SummaryPlant basic helix-loop-helix (bHLH) transcription factors play essential roles in abiotic stress tolerance. However, most bHLHs have not been functionally characterized. Here, we characterized the functional role of a bHLH transcription factor from Arabidopsis, AtbHLH112, in response to abiotic stress.AtbHLH112 is a nuclear-localized protein, and its nuclear localization is induced by salt, drought and abscisic acid (ABA). In addition, AtbHLH112 serves as a transcriptional activator, with the activation domain located at its N-terminus.In addition to binding to the E-box motifs of stress-responsive genes, AtbHLH112 binds to a novel motif with the sequence 'GG[GT]CC[GT] [GA][TA]C' (GCG-box). Gain-and loss-offunction analyses showed that the transcript level of AtbHLH112 is positively correlated with salt and drought tolerance. AtbHLH112 mediates stress tolerance by increasing the expression of P5CS genes and reducing the expression of P5CDH and ProDH genes to increase proline levels. AtbHLH112 also increases the expression of POD and SOD genes to improve reactive oxygen species (ROS) scavenging ability.We present a model suggesting that AtbHLH112 is a transcriptional activator that regulates the expression of genes via binding to their GCG-or E-boxes to mediate physiological responses, including proline biosynthesis and ROS scavenging pathways, to enhance stress tolerance.

The 37K protein of Chinese wheat mosaic virus (CWMV) belongs to the 30K superfamily of plant virus movement proteins. CWMV 37K trans-complemented the cell-to-cell spread of a movement-defective Potato virus X. CWMV 37K fused to enhanced green fluorescent protein localized to plasmodesmata and formed endoplasmic reticulum (ER)-derived vesicular and large aggregate structures. CWMV 37K has two putative N-terminal transmembrane domains (TMDs). Mutations disrupting TMD1 or TMD2 impaired 37K movement function; those mutants were unable to form ER-derived structures but instead accumulated in the ER. Treatment with Brefeldin A or overexpression of the dominant negative mutant of Sar1 retained 37K in the ER, indicating that ER export of 37K is dependent on the secretory pathway. Moreover, CWMV 37K interacted with pectin methylesterases and mutations in TMD1 or TMD2 impaired this interaction in planta. The results suggest that the two TMDs regulate the movement function and intracellular transport of 37K.

Syringe infiltration is an important transient transformation method that is widely used in many molecular studies. Owing to the wide use of syringe agroinfiltration, it is important and necessary to improve its transformation efficiency. Here, we studied the factors influencing the transformation efficiency of syringe agroinfiltration. The pCAMBIA1301 was transformed into Nicotiana benthamiana leaves for investigation. The effects of 5-azacytidine (AzaC), Ascorbate acid (ASC) and Tween-20 on transformation were studied. The β-glucuronidase (GUS) expression and GUS activity were respectively measured to determine the transformation efficiency. AzaC, ASC and Tween-20 all significantly affected the transformation efficiency of agroinfiltration, and the optimal concentrations of AzaC, ASC and Tween-20 for the transgene expression were identified. Our results showed that 20 μM AzaC, 0.56 mM ASC and 0.03% (v/v) Tween-20 is the optimal concentration that could significantly improve the transformation efficiency of agroinfiltration. Furthermore, a combined supplement of 20 μM AzaC, 0.56 mM ASC and 0.03% Tween-20 improves the expression of transgene better than any one factor alone, increasing the transgene expression by more than 6-fold. Thus, an optimized syringe agroinfiltration was developed here, which might be a powerful method in transient transformation analysis.

Like other members of the family Reoviridae, rice black-streaked dwarf virus (RBSDV, genus Fijivirus) is thought to replicate and assemble within cytoplasmic viral inclusion bodies, commonly called viroplasms. RBSDV P9-1 is the key protein for the formation of viroplasms, but little is known about the other proteins of the viroplasm or the molecular interactions amongst its components. RBSDV non-structural proteins were screened for their association with P9-1 using a co-immunoprecipitation assay. Only P6 was found to directly interact with P9-1, an interaction that was confirmed by bimolecular fluorescence complementation assay in Spodoptera frugiperda (Sf9) cells. Immunoelectron microscopy showed that P6 and P9-1 co-localized in electron-dense inclusion bodies, indicating that P6 is a constituent of the viroplasm. In addition, non-structural protein P5 also localized to viroplasms and interacted with P6. In Sf9 cells, P6 was diffusely distributed throughout the cytoplasm when expressed alone, but localized to inclusions when coexpressed with P9-1, suggesting that P6 is recruited to viral inclusion bodies by binding to P9-1. P5 localized to the inclusions formed by P9-1 when co-expressed with P6 but did not when P6 was absent, suggesting that P5 is recruited to viroplasms by binding to P6. This study provides a model by which viral non-structural proteins are recruited to RBSDV viroplasms.

Rice black-streaked dwarf virus (RBSDV), a plant-infecting reovirus (genus Fijivirus), generally induces virus-containing tubules in infected cells. The nonstructural protein P7-1, encoded by the first open reading frame of segment 7, is involved in forming the structural matrix of these tubules. In experiments to investigate the subcellular localization of P7-1 in Nicotiana benthamiana epidermal cells, fluorescence of P7-1:eGFP was observed in the nucleus, cytoplasm and cell periphery, and in punctate points along the cell wall of plasmolyzed cells. Co-localization with plasmodesmata-located protein 1 showed that P7-1 formed the punctate points at plasmodesmata. Mutational analysis demonstrated that transmembrane domain 1 and adjacent residues were necessary and sufficient for P7-1 to form punctate structures at the cell wall in the plasmolyzed cells. Chemical drug and protein inhibitor treatments indicated that P7-1 utilized the ER-to-Golgi secretory pathway and the actomyosin motility system for its intracellular transport. The plasmodesmatal localization of RBSDV P7-1 is therefore dependent on the secretory pathway and the actomyosin motility system.

Gliomas are the most frequent primary malignant brain tumors of the central nervous system, causing significant impairment and death. There is mounting evidence that N7 methylguanosine (m7G) RNA dysmethylation plays a significant role in the development and progression of cancer. However, the expression patterns and function of the m7G RNA methylation regulator in gliomas are yet unknown. The goal of this study was to examine the expression patterns of 31 critical regulators linked with m7G RNA methylation and their prognostic significance in gliomas. To begin, we systematically analyzed patient clinical and prognostic data and mRNA gene expression data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. We found that 17 key regulators of m7G RNA methylation showed significantly higher expression levels in gliomas. We then divided the sample into two subgroups by consensus clustering. Cluster 2 had a poorer prognosis than cluster 1 and was associated with a higher histological grade. In addition, cluster 2 was significantly enriched for cancer-related pathways. Based on this discovery, we developed a risk model involving three m7G methylation regulators. Patients were divided into high-risk and low-risk groups based on risk scores. Overall survival (OS) was significantly lower in the high-risk group than in the low-risk group. Further analysis showed that the risk score was an independent prognostic factor for gliomas.

The homeodomain-leucine zipper (HD-Zip) proteins play crucial roles in plant developmental and environmental responses. However, how they mediate gene expression to facilitate abiotic stress tolerance remains unknown. In the present study, we characterized BpHOX2 (encoding a HD-Zip I family protein) from birch (Betula platyphylla). BpHOX2 is predominately expressed in mature stems and leaves, but expressed at a low level in apical buds and roots, suggesting that it has tissue-specific characteristics. BpHOX2 expression was highly induced by osmotic and salt, but only slightly induced by abscisic acid. Overexpression of BpHOX2 markedly improved osmotic tolerance, while knockdown of BpHOX2 increased sensitivity to osmotic stress. BpHOX2 could induce the expression of pyrroline-5-carboxylate synthase, peroxidase, and superoxide dismutase genes to improve proline levels and the reactive oxygen species scavenging capability. Chromatin immunoprecipitation sequencing combined with RNA sequencing showed that BpHOX2 could bind to at least four cis-acting elements, including dehydrationresponsive element "RCCGAC", Myb-p binding box "CCWACC," and two novel cis-acting elements with the sequences of "AAGAAG" and "TACGTG" (termed HBS1 and HBS2, respectively) to regulate gene expression. Our results suggested that BpHOX2 is a transcription factor that binds to different cis-acting elements to regulate gene expression, ultimately improving osmotic tolerance in birch.

BackgroundPostoperative cerebral edema is common in patients with meningioma. It is of great clinical significance to predict the postoperative cerebral edema exacerbation (CEE) for the development of individual treatment programs in patients with meningioma.ObjectiveTo evaluate the value of three-dimensional radiomics Features from Multi-Parameter MRI in predicting the postoperative CEE in patients with meningioma.MethodsA total of 136 meningioma patients with complete clinical and radiological data were collected for this retrospective study, and they were randomly divided into primary and validation cohorts. Three-dimensional radiomics features were extracted from multisequence MR images, and then screened through Wilcoxon rank sum test, elastic net and recursive feature elimination algorithms. A radiomics signature was established based support vector machine method. By combining clinical with the radiomics signature, a clin-radiomics combined model was constructed for individual CEE prediction.ResultsThree significance radiomics features were selected to construct a radiomics signature, with areas under the curves (AUCs) of 0.86 and 0.800 in the primary and validation cohorts, respectively. Two clinical characteristics (peritumoral edema and tumor size) and radiomics signature were determined to establish the clin-radiomics combined model, with an AUC of 0.91 in the primary cohort and 0.83 in the validation cohort. The clin-radiomics combined model showed good discrimination, calibration, and clinically useful for postoperative CEE prediction.ConclusionsBy integrating clinical characteristics with radiomics signature, the clin-radiomics combined model could assist in postoperative CEE prediction before surgery, and provide a basis for surgical treatment decisions in patients with meningioma.