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Elucidating regulators and molecular mechanisms underlying gene transcriptional and post-transcriptional co-regulatory network is key to understand plant cold-stress responses. Previous studies were mainly conducted on single species and whether the regulators and mechanisms are conserved across different species remains elusive. Here, we selected three species that diverged at early evolution of rosids (93–115 million years ago) and integrated phylotranscriptome and ChIP/DAP-seq datasets to identify cold-responsive regulators and their regulatory networks. First, we found over ten thousand cold-responsive genes including differentially expressed genes (DEGs) and alternative splicing genes (DASGs) in each species. Among the DEGs, genes encoding a set of transcription factors (TFs) (AP2/ERF, MYB, WRKY, NAC, etc.) and RNA binding proteins (RBPs) (Ribosomal, RRM, DEAD, Helicase_C, etc.) are conserved in cold responses in rosids. Compared to TFs, RBPs show a delayed cold-responsive pattern, likely suggesting a hierarchical regulation of DEGs and DASGs. Between DEGs and DASGs, we identified 259 overlapping DE-DASG orthogroups and interestingly, pathway analysis on each dataset of DEGs, DASGs, and DE-DASGs coincidently shows an enrichment of circadian rhythm. Evidentially, many circadian components are cold-regulated at both transcriptional and post-transcriptional levels. Moreover, we reasoned 226 cold-responsive genes regulated by at least two of five circadian components (CCA1, LHY, RV4, RVE8, and RVE7) in rosids. Finally, we unveiled a conserved hierarchical network in dynamic transcriptional and post-transcriptional regulation of cold-responsive genes launched by circadian components in rosids. Together, our results provide insights into core regulators and mechanisms underlying cold-responsive regulatory network across rosids, despite a long evolutionary history.

Post-stroke depression (PSD) is the most common emotional problem following a stroke. White matter hyperintensities (WMHs) are often reported in patients with a stroke, and are often divided into deep WMHs (DWMHs) and periventricular WMHs (PVWMHs). The relationship between WMHs and PSD remains controversial. This review aims to resolve this controversy.A systematic search of electronic databases was conducted for studies. We extracted the relevant data and evaluated the study quality by using the Newcastle-Ottawa Scale. We pooled odds ratios (OR) for the same type of WMHs that were present in the relevant PSD period.15 studies (n = 4133 patients) met our inclusion criteria. In the acute phase, WMHs, DWMHs, severe WMHs, and severe DWMHs were not significant risk factors for incident depression, but PVWMHs (pooled OR, 1.21; 95 % CI, 1.01-1.44) and severe PVWMHs (pooled OR, 1.72; 95 % CI, 1.12-2.65) had a significant association with PSD. In the subacute phase, DWMHs, DWMHs, and severe WMHs were not significantly associated with PSD, but PVWMHs (pooled OR, 2.44; 95 % CI, 1.25-4.76) showed a significant association with PSD. In the chronic phase, severe PVWMHs had no significant association with PSD, while WMHs (pooled OR, 1.063; 95 % CI, 1.03-1.09), DWMHs (pooled OR, 1.40; 95 % CI, 1.11-1.76), PVWMHs (pooled OR, 1.28; 95 % CI, 1.11-1.48), and severe DWMHs (pooled OR, 1.52; 95 % CI, 1.12-2.05) showed a significant association with PSD.We found a significant association between WMHs/DWMHs/PVWMHs and PSD in the chronic post-stroke phase. PVWMHs had a stronger correlation with PSD in each period after stroke than WMHs and DWMHs. High-quality prospective studies are still needed to fully resolve this relationship.

Through the investigation of Qinglong mining area and adjacent karst underground river system, mining activities and water-rock interactions are found to control the hydrogeochemical evolution of karst underground water. Along the flow direction of the karst underground river, the hydro-chemical type is converted from HCO

Although some nucleotide binding, leucine-rich repeat immune receptor (NLR) proteins conferring resistance to specific viruses have been identified in dicot plants, NLR proteins involved in viral resistance have not been described in monocots. We have used map-based cloning to isolate the CC-NB-LRR (CNL) Barley stripe mosaic virus (BSMV) resistance gene barley stripe resistance 1 (BSR1) from Brachypodium distachyon Bd3-1 inbred line. Stable BSR1 transgenic Brachypodium line Bd21-3, barley (Golden Promise) and wheat (Kenong 199) plants developed resistance against BSMV ND18 strain. Allelic variation analyses indicated that BSR1 is present in several Brachypodium accessions collected from countries in the Middle East. Protein domain swaps revealed that the intact LRR domain and the C-terminus of BSR1 are required for resistance. BSR1 interacts with the BSMV ND18 TGB1 protein in planta and shows temperature-sensitive antiviral resistance. The R

Upon stress challenges, proteins/RNAs undergo liquid–liquid phase separation (LLPS) to fine-tune cell physiology and metabolism to help cells adapt to adverse environments. The formation of LLPS has been recently linked with intracellular pH, and maintaining proper intracellular pH homeostasis is known to be essential for the survival of organisms. However, organisms are constantly exposed to diverse stresses, which are accompanied by alterations in the intracellular pH. Aging processes and human diseases are also intimately linked with intracellular pH alterations. In this review, we summarize stress-, aging-, and cancer-associated pH changes together with the mechanisms by which cells regulate cytosolic pH homeostasis. How critical cell components undergo LLPS in response to pH alterations is also discussed, along with the functional roles of intracellular pH fluctuation in the regulation of LLPS. Further studies investigating the interplay of pH with other stressors in LLPS regulation and identifying protein responses to different pH levels will provide an in-depth understanding of the mechanisms underlying pH-driven LLPS in cell adaptation. Moreover, deciphering aging and disease-associated pH changes that influence LLPS condensate formation could lead to a deeper understanding of the functional roles of biomolecular condensates in aging and aging-related diseases.

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-AC ), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.

Salicylic acid (SA) acts as a signaling molecule to perceive and defend against pathogen infections. Accordingly, pathogens evolve versatile strategies to disrupt the SA-mediated signal transduction, and how plant viruses manipulate the SA-dependent defense responses requires further characterization. Here, we show that barley stripe mosaic virus (BSMV) infection activates the SA-mediated defense signaling pathway and upregulates the expression of Nicotiana benthamiana thioredoxin h-type 1 (NbTRXh1). The γb protein interacts directly with NbTRXh1 in vivo and in vitro. The overexpression of NbTRXh1, but not a reductase-defective mutant, impedes BSMV infection, whereas low NbTRXh1 expression level results in increased viral accumulation. Similar with its orthologs in Arabidopsis (Arabidopsis thaliana), NbTRXh1 also plays an essential role in SA signaling transduction in N. benthamiana. To counteract NbTRXh1-mediated defenses, the BSMV γb protein targets NbTRXh1 to dampen its reductase activity, thereby impairing downstream SA defense gene expression to optimize viral cell-to-cell movement. We also found that NbTRXh1-mediated resistance defends against lychnis ringspot virus, beet black scorch virus, and beet necrotic yellow vein virus. Taken together, our results reveal a role for the multifunctional γb protein in counteracting plant defense responses and an expanded broad-spectrum antibiotic role of the SA signaling pathway.

Salicylic acid (SA) acts as a signaling molecule to perceive and defend against pathogen infections. Accordingly, pathogens evolve versatile strategies to disrupt the SA-mediated signal transduction. However, it is necessary to further characterize how plant viruses manipulate the SA-dependent defense responses. Here, we show that Barley stripe mosaic virus (BSMV) infection activates SA-mediated defense signaling pathway and upregulates the expression of Nicotiana benthamiana thioredoxin h-type 1 (NbTRXh1). The γb protein interacts directly with NbTRXh1 in vivo and in vitro. Overexpression of NbTRXh1, but not a reductase-defective mutant, impedes BSMV infection, whereas low NbTRXh1 expression level results in increased viral accumulation. Similar with its orthologues in Arabidopsis, NbTRXh1 also plays an essential role in SA signaling transduction in N. benthamiana. To counteract NbTRXh1-mediated defenses, the BSMV γb protein targets NbTRXh1 to dampen its reductase activity and thereby impairing downstream SA defense genes expression to optimize viral cell-to-cell movement. We also found that NbTRXh1-mediated resistance defends against Lychnis ringspot virus, Beet black scorch virus, and Beet necrotic yellow vein virus. Taken together, our results reveal a novel role for the multifunctional γb protein in counteracting plant defense responses, and broadens the broad-spectrum antibiotic role of SA signaling pathway.One sentence summaryBSMV γb protein impairs NbTRXh1 reductase activity and dampen downstream SA-related genes expression to facilitate viral cell-to-cell movement.

The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH-dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC-mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2-Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC-mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.

Background Puccinellia tenuiflora is the most saline-alkali tolerant plant in the Songnen Plain, one of the three largest soda saline-alkali lands worldwide. Here, we investigated the physicochemical properties of saline-alkali soils from the Songnen Plain and sequenced the transcriptomes of germinated P. tenuiflora seedlings under long-term treatment (from seed soaking) with saline-alkali soil extracts. Results We found that the soils from Songnen Plain were reasonably rich in salts and alkali; moreover, the soils were severely deficient in nitrogen [N], phosphorus [P], potassium [K] and several other mineral elements. This finding demonstrated that P. tenuiflora can survive from not only high saline-alkali stress but also a lack of essential mineral elements. To explore the saline-alkali tolerance mechanism, transcriptional analyses of P. tenuiflora plants treated with water extracts from the saline-alkali soils was performed. Interestingly, unigenes involved in the uptake of N, P, K and the micronutrients were found to be significantly upregulated, which indicated the existence of an efficient nutrition-uptake system in P. tenuiflora. Compared with P. tenuiflora, the rice Oryza sativa was hypersensitive to saline-alkali stress. The results obtained using a noninvasive microtest techniques confirmed that the uptake of NO3- and NH4+ and the regulatory flux of Na+ and H+ were significantly higher in the roots of P. tenuiflora than in those of O. sativa. In the corresponding physiological experiments, the application of additional nutrition elements significantly eliminated the sensitive symptoms of rice to saline-alkali soil extracts. Conclusions Our results imply that the survival of P. tenuiflora in saline-alkali soils is due to a combination of at least two regulatory mechanisms and the high nutrient uptake capacity of P. tenuiflora plays a pivotal role in its adaptation to those stress. Taken together, our results highlight the role of nutrition uptake in saline-alkali stress tolerance in plants. Electronic supplementary material The online version of this article (10.1186/s12864-019-5860-5) contains supplementary material, which is available to authorized users.

Lithium hexafluorophosphate (LiPF6) is one of the leading electrolytes in lithium-ion batteries, and its usage has increased tremendously in the past few years. Little is known, however, about its potential environmental and biological impacts. In order to improve our understanding of the cytotoxicity of LiPF6 and the specific cellular response mechanisms to it, we performed a genome-wide screen using a yeast (Saccharomyces cerevisiae) deletion mutant collection and identified 75 gene deletion mutants that showed LiPF6 sensitivity. Among these, genes associated with mitochondria showed the most enrichment. We also found that LiPF6 is more toxic to yeast than lithium chloride (LiCl) or sodium hexafluorophosphate (NaPF6). Physiological analysis showed that a high concentration of LiPF6 caused mitochondrial damage, reactive oxygen species (ROS) accumulation, and ATP content changes. Compared with the results of previous genome-wide screening for LiCl-sensitive mutants, we found that oxidative phosphorylation-related mutants were specifically hypersensitive to LiPF6. In these deletion mutants, LiPF6 treatment resulted in higher ROS production and reduced ATP levels, suggesting that oxidative phosphorylation-related genes were important for counteracting LiPF6-induced toxicity. Taken together, our results identified genes specifically involved in LiPF6-modulated toxicity, and demonstrated that oxidative stress and ATP imbalance maybe the driving factors in governing LiPF6-induced toxicity.

Yeast (Saccharomyces cerevisiae) has been used as one of the main model systems for studying molecular mechanisms underlying cellular aging. A major technical challenge in studying aging in yeast is the isolation of aged cells from exponentially growing cell cultures, since aged cells in such cultures are rare. Several methods for isolating aged cells have been developed to achieve this. Here, we describe a biotin-streptavidin affinity purification protocol for isolating aged yeast cells. It consists of three main steps: biotinylation of yeast cells, culturing cells to the desired age, and harvesting the aged cells using streptavidin-coated magnetic microbeads. The isolated aged cells can be used for microscopy, biochemistry, or molecular biology analysis.

Phenomic studies can provide a systemic overview of the network of interactions between phenotypes, genotypes, and environmental factors. Yeast (Saccharomyces cerevisiae) is one of the most important model organisms for phenomic studies due to the availability of a large variety of genome-wide strain collections. We describe a detailed protocol for performing a yeast phenomic screen for evaluation of protein colocalization via a genome-wide imaging-based screening approach utilizing a GFP-tagged yeast strain collection.

Phenomic studies can provide a systemic overview of the network of interactions between phenotypes, genotypes, and environmental factors. Yeast (Saccharomyces cerevisiae) is one of the most important model organisms for phenomic studies due to the availability of a large variety of genome-wide strain collections. We describe a detailed protocol for performing a yeast phenomic screen for evaluation of protein colocalization via a genome-wide imaging-based screening approach utilizing a GFP-tagged yeast strain collection.

Yeast (Saccharomyces cerevisiae) has been used as one of the main model systems for studying molecular mechanisms underlying cellular aging. A major technical challenge in studying aging in yeast is the isolation of aged cells from exponentially growing cell cultures, since aged cells in such cultures are rare. Several methods for isolating aged cells have been developed to achieve this. Here, we describe a biotin-streptavidin affinity purification protocol for isolating aged yeast cells. It consists of three main steps: biotinylation of yeast cells, culturing cells to the desired age, and harvesting the aged cells using streptavidin-coated magnetic microbeads. The isolated aged cells can be used for microscopy, biochemistry, or molecular biology analysis.

Nine genera of viruses in five different families use triple gene block (TGB) proteins for virus movement. The TGB modules fall into two classes: hordei-like and potex-like. Although TGB-mediated viral movement has been extensively studied, determination of the constituents of the viral ribonucleoprotein (vRNP) movement complexes and the mechanisms underlying their involvement in vRNP-mediated movement are far from complete. In the current study, immunoprecipitation of TGB1 protein complexes formed during Barley stripe mosaic virus (BSMV) infection revealed the presence of the γb protein in the products. Further experiments demonstrated that TGB1 interacts with γb in vitro and in vivo, and that γb-TGB1 localizes at the periphery of chloroplasts and plasmodesmata (PD). Subcellular localization analyses of the γb protein in Nicotiana benthamiana epidermal cells indicated that in addition to chloroplast localization, γb also targets the ER, actin filaments and PD at different stages of viral infection. By tracking γb localization during BSMV infection, we demonstrated that γb is required for efficient cell-to-cell movement. The N-terminus of γb interacts with the TGB1 ATPase/helicase domain and enhances ATPase activity of the domain. Inactivation of the TGB1 ATPase activity also significantly impaired PD targeting. In vitro translation together with co-immunoprecipitation (co-IP) analyses revealed that TGB1-TGB3-TGB2 complex formation is enhanced by ATP hydrolysis. The γb protein positively regulates complex formation in the presence of ATP, suggesting that γb has a novel role in BSMV cell-to-cell movement by directly promoting TGB1 ATPase-mediated vRNP movement complex assembly. We further demonstrated that elimination of ATPase activity abrogates PD and actin targeting of Potato virus X (PVX) and Beet necrotic yellow vein virus (BNYVV) TGB1 proteins. These results expand our understanding of the multifunctional roles of γb and provide new insight into the functions of TGB1 ATPase domains in the movement of TGB-encoding viruses., Author summary Plant viruses employ varied movement strategies to mediate local and systemic infections. Viral ribonucleoprotein (vRNP) movement complexes comprising either the hordei- or potex-like triple gene block (TGB) and viral RNAs represent important models for plant virus movement. However, the constituents of viral ribonucleoprotein (vRNP) movement complexes as well as their biological significance in vRNP assembly and subsequent interactions are far from complete. Additionally, the mechanistic roles of the highly conserved TGB1 ATPase domain in vRNP-mediated movement remain an enigma. Here, we demonstrate that the γb protein acts as a novel positive regulator of BSMV cell-to-cell movement by directly interacting with the TGB1 protein. In vitro biochemical assays verified an essential role of TGB1 ATPase-mediated ATP hydrolysis in assembly of vRNP movement complexes, a process that can be further enhanced by the γb protein. We also extend our studies of BSMV TGB1 ATPase to those of PVX and BNYVV, and suggest a model for an evolutionally conserved mode of energy-coupled vRNP movement complex assembly among different TGB-encoding viruses. Our results address the knowledge gap between TGB1 ATPase activity and vRNP movement complex assembly and expand our understanding of the multifaceted roles of γb in BSMV infection.

Stress granules (SGs) are nonmembrane assemblies formed in cells in response to stress conditions. SGs mainly contain untranslated mRNA and a variety of proteins. RNAs and scaffold proteins with intrinsically disordered regions or RNA‐binding domains are essential for the assembly of SGs, and multivalent macromolecular interactions among these components are thought to be the driving forces for SG assembly. The SG assembly process includes regulation through post‐translational modification and involvement of the cytoskeletal system. During aging, many intracellular bioprocesses become disrupted by factors such as cellular environmental changes, mitochondrial dysfunction, and decline in the protein quality control system. Such changes could lead to the formation of aberrant SGs, as well as alterations in their maintenance, disassembly, and clearance. These aberrant SGs might in turn promote aging and aging‐associated diseases. In this paper, we first review the latest progress on the molecular mechanisms underlying SG assembly and SG functioning under stress conditions. Then, we provide a detailed discussion of the relevance of SGs to aging and aging‐associated diseases., Stress granules are sensitive to environmental changes. Age‐dependent cellular changes and breakdown of mitochondrial function and protein quality control system would lead to defects in maintaining normal stress granule assembly, dynamics, disassembly, and clearance. This in turn could promote aging and aging‐associated diseases. In this paper, we provide a detailed discussion of the relevance of stress granules to aging and aging‐associated diseases. And it seems that maintaining a proper stress granule dynamic might be a potential strategy to delay aging and increase lifespan.

The Barley stripe mosaic virus (BSMV) γb protein is a viral suppressor of RNA silencing (VSR) and symptom determinant. However, it is unclear how post-translational modification affects the different functions of γb. Here, we demonstrate that γb is phosphorylated at Ser-96 by a PKA-like kinase in vivo and in vitro. Mutant viruses containing a nonphosphorylatable substitution (BSMVS96A or BSMVS96R ) exhibited reduced viral accumulation in Nicotiana benthamiana due to transient induction of the cell death response that constrained the virus to necrotic areas. By contrast, a BSMVS96D mutant virus that mimics γb phosphorylation spread similarly to the wild-type virus. Furthermore, the S96A mutant had reduced local and systemic γb VSR activity due to having compromised its binding activity to 21-bp dsRNA. However, overexpression of other VSRs in trans or in cis failed to rescue the necrosis induced by BSMVS96A , demonstrating that suppression of cell death by γb phosphorylation is functionally distinct from its RNA silencing suppressor activities. These results provide new insights into the function of γb phosphorylation in regulating RNA silencing and the BSMV-induced host cell death response, and contribute to our understanding of how the virus optimizes the balance between viral replication and virus survival in the host plants during virus infection.

Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA-binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear-cytoplasmic trafficking is required for BSMV cell-to-cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95-104) and a nuclear localization signal (NLS) (amino acids 227-238). NoLS mutations reduced BSMV cell-to-cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine-arginine-rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV-infected cells, Fib2 accumulation increased by about 60%-70% and co-localized with TGB1 in the plasmodesmata. In addition, BSMV cell-to-cell movement in fib2 knockdown transgenic plants was reduced to less than one-third of that of non-transgenic plants. Fib2 also co-localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1-Fib2 interactions play a direct role in cell-to-cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell-to-cell movement.

Chloroplasts are multifunctional organelles whose morphology is affected by environmental stresses. Although the three-dimensional (3D) architecture of thylakoid membranes has been reported previously, a 3D visualization of chloroplast under stress has not been explored. In this work, we used a positive-strand RNA ((+)RNA) virus, barley stripe mosaic virus (BSMV) to observe chloroplast structural changes during infection by electron tomography. The analyses revealed remodeling of the chloroplast membranes, characterized by the clustering of outer membrane-invaginated spherules in inner membrane-derived packets. Diverse morphologies of cytoplasmic invaginations (CIs) were evident with spherules at the periphery and different sized openings connecting the CIs to the cytoplasm. Immunoelectron microscopy of these viral components verified that the aberrant membrane structures were sites for BSMV replication. The BSMV αa replication protein localized at the surface of the chloroplasts and played a prominent role in eliciting chloroplast membrane rearrangements. In sum, our results have revealed the 3D structure of the chloroplasts induced by BSMV infection. These findings contribute to our understanding of chloroplast morphological changes under stress conditions and during assembly of plant (+)RNA virus replication complexes.

Barley stripe mosaic virus (BSMV) is the type member of the genus Hordeivirus . Brachypodium distachyon line Bd3-1 shows resistance to the BSMV ND18 strain, but is susceptible to an ND18 double mutant (β ND TGB1 R390K, T392K ) in which lysine is substituted for an arginine at position 390 and for threonine at position 392 of the triple gene block 1 (TGB1) protein. In order to understand differences in gene expression following infection with ND18 and double mutant ND18, Bd3-1 seedlings were subjected to RNA-seq analyses at 1, 6, and 14 days post inoculation (dpi). The results revealed that basal immunity genes involved in cellulose synthesis and pathogenesis-related protein biosynthesis were enhanced in incompatible interactions between Bd3-1 and ND18. Most of the differentially expressed transcripts are related to trehalose biosynthesis, ethylene, jasmonic acid metabolism, protein phosphorylation, protein ubiquitination, transcriptional regulation, and transport process, as well as pathogenesis-related protein biosynthesis. In compatible interactions between Bd3-1 and ND18 mutant, Bd3-1 developed weak basal resistance responses to the virus. Many genes involved in cellulose biosynthesis, protein amino acid phosphorylation, protein biosynthesis, protein glycosylation, glycolysis and cellular macromolecular complex assembly that may be related to virus replication, assembly and movement were up-regulated. Some genes involved in oxidative stress responses were also up-regulated at 14 dpi. BSMV ND18 mutant infection suppressed expression of genes functioning in regulation of transcription, protein kinase, cellular nitrogen compound biosynthetic process and photosynthesis. Differential expression patterns between compatible and incompatible interactions in Bd3-1 to the two BSMV strains provide important clues for understanding mechanism of resistance to BMSV in the model plant Brachypodium .

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis., Author summary The appropriate amount and balance of four dNTPs are crucial for all cells correctly copying and passing on their genetic material generation by generation. Eukaryotes have developed an alert and response system to deal with the disturbance. Here, we uncovered a second-level effector branch. It is activated by the upstream surveillance kinase cascade, which can induce the expression of dNTP-producing enzymes. It can also reduce the inhibitor of these enzymes to further boost their activity according to the degrees of threats. These findings suggest a multi-level response system to guarantee the appropriate dNTP supply, which is essential to maintain genetic stability under various environmental challenges.

Figure S6. Additional analysis of DEGs. (a) Heatmap of 278 transport unigenes that share a significantly upregulated pattern. (b) Heatmap of 272 oxidation-reduction process unigenes that share a significantly upregulated pattern. (c) Heatmap of 271 organic acid metabolic process unigenes that share a significantly upregulated pattern. (PDF 162 kb)

Figure S2. BLAST results from the Nr database. (a) Similarity distribution. (b) Best-hit species classification. (c) E-value distribution. (PDF 120 kb)

Figure S8. Correlation analysis of 19 randomly selected DEGs based on the qRT-PCR and RNA-seq data. Pearson correlation coefficients are shown (R2 = 0.8073) (P < 0.001). (PDF 78 kb)

Figure S7. Organic acid metabolism under saline-alkali conditions; the red font indicates the upregulated unigenes. (PDF 107 kb)

Figure S4. KEGG pathways of assembled unigenes from P. tenuiflora. The x-axis indicates the categories, and the y-axis indicates the number of unigenes. (PDF 189 kb)

Figure S5. COG classifications of assembled unigenes from P. tenuiflora. Of the 42,276 de novo-assembled unigenes, 16,829 were annotated and separated into 23 categories. (PDF 141 kb)

Highlight CK2 phosphorylation of the TGB1 protein has a critical role in promoting barley stripe mosaic virus movement in monocots and dicots by affecting the interactions between TGB1 and TGB3 proteins., The barley stripe mosaic virus (BSMV) triple gene block 1 (TGB1) protein is required for virus cell-to-cell movement. However, little information is available about how these activities are regulated by post-translational modifications. In this study, we showed that the BSMV Xinjiang strain TGB1 (XJTGB1) is phosphorylated in vivo and in vitro by protein kinase CK2 from barley and Nicotiana benthamiana. Liquid chromatography tandem mass spectrometry analysis and in vitro phosphorylation assays demonstrated that Thr-401 is the major phosphorylation site of the XJTGB1 protein, and suggested that a Thr-395 kinase docking site supports Thr-401 phosphorylation. Substitution of Thr-395 with alanine (T395A) only moderately impaired virus cell-to-cell movement and systemic infection. In contrast, the Thr-401 alanine (T401A) virus mutant was unable to systemically infect N. benthamiana but had only minor effects in monocot hosts. Substitution of Thr-395 or Thr-401 with aspartic acid interfered with monocot and dicot cell-to-cell movement and the plants failed to develop systemic infections. However, virus derivatives with single glutamic acid substitutions at Thr-395 and Thr-401 developed nearly normal systemic infections in the monocot hosts but were unable to infect N. benthamiana systemically, and none of the double mutants was able to infect dicot and monocot hosts. The mutant XJTGB1T395A/T401A weakened in vitro interactions between XJTGB1 and XJTGB3 proteins but had little effect on XJTGB1 RNA-binding ability. Taken together, our results support a critical role of CK2 phosphorylation in the movement of BSMV in monocots and dicots, and provide new insights into the roles of phosphorylation in TGB protein functions.

All well-characterized positive-strand RNA viruses[(+)RNA viruses] induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remain elusive. We report here the biogenesis and topology of the Beet black scorch virus (BBSV) replication complex. Distinct cytopathological changes typical of endoplasmic reticulum (ER) aggregation and vesiculation were observed in BBSV-infected Nicotiana benthamiana cells. Immunogold labeling of the auxiliary replication protein p23 and double-stranded RNA (dsRNA) revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that p23 plays a crucial role in mediating the ER rearrangement. Three-dimensional electron tomographic analysis revealed the formation of multiple ER-originated vesicle packets. Each vesicle packet enclosed a few to hundreds of independent spherules that were invaginations of the ER membranes into the lumen. Strikingly, these vesicle packets were connected to each other via tubules, a rearrangement event that is rare among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which showed diverse structures. Our results provide the first, to our knowledge, three-dimensional ultrastructural analysis of membrane-bound VRCs of a plant (+)RNA virus and should help to achieve a better mechanistic understanding of the organization and microenvironment of plant (+)RNA virus replication complexes. IMPORTANCE Assembly of virus replication complexes for all known positive-strand RNA viruses depends on the extensive remodeling of host intracellular membranes. Beet black scorch virus , a necrovirus in the family Tombusviridae , invaginates the endoplasmic reticulum (ER) membranes to form spherules in infected cells. Double-stranded RNAs, the viral replication intermediate, and the viral auxiliary replication protein p23 are all localized within such viral spherules, indicating that these are the sites for generating progeny viral RNAs. Furthermore, the BBSV p23 protein could to some extent reorganize the ER when transiently expressed in N. benthamiana . Electron tomographic analysis resolves the three-dimensional (3D) architecture of such spherules, which are connected to the cytoplasm via a neck-like structure. Strikingly, different numbers of spherules are enclosed in ER-originated vesicle packets that are connected to each other via tubule-like structures. Our results have significant implications for further understanding the mechanisms underlying the replication of positive-strand RNA viruses.

International audience; Positive-sense (+) RNA viruses represent the most abundant group of viruses and are dependent on the host cell machinery to replicate. One remarkable feature that occurs after (+) RNA virus entry into cells is the remodeling of host endomembranes, leading to the formation of viral replication factories. Recently, rapid progress in three-dimensional (3D) imaging technologies, such as electron tomography (ET) and focused ion beam-scanning electron microscopy (FIB-SEM), has enabled researchers to visualize the novel membrane structures induced by viruses at high resolution. These 3D imaging technologies provide new mechanistic insights into the viral infection cycle. In this review, we summarize the latest reports on the cellular remodeling that occurs during plant virus infection; in particular, we focus on studies that provide 3D architectural information on viral replication factories. We also outline the mechanisms underlying the formation of these membranous structures and discuss possible future research directions.

Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA‐binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear‐cytoplasmic trafficking is required for BSMV cell‐to‐cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95–104) and a nuclear localization signal (NLS) (amino acids 227–238). NoLS mutations reduced BSMV cell‐to‐cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine–arginine‐rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV‐infected cells, Fib2 accumulation increased by about 60%–70% and co‐localized with TGB1 in the plasmodesmata. In addition, BSMV cell‐to‐cell movement in fib2 knockdown transgenic plants was reduced to less than one‐third of that of non‐transgenic plants. Fib2 also co‐localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1–Fib2 interactions play a direct role in cell‐to‐cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell‐to‐cell movement.