transport and PSII Photoprotection Determine the Salinity Tolerance of Amphidiploid Brassicas.

Brassica species show varying levels of resistance to salt stress. To understand the genetics underlying these differential stress tolerance patterns in Brassicas, we exposed two widely cultivated amphidiploid Brassica species having different genomes, Brassica juncea (AABB, n = 18) and Brassica napus (AACC, n = 19), to elevated levels of NaCl concentration (300 mM, half the salinity of seawater). B. juncea produced more biomass, an increased chlorophyll content, and fewer accumulated sodium (Na+) and chloride (Cl-) ions in its photosynthesizing tissues. Chlorophyll fluorescence assays revealed that the reaction centers of PSII of B. juncea were more photoprotected and hence more active than those of B. napus under NaCl stress, which, in turn, resulted in a better PSII quantum efficiency, better utilization of photochemical energy with significantly reduced energy loss, and higher electron transport rates, even under stressful conditions. The expression of key genes responsible for salt tolerance (NHX1 and AVP1, which are nuclear-encoded) and photosynthesis (psbA, psaA, petB, and rbcL, which are chloroplast-encoded) were monitored for their genetic differences underlying stress tolerance. Under NaCl stress, the expression of NHX1, D1, and Rubisco increased several folds in B. juncea plants compared to B. napus, highlighting differences in genetics between these two Brassicas. The higher photosynthetic potential under stress suggests that B. juncea is a promising candidate for genetic modifications and its cultivation on marginal lands.

Cell Entry of Avian Reovirus Modulated by Cell-Surface Annexin A2 and Adhesion G Protein-Coupled Receptor Latrophilin-2 Triggers Src and p38 MAPK Signaling Enhancing Caveolin-1- and Dynamin 2-Dependent Endocytosis.

The specifics of cell receptor-modulated avian reovirus (ARV) entry remain unknown. By using a viral overlay protein-binding assay (VOPBA) and an in-gel digestion coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we determined that cell-surface annexin A2 (AnxA2) and adhesion G protein-coupled receptor Latrophilin-2 (ADGRL2) modulate ARV entry. Direct interaction between the ARV σC protein and AnxA2 and ADGRL2 in Vero and DF-1 cells was demonstrated in situ by proximity ligation assays. By using short hairpin RNAs (shRNAs) to silence the endogenous AnxA2 and ADGRL2 genes, ARV entry could be efficiently blocked. A significant decrease in virus yields and the intracellular specific signal for σC protein was observed in Vero cells preincubated with the specific AnxA2 and ADGRL2 monoclonal antibodies, indicating that AnxA2 and ADGRL2 are involved in modulating ARV entry. Furthermore, we found that cells pretreated with the AnxA2/S100A10 heterotetramer (A2t) inhibitor A2ti-1 suppressed ARV-mediated activation of Src and p38 mitogen-activated protein kinase (MAPK), demonstrating that Src and p38 MAPK serve as downstream molecules of cell-surface AnxA2 signaling. Our results reveal that suppression of cell-surface AnxA2 with the A2ti-1 inhibitor increased Csk-Cbp interaction, suggesting that ARV entry suppresses Cbp-mediated relocation of Csk to the membrane, thereby activating Src. Furthermore, reciprocal coimmunoprecipitation assays revealed that σC can interact with signaling molecules, lipid raft, and vimentin. The current study provides novel insights into cell-surface AnxA2- and ADGRL2-modulated cell entry of ARV which triggers Src and p38 MAPK signaling to enhance caveolin-1-, dynamin 2-, and lipid raft-dependent endocytosis. IMPORTANCE By analyzing results from VOPBA and LC-MS/MS, we have determined that cell-surface AnxA2 and ADGRL2 modulate ARV entry. After ARV binding to receptors, Src and p38 MAPK signaling were triggered and, in turn, increased the phosphorylation of caveolin-1 (Tyr14) and upregulated dynamin 2 expression to facilitate caveolin-1-mediated and dynamin 2-dependent endocytosis. In this work, we demonstrated that ARV triggers Src activation by impeding Cbp-mediated relocation of Csk to the membrane in the early stages of the life cycle. This work provides better insight into cell-surface AnxA2 and ADGRL2, which upregulate Src and p38MAPK signaling pathways to enhance ARV entry and productive infection.

Oncolytic Avian Reovirus p17-Modulated Inhibition of mTORC1 by Enhancement of Endogenous mTORC1 Inhibitors Binding to mTORC1 To Disrupt Its Assembly and Accumulation on Lysosomes.

The mechanism by which avian reovirus (ARV)-modulated suppression of mTORC1 triggers autophagy remains largely unknown. In this work, we determined that p17 functions as a negative regulator of mTORC1. This study suggest novel mechanisms whereby p17-modulated inhibition of mTORC1 occurs via upregulation of p53, inactivation of Akt, and enhancement of binding of the endogenous mTORC1 inhibitors (PRAS40, FKBP38, and FKPP12) to mTORC1 to disrupt its assembly and accumulation on lysosomes. p17-modulated inhibition of Akt leads to activation of the downstream targets PRAS40 and TSC2, which results in mTORC1 inhibition, thereby triggering autophagy and translation shutoff, which is favorable for virus replication. p17 impairs the interaction of mTORC1 with its activator Rheb, which promotes FKBP38 interaction with mTORC1. It is worth noting that p17 activates ULK1 and Beclin1 and increases the formation of the Beclin 1/class III PI3K complex. These effects could be reversed in the presence of insulin or depletion of p53. Furthermore, we found that p17 induces autophagy in cancer cell lines by upregulating the p53/PTEN pathway, which inactivates Akt and mTORC1. This study highlights p17-modulated inhibition of Akt and mTORC1, which triggers autophagy and translation shutoff by positively modulating the tumor suppressors p53 and TSC2 and endogenous mTORC1 inhibitors. IMPORTANCE The mechanisms by which p17-modulated inhibition of mTORC1 induces autophagy and translation shutoff is elucidated. In this work, we determined that p17 serves as a negative regulator of mTORC1. This study provides several lines of conclusive evidence demonstrating that p17-modulated inhibition of mTORC1 occurs via upregulation of the p53/PTEN pathway, downregulation of the Akt/Rheb/mTORC1 pathway, enhancement of binding of the endogenous mTORC1 inhibitors to mTORC1 to disrupt its assembly, and suppression of mTORC1 accumulation on lysosomes. This work provides valuable information for better insights into p17-modulated inhibition of mTORC1, which induces autophagy and translation shutoff to benefit virus replication.

Oncolytic avian reovirus σA-modulated fatty acid metabolism through the PSMB6/Akt/SREBP1/acetyl-CoA carboxylase pathway to increase energy production for virus replication.

We have demonstrated previously that the σA protein of avian reovirus (ARV) functions as an activator of cellular energy, which upregulates glycolysis and the TCA cycle for virus replication. To date, there is no report with respect to σA-modulated regulation of cellular fatty acid metabolism. This study reveals that the σA protein of ARV inhibits fatty acids synthesis and enhance fatty acid oxidation by upregulating PSMB6, which suppresses Akt, sterol regulatory element-binding protein 1 (SREBP1), acetyl-coA carboxylase α (ACC1), and acetyl-coA carboxylase ß (ACC2). SREBP1 is a transcription factor involved in fatty acid and cholesterol biosynthesis. Overexpression of SREBP1 reversed σA-modulated suppression of ACC1 and ACC2. In this work, a fluorescence resonance energy transfer-based genetically encoded indicator, Ateams, was used to study σA-modulated inhibition of fatty acids synthesis which enhances cellular ATP levels in Vero cells and human cancer cell lines (A549 and HeLa). By using Ateams, we demonstrated that σA-modulated inhibition of Akt, SREBP1, ACC1, and ACC2 leads to increased levels of ATP in mammalian and human cancer cells. Furthermore, knockdown of PSMB6 or overexpression of SREBP1 reversed σA-modulated increased levels of ATP in cells, indicating that PSMB6 and SREBP1 play important roles in ARV σA-modulated cellular fatty acid metabolism. Furthermore, we found that σA R155/273A mutant protein loses its ability to enter the nucleolus, which impairs its ability to regulate fatty acid metabolism and does not increase ATP formation, suggesting that nucleolus entry of σA is critical for regulating cellular fatty acid metabolism to generate more energy for virus replication. Collectively, this study provides novel insights into σA-modulated inhibition of fatty acid synthesis and enhancement of fatty acid oxidation to produce more energy for virus replication through the PSMB6/Akt/SREBP1/ACC pathway.

Impact of Nanomaterials on the Regulation of Gene Expression and Metabolomics of Plants under Salt Stress.

Plant salinity resistance results from a combination of responses at the physiological, molecular, cellular, and metabolic levels. This article focuses on plant stress tolerance mechanisms for controlling ion homeostasis, stress signaling, hormone metabolism, anti-oxidative enzymes, and osmotic balance after nanoparticle applications. Nanoparticles are used as an emerging tool to stimulate specific biochemical reactions related to plant ecophysiological output because of their small size, increased surface area and absorption rate, efficient catalysis of reactions, and adequate reactive sites. Regulated ecophysiological control in saline environments could play a crucial role in plant growth promotion and survival of plants under suboptimal conditions. Plant biologists are seeking to develop a broad profile of genes and proteins that contribute to plant salt resistance. These plant metabolic profiles can be developed due to advancements in genomic, proteomic, metabolomic, and transcriptomic techniques. In order to quantify plant stress responses, transmembrane ion transport, sensors and receptors in signaling transduction, and metabolites involved in the energy supply require thorough study. In addition, more research is needed on the plant salinity stress response based on molecular interactions in response to nanoparticle treatment. The application of nanoparticles as an aspect of genetic engineering for the generation of salt-tolerant plants is a promising area of research. This review article addresses the use of nanoparticles in plant breeding and genetic engineering techniques to develop salt-tolerant crops.

p17-Modulated Hsp90/Cdc37 Complex Governs Oncolytic Avian Reovirus Replication by Chaperoning p17, Which Promotes Viral Protein Synthesis and Accumulation of Viral Proteins σC and σA in Viral Factories.

In this work we have determined that heat shock protein 90 (Hsp90) is essential for avian reovirus (ARV) replication by chaperoning the ARV p17 protein. p17 modulates the formation of the Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation. Inhibition of the Hsp90/Cdc37 complex by inhibitors (17-N-allylamino-17-demethoxygeldanamycin 17-AGG, and celastrol) or short hairpin RNAs (shRNAs) significantly reduced expression levels of viral proteins and virus yield, suggesting that the Hsp90/Cdc37 chaperone complex functions in virus replication. The expression levels of p17 were decreased at the examined time points (2 to 7 h and 7 to 16 h) in 17-AAG-treated cells in a dose-dependent manner while the expression levels of viral proteins σA, σC, and σNS were decreased at the examined time point (7 to 16 h). Interestingly, the expression levels of σC, σA, and σNS proteins increased along with coexpression of p17 protein. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability, maturation, and virus production. Virus factories of ARV are composed of nonstructural proteins σNS and µNS. We found that the Hsp90/Cdc37 chaperone complex plays an important role in accumulation of the outer-capsid protein σC, inner core protein σA, and nonstructural protein σNS of ARV in viral factories. Depletion of Hsp90 inhibited σA, σC, and p17 proteins colocalized with σNS in viral factories. This study provides novel insights into p17-modulated formation of the Hsp90/Cdc37 chaperone complex governing virus replication via stabilization and maturation of viral proteins and accumulation of viral proteins in viral factories for virus assembly. IMPORTANCE Molecular mechanisms that control stabilization of ARV proteins and the intermolecular interactions among inclusion components remain largely unknown. Here, we show that the ARV p17 is an Hsp90 client protein. The Hsp90/Cdc37 chaperone complex is essential for ARV replication by protecting p17 chaperone from ubiquitin-proteasome degradation. p17 modulates the formation of Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation, suggesting a feedback loop between p17 and the Hsp90/Cdc37 chaperone complex. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability and virus production. Depletion of Hsp90 prevented viral proteins σA, σC, and p17 from colocalizing with σNS in viral factories. Our findings elucidate that the Hsp90/Cdc37 complex chaperones p17, which, in turn, promotes the synthesis of viral proteins σA, σC, and σNS and facilitates accumulation of the outer-capsid protein σC and inner core protein σA in viral factories for virus assembly.

Molecular chaperone TRiC governs avian reovirus replication by protecting outer-capsid protein σC and inner core protein σA and non-structural protein σNS from ubiquitin- proteasome degradation.

Avian reoviruses (ARVs) are important pathogens that cause considerable economic losses in poultry farming. To date, host factors that control stabilization of ARV proteins remain largely unknown. In this work we determined that the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC) is essential for avian reovirus (ARV) replication by stabilizing outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV. TriC serves as a chaperone of viral proteins and prevent their degradation via the ubiquitin-proteasome pathway. Furthermore, reciprocal co-immunoprecipitation assays confirmed the association of viral proteins (σA, σC, and σNS) with TRiC. Immunofluorescence staining indicated that the TRiC chaperonins (CCT2 and CCT5) are colocalized with viral proteins σC, σA, and σNS of ARV. In this study, inhibition of TRiC chaperonins (CCT2 and CCT5) by the inhibitor HSF1A or shRNAs significantly reduced expression levels of the σC, σA, and σNS proteins of ARV as well as virus yield, suggesting that the TRiC complex functions in stabilization of viral proteins and virus replication. This study provides novel insights into TRiC chaperonin governing virus replication via stabilization of outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV.

Editing the plastid genome of recalcitrant plant species.

Transformation of the chloroplast genome offers key advantages over traditional methods for generating transgenic plants, but this approach is limited to a few plant species. Nakazato et al. have developed a novel technique that will help to extend the technology to other plant species that are recalcitrant to current tissue culture-based chloroplast transformation protocols.

Effects of Salinity Stress on Chloroplast Structure and Function.

Salinity is a growing problem affecting soils and agriculture in many parts of the world. The presence of salt in plant cells disrupts many basic metabolic processes, contributing to severe negative effects on plant development and growth. This review focuses on the effects of salinity on chloroplasts, including the structures and function of these organelles. Chloroplasts house various important biochemical reactions, including photosynthesis, most of which are considered essential for plant survival. Salinity can affect these reactions in a number of ways, for example, by changing the chloroplast size, number, lamellar organization, lipid and starch accumulation, and interfering with cross-membrane transportation. Research has shown that maintenance of the normal chloroplast physiology is necessary for the survival of the entire plant. Many plant species have evolved different mechanisms to withstand the harmful effects of salt-induced toxicity on their chloroplasts and its machinery. The differences depend on the plant species and growth stage and can be quite different between salt-sensitive (glycophyte) and salt-tolerant (halophyte) plants. Salt stress tolerance is a complex trait, and many aspects of salt tolerance in plants are not entirely clear yet. In this review, we discuss the different mechanisms of salt stress tolerance in plants with a special focus on chloroplast structure and its functions, including the underlying differences between glycophytes and halophytes.

Analysis of Gene Expression Changes in Plants Grown in Salty Soil in Response to Inoculation with Halophilic Bacteria.

Soil salinity is an increasing problem facing agriculture in many parts of the world. Climate change and irrigation practices have led to decreased yields of some farmland due to increased salt levels in the soil. Plants that have tolerance to salt are thus needed to feed the world's population. One approach addressing this problem is genetic engineering to introduce genes encoding salinity, but this approach has limitations. Another fairly new approach is the isolation and development of salt-tolerant (halophilic) plant-associated bacteria. These bacteria are used as inoculants to stimulate plant growth. Several reports are now available, demonstrating how the use of halophilic inoculants enhance plant growth in salty soil. However, the mechanisms for this growth stimulation are as yet not clear. Enhanced growth in response to bacterial inoculation is expected to be associated with changes in plant gene expression. In this review, we discuss the current literature and approaches for analyzing altered plant gene expression in response to inoculation with halophilic bacteria. Additionally, challenges and limitations to current approaches are analyzed. A further understanding of the molecular mechanisms involved in enhanced plant growth when inoculated with salt-tolerant bacteria will significantly improve agriculture in areas affected by saline soils.

Plastid Transcriptomics: An Important Tool For Plastid Functional Genomics.

Plastids in higher plants carry out specialized roles such as photosynthesis, nitrogen assimilation, biosynthesis of amino acids, fatty acids, isoprenoids, and various metabolites. Plastids arise from undifferentiated precursors known as proplastids, which are found in the root and shoot meristems. They are highly dynamic as they change their number, morphology, and physiology according to the tissue they are present. In addition to housing various metabolic activities, plastids also serve as a global sensor for both internal and external environmental cues including different stresses, and help plants to respond/adjust accordingly. They relay information to the nucleus, which then responds by changing the expression levels of specific genes. It has been shown that plants with impaired plastid functions exhibit abnormalities. One of the sources emanating these signals to the nucleus is plastid transcription. Normal plastid functioning is therefore critical for plant survival. Despite immense significance for plant acclimation, the plastid transcriptome is largely an unstudied research area. In this review, we discuss the importance of plastid transcriptomics for the acclimation of plants under changing environmental conditions and summarize the key literature published in this field.

Aspirin and 5-Aminoimidazole-4-carboxamide Riboside Attenuate Bovine Ephemeral Fever Virus Replication by Inhibiting BEFV-Induced Autophagy.

Recent study in our laboratory has demonstrated that BEFV-induced autophagy via activation of the PI3K/Akt/NF-κB and Src/JNK pathways and suppression of the PI3K-AKt-mTORC1 pathway is beneficial for virus replication. In the current study, we found that both aspirin and 5-aminoimidazole-4-carboxamide-1-ß-riboside (AICAR) siginificantly attenuated virus replication by inhibiting BEFV-induced autophagy via suppressing the BEFV-activated PI3K/Akt/NF-κB and Src/JNK pathways as well as inducing reversion of the BEFV-suppressed PI3K-Akt-mTORC1 pathway. AICAR reversed the BEFV-activated PI3K/Akt/NF-κB and Src/JNK pathways at the early to late stages of infection and induced reversion of the BEFV-suppressed PI3K-AKt-mTORC1 pathway at the late stage of infection. Our findings reveal that inhibition of BEFV-induced autophagy by AICAR is independent of AMPK. Furthermore, we found that AICAR transcriptionally downregulates the ATG related genes ULK1, Beclin 1, and LC3 and enhances Atg7 degradation by the proteasome pathway. Aspirin suppresses virus replication by inhibiting BEFV-induced autophagy. It directly suppressed the NF-κB pathway and reversed the BEFV-activated Src/JNK pathway at the early stage of infection and reversed the BEFV-suppressed PI3K/Akt/mTOR pathway at the late stage of infection. The current study provides mechanistic insights into the effects of aspirin and AICAR on BEFV replication through suppression of BEFV-induced autophagy.

Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine.

To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 107 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (107) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4+ T cells and CD4+/CD8+ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.

Plant Organelle DNA Maintenance.

Plant cells contain two double membrane bound organelles, plastids and mitochondria, that contain their own genomes. There is a very large variation in the sizes of mitochondrial genomes in higher plants, while the plastid genome remains relatively uniform across different species. One of the curious features of the organelle DNA is that it exists in a high copy number per mitochondria or chloroplast, which varies greatly in different tissues during plant development. The variations in copy number, morphology and genomic content reflect the diversity in organelle functions. The link between the metabolic needs of a cell and the capacity of mitochondria and chloroplasts to fulfill this demand is thought to act as a selective force on the number of organelles and genome copies per organelle. However, it is not yet clear how the activities of mitochondria and chloroplasts are coordinated in response to cellular and environmental cues. The relationship between genome copy number variation and the mechanism(s) by which the genomes are maintained through different developmental stages are yet to be fully understood. This Special Issue has several contributions that address current knowledge of higher plant organelle DNA. Here we briefly introduce these articles that discuss the importance of different aspects of the organelle genome in higher plants.