CRISPR/Cas9-mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV-U1 strain in regenerated Nicotiana tabacum.

CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.

Identification of two additional plasmodesmata localization domains in the tobacco mosaic virus cell-to-cell-movement protein.

Despite decades of intensive studies, the failure to identify plasmodesmata (PD) localization sequences has constrained our understanding of Tobacco mosaic virus (TMV) movement. Recently, we identified the first PD localization signal (major PLS) in the TMV movement protein (MP), which encompasses the first 50 amino acid residues of the MP. Although the major PLS is sufficient for PD targeting, the efficiency is lower than the full-length TMV MP. To address this efficiency gap, we identified two additional PLS domains encompassing amino acid residues 61 to 80, and 147 to 170 of the MP and showed that these two domains target to PD, but do not transit to adjacent cells. We also demonstrated that the MP61-80 fragment interacts with Arabidopsis synaptotagmin A, which was also shown to interact with the major TMV MP PLS. Therefore, our findings have provided new insights to more fully understand the mechanism underlying plasmodesmal targeting of TMV MP.

Transcriptomic profile of tobacco in response to Tomato zonate spot orthotospovirus infection.

BACKGROUND: Tomato zonate spot virus (TZSV), a dominant species of thrips-transmitted orthotospoviruses in Yunnan and Guangxi provinces in China, causes significant loss of yield in lots of crops and is a major threat to incomes of rural families. However, the detailed molecular mechanism of crop disease caused by TZSV remains obscure. METHODS: Next-generation sequencing (NGS)-based transcriptome analysis (RNA-seq) was performed to investigate and compare the gene expression changes in systemic leaves of tobacco upon infection with TZSV and mock-inoculated plants as a control. RESULTS: De novo assembly and analysis of tobacco transcriptome data by RNA-Seq identified 135,395 unigenes. 2102 differentially expressed genes (DEGs) were obtained in tobacco with TZSV infection, among which 1518 DEGs were induced and 584 were repressed. Gene Ontology enrichment analysis revealed that these DEGs were associated with multiple biological functions, including metabolic process, oxidation-reduction process, photosynthesis process, protein kinase activity. The KEGG pathway analysis of these DEGs indicated that pathogenesis caused by TZSV may affect multiple processes including primary and secondary metabolism, photosynthesis and plant-pathogen interactions. CONCLUSION: Our global survey of transcriptional changes in TZSV infected tobacco provides crucial information into the precise molecular mechanisms underlying pathogenesis and symptom development. This is the first report on the relationships in the TZSV-plant interaction using transcriptome analysis. Findings of present study will significantly help enhance our understanding of the complicated mechanisms of plant responses to orthotospoviral infection.

Diethanolamine-modified magnetic fluorescent Fe3O4@ZnS nanoparticles for ultrasensitive detection and removal of Cu2+.

Currently, growing attention has been paid to the sensitive determination and removal of Cu2+ because excessive levels of Cu2+ could do harm to organisms. Herein, a novel diethanolamine-modified magnetic fluorescent Fe3O4@ZnS nanoparticle (MFNP) for simultaneous detection and removal of Cu2+ was designed and synthesized through dithiocarbamate linkage strategy. The characterization of MFNP was confirmed by transmission electron microscope (TEM), infrared (IR) and emission spectra. The results showed that MFNP could quantificationally detect Cu2+ with high sensitivity and selectivity under a broad pH range (pH 4.5-9). The removal of Cu2+ was achieved by the aggregation-induced sedimentation (AIS) strategy and by external magnetic field.

Characterization of the CMS genetic regulation through comparative complete mitochondrial genome sequencing in Nicotiana tabacum.

Mitochondrial genomes (mitogenomes) of flowering plants vary greatly in structure and size, which can lead to frequent gene mutation, rearrangement, or recombination, then result in the cytoplasmic male sterile (CMS) mutants. In tobacco (Nicotiana tabacum), suaCMS lines are widely used in heterosis breeding; however, the related genetic regulations are not very clear. In this study, the cytological observation indicated that the pollen abortion of tobacco suaCMS(HD) occurred at the very early stage of the stamen primordia differentiation. In this study, the complete mitochondrial genomes of suaCMS(HD) and its maintainer HD were sequenced using the PacBio and Illumina Hiseq technology. The total length of the assembled mitogenomes of suaCMS(HD) and HD was 494,317 bp and 430,694 bp, respectively. Comparative analysis indicated that the expanded 64 K bases in suaCMS(HD) were mainly located in noncoding regions, and 23 and 21 big syntenic blocks (>5000 bp) were found in suaCMS(HD) and HD with a series of repeats. Electron transport chain-related genes were highly conserved in two mitogenomes, except five genes (ATP4, ATP6, COX2, CcmFC, and SDH3) with substantial substitutions. Three suaCMS(HD)-specific genes, orf261, orf291, and orf433, were screened. Sequence analysis and RT-PCR verification showed that they were unique to suaCMS(HD). Further gene location analysis and protein property prediction indicated that all the three genes were likely candidates for suaCMS(HD). This study provides new insight into understanding the suaCMS mechanism and is useful for improving tobacco breeding.

Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.

Label-free electrochemical immunosensor for prostate-specific antigen based on silver hybridized mesoporous silica nanoparticles.

Due to high sensitivity and good selectivity, electrochemical immunosensor is often used to detect tumor markers. Prostate-specific antigen (PSA) is the most validated tumor marker for prostate cancer. In this work, mesoporous silica nanoparticles (MSNs) were used to increase the fixation capacity of primary antibody, and silver nanoparticles (Ag NPs) were used to enhance the electron transfer rates. Silver hybridized mesoporous silica nanoparticles (Ag@MSNs) were synthesized and used as electrode material. Hydroquinone (HQ) generated a stable electrochemical signal and was used as a mediator. Based on the specific antibody-antigen interaction, a label-free immunosensor was developed for the sensing of PSA. The current method allows us to detect PSA over a wide concentration range from 0.05 to 50.0 ng ml(-1) with a detection limit of 15 pg ml(-1). The proposed immunosensor was used to determine PSA in human serum with satisfactory results.

Development of a novel water-soluble magnetic fluorescent nanoparticle for the selective detection and removal of Cu2+.

Recently, much attention has been paid to the selective detection and removal of Cu2+ because an excess of Cu2+ can harm the environment and living systems. Herein, we developed a novel water-soluble di-2-picolylamine/proline co-modified Fe3O4@ZnS magnetic fluorescent nanoparticle (MFNP-Cu) for the selective detection and removal of Cu2+ through a dithiocarbamate linkage strategy. The characterization of MFNP-Cu was confirmed by x-ray diffraction (XRD), transmission electron microscope (TEM), magnetization hysteresis loops, infrared (IR) and emission spectra. The results showed that MFNP-Cu could quantifiably detect Cu2+ with high sensitivity and selectivity over a broad pH range (pH 4.1-9). The maximum adsorption capacity of MFNP-Cu was calculated to be about 517.9 mg g(-1), which is higher than previously reported. This excellent property was investigated by kinetics equilibrium and thermodynamic studies. Moreover, the removal properties of MFNP-Cu toward Cu2+ from contaminated water samples was achieved by an external magnetic field.

Label-free electrochemical immunosensor based on graphene/methylene blue nanocomposite.

For the specificity of prostate cancer markers, prostate specific antigen (PSA) has been widely used in prostate cancer screening, diagnosis, and treatment after monitoring. In normal male serum, PSA can only be detected in traces of 0-4 ng mL(-1). In this paper, we constructed an electrochemical immunosensor for PSA detection using a nanocomposite film of graphene sheets-methylene blue-chitosan (GS-MB-CS) as electrode material. The nanocomposite film showed high binding affinity to the electrode and was used to immobilize the antibody of PSA. The modification procedure was monitored by cyclic voltammetry (CV). An amperometric biosensor was easily developed based on the response of peak current to the capture of PSA induced by specific antigen-antibody reactions. Under optimum conditions, the amperometric signal decreased linearly with PSA concentration (0.05-5.00 ng mL(-1)). A low limit of detection (13 pg mL(-1)) and a high selectivity are obtained. Moreover, the prepared immunosensor was applied for the analysis of PSA in serum samples with satisfactory results. The proposed method may have a promising future in biochemical assays for high selectivity, good reproducibility, and stability.

Removal of o-nitrobenzoic acid by adsorption on to a new organoclay: montmorillonite modified with HDTMA microemulsion.

A new organoclay, consisting of montmorillonite modified by a hexadecyl trimethyl ammonium (HDTMA) microemulsion, was synthesized, characterized and used as an adsorbent for the removal of o-nitrobenzoic acid from aqueous solution. Adsorption kinetics, isotherms and effects of operating variables, such as adsorbent dosage, ionic strength and initial solution pH, were also investigated. The results of Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis and BET surface area determination indicated that HDTMA molecules had entered into the interlayer of the montmorillonite. The optimized experimental conditions for the adsorption of o-nitrobenzoic acid by montmorillonite modified by HDTMA microemulsion were 0.5 g adsorbent dosage, 0.4 mL of 0.1 mol lbL(-1) CaCl2 solution, initial solution pH of 6.0 and contact time of 6 h. The adsorption isotherms of o-nitrobenzoic acid fitted the Langmuir model well (R2 = 0.9880). The adsorption kinetics data fitted the pseudo-second-order equation (R2 = 0.9999). These above results indicate that montmorillonite modified by an HDTMA microemulsion can be used as adsorbent for o-nitrobenzoic acid because of its high adsorption capacity and low cost.

[Synthesis and characterization of CTMAB and PDMDAAC modified organobentonite].

The natural bentonite was purified and changed to sodium form by NaCl via exchange reaction. Their characteristics, such as swelling volume, swelling value, colloid valence, ethylene blue adsorbed and cation exchange capacity, were measured. The results indicate that the property of Na-bentonite is better than that of natural bentonite. Using cetyltrimethylammonium bromide (CTMAB) and homopolymer of dimethyldiallyammomium chloride (PDMDAAC) as organo-intercalating reagents, two organic modified bentonites were prepared and characterized by Fourier transform infrared (FTIR), X-ray powder diffraction (XRD) and BET surface area. The XRD results showed that the CTMAB-bentonite and PDMDAAC-bentonite had typical X-ray diffraction peaks, and the d001 values increased to 1.89 and 1.45 nm, respectively. Combined with the results of FTIR, the modified reagents had been intercalated to the layer of bentonite. The BET areas, pore volumes and average pore diameters of the two organo-bentonites were decreased as compared to that of Na-bentonite.

Teacher Online Informal Learning as a Means to Innovative Teaching During Home Quarantine in the COVID-19 Pandemic.

The home quarantine in the COVID-19 pandemic has created challenges for teaching across the world and called for innovative teaching, as well as teachers' learning. Given the rapid development of teachers' online learning and teaching, identifying effective ways to facilitate innovative teaching under such challenging conditions is a critical issue. Although researchers have realized that workplace informal learning (IL) increasingly reveals its potential value to individual development, the relationship between IL and innovation has been under-explored. The purpose of this study was to evaluate the impact of IL on innovative teaching, through the mediating roles of three types of teaching-related efficacy, with a particular focus on college teachers and online context. A sample of 479 Chinese college teachers was randomly selected to participate in the survey. The results showed that teachers' online IL in pandemic improved their personal teaching efficacy and ICT efficacy (information and communication technology efficacy), and then facilitated their innovative teaching without differences of gender and teaching-age effect. Whereas, general teaching efficacy was not a mediator between online IL and innovative teaching. Hence, we proposed a can-do motivating model of teacher efficacy in fostering innovative teaching through informal learning. It implies three properties of teachers' online IL: social interaction, autonomous learning and novelty-seeking. It also revealed that innovative teaching can be driven in COVID-19 pandemic, mainly by learning domain-specific knowledge and skills, thus enhancing personal teaching efficacy and ICT efficacy in online teaching context.

A Motivational Mechanism Framework for Teachers' Online Informal Learning and Innovation During the COVID-19 Pandemic.

Online informal learning (IL) spreads quickly in the COVID-19 Pandemic. Studies have predicted that both online and workplace IL have potential value to individual and organization development, whereas the study on its link with innovation remains scarce. IL is an individualized learning pattern different from formal learning, and its functioning mechanism on innovation will deepen our understanding of the relationship between learning and innovation. Self-efficacy and autonomous motivation are considered as two streams of motivational mediating mechanisms to innovation. However, previous studies have proceeded largely in separation from each other. Researchers highlight the need to develop a more fine-grained theory of motivation and innovation. In addressing these literature gaps, this paper takes college teachers as the sample and focuses on the motivational mediating mechanism between online IL and innovation. The results showed that teachers IL could positively predict innovative teaching performance. Personal teaching efficacy and autonomous motivation played as sequential mediators on the link between IL and innovative teaching performance. This study extends the literature of IL-innovation relationship and enriches understanding of cognition-oriented motivation theory, highlighting one's internal autonomous construction is the key to innovation. Theoretical and practical implications for psychological empowerment are discussed.

The complete chloroplast genome sequence of Nicotiana debneyi (Solanaceae).

In present study, we report the complete chloroplast genome of Nicotiana debneyi, a species endemic to eastern coast of Australia. The total genome size of N.debneyi is 156,073 bp in length, containing a large single-copy region of 86,672 bp, a small single-copy region of 18,581 bp, and two inverted repeat regions of 25,410 bp. The all GC content of N.debneyi chloroplast genome is 38.4%. It encodes a total of 129 unique genes, including 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes, of which seven tRNA, four rRNA and seven protein-coding genes are duplicated in the IR. Sixteen genes contain a single intron, and only two genes have two introns. Phylogenetic analysis results strongly supported that N.debneyi was closely related to Nicotiana sylvestris and Nicotiana tabacum.

Cloning, structural features, and expression analysis of resistance gene analogs in tobacco.

Using degenerate primers based on the conserved nucleotide binding site (NBS) and protein kinase domain (PKD), 100 resistance gene analogs (RGAs) were isolated from tobacco variety Nicotiana repanda. BLASTx search against the GenBank database revealed that 27 belong to the NBS class and 73 belong to the protein kinase (PK) class. Cluster analysis and multiple sequence alignment of the deduced protein sequences indicate that RGAs of the NBS class can be divided into two groups: toll/interleukin receptor (TIR) and non-TIR types. Both types possess 6 conserved motifs (P-loop, RNBS-A, Kinase-2, RNBS-B, RNBS-C, GLPL). Based on their sequence similarity, the tobacco RGAs of the PK class were assigned to 8 subclasses. We examined their expression after infection with either Tobacco mosaic virus (TMV) or the tobacco black shank pathogen (Phytophthora parasitica var. nicotianae). The expression levels of 4 RGAs of the PK class were significantly elevated by TMV and 1 RGA of the PK class and 3 RGAs of the NBS class were up-regulated by P. parasitica var. nicotianae. The expression of two RGAs of the PK class was induced by P. parasitica var. nicotianae. Infection by either TMV or P. parasitica var. nicotianae enhanced the expression of NtRGA2, a RGA of the PK class. The present study shows that RGAs are abundant in the tobacco genome and the identification of tobacco RGAs induced by pathogens should provide valuable information for cloning related resistance genes in tobacco.

Adsorption of heavy metals by l-cysteine intercalated layered double hydroxide: Kinetic, isothermal and mechanistic studies.

To understand the interaction mechanisms between heavy metals and functional groups modified nanomaterials, the l-cysteine (Cys) intercalated MgAl-layered double hydroxide (MgAl-Cys-LDH) was designed by a facile co-precipitation method and used to remove Cu(II), Pb(II) and Cd(II) in water solutions. The XRD, FTIR, SEM, TEM, EDS and XPS characterization analyses proved the carboxyl, thio and amido groups were successfully introduced into MgAl-LDH. The possible mechanisms were analyzed by the XPS and XRD spectra and involved the precipitation of metal hydroxides or sulfides, surface complexation with abundant surface groups, and the isomorphic substitution of Mg(II). The adsorption kinetics and isotherms data of Cu(II), Pb(II) and Cd(II) on MgAl-Cys-LDH were well described by the pseudo-second-order kinetic equation and the Langmuir model. The influence factors such as initial solution pH of heavy metals, dosage of MgAl-Cys-LDH, adsorption time and various types of water were investigated, and the results suggested that MgAl-Cys-LDH had potential applications in real wastewater treatment containing heavy metals.

Phosphate adsorption performance and mechanisms by nanoporous biochar-iron oxides from aqueous solutions.

To evaluate the adsorption mechanism and performance of phosphate onto the composite of low-cost biochar and iron oxide, four biochar-iron oxides, namely biochar-magnetite (BC-M), biochar-ferrihydrite (BC-F), biochar-goethite (BC-G), and biochar-hematite (BC-H), were prepared by fabricating iron oxide to porous biochar. The biochar-iron oxides had huge surface areas of 691-864 m2/g and average pore diameters of 3.4-4.0 nm. Based on the characterization analysis of FTIR, XRD, XPS, and zeta potential, the interactions of electrostatic attraction, ligand exchange, and deposition dominated the phosphate adsorption onto biochar-iron oxides. The maximum adsorption capacity of phosphate followed the order of BC-G > BC-F > BC-H > BC-M. The isotherm data of BC-M and BC-H were well fitted by the Langmuir and Freundlich models, while those of BC-G and BC-F followed the Langmuir model. In addition, BC-M, BC-F, BC-G, and BC-H owned excellent regeneration ability and adsorption performance in practical (simulated) wastewater environment. Then the biochar-iron oxides exerted extensive and satisfactory prospect in wastewater remediation and recycling application in soil.

Adsorption of phosphate from aqueous solution by vegetable biochar/layered double oxides: Fast removal and mechanistic studies.

Two biochars, from Chinese cabbage (Cc, Brassica rapa pekinensis) and rape (Ra, Brassia campestris L.), were used to prepare biochar/Mg-Al layered double oxides (LDOs) as adsorbents for phosphate removal from aqueous solution. The biochar/LDOs were horizontally alternated lamellar particles and had abundant groups of oxides and biochars. The phosphate removal percentage remained above 92% at a pH range of 2-10, and above 95% during the first 5 min for 50 mg/L phosphate by 0.05 g biochar/LDOs. The adsorption kinetics and isotherms data were well fitted by the pseudo-second-order kinetic equation, as well as by the Freundlich and Langmuir models. Based on FTIR, XPS, and zeta potential analysis, the interaction mechanisms were defined as "memory effect", electrostatic attraction, surface complexation, and anion exchange. The results indicate that vegetable biochar/LDOs can be considered a novel and efficient sorbent for phosphate removal from water or wastewater.

Efficient and fast removal of Pb2+ and Cd2+ from an aqueous solution using a chitosan/Mg-Al-layered double hydroxide nanocomposite.

A new nanocomposite based on chitosan (CS) and Mg-Al-layered double hydroxide (Mg-Al-LDH) was prepared using an emulsion-crosslinking method. CS was immobilized within the Mg-Al-LDH matrix to form the CS-LDH after crosslinking by epichlorohydrin. The characterization of XPS, SEM, TEM, FTIR, and BET analysis showed that the CS-LDH had a high specific surface area and contained many different functional groups. The adsorption capacity of the CS-LDH was evaluated by adsorbing Pb2+ and Cd2+ as representative heavy metals. Batch adsorption method was used to investigate the effects of the amount of adsorbent, pH of the initial solution, and contact time. We also examined the adsorption kinetics, isotherms, and mechanisms. The capacity of the CS-LDH for Pb2+ and Cd2+ adsorption was higher than that of CS and the Mg-Al-LDH, and the isothermal data followed the Langmuir isotherm model. The adsorption equilibrium was quickly achieved and the kinetic data obeyed the pseudo-second-order kinetic model. The adsorption process was almost not affected by solution pH above 3. The CS-LDH and heavy metals interacted via the following mechanisms: precipitation, surface complexation, and isomorphic substitution.