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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Non-Structural Protein NSm of Tomato Spotted Wilt Virus Is an Avirulence Factor Recognized by Resistance Genes of Tobacco and Tomato via Different Elicitor Active Sites.

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

Removal of Cu2+, Cd2+ and Pb2+ from aqueous solutions by magnetic alginate microsphere based on Fe3O4/MgAl-layered double hydroxide.

In this work, the magnetic alginate microsphere of Fe3O4/MgAl-LDH (Fe3O4/LDH-AM) was prepared by immobilizing the Fe3O4/LDH with calcium alginate (CA) and was used to remove Cd2+, Pb2+, and Cu2+ from aqueous solutions. The obtained Fe3O4/LDH-AM was characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, and Brunauer-Emmett-Teller surface area determination. The results indicated that the surface groups of the alginate and LDH were retained and so was the crystal structure in the alginate microsphere. The adsorption performance of the Fe3O4/LDH-AM for Cd2+, Pb2+, and Cu2+ in aqueous solutions was evaluated by batch and column adsorption experiments. The effects of adsorption conditions, kinetics, isotherms, mechanisms, and potential applications were investigated. The adsorption kinetic data conformed to the pseudo-second-order kinetic equation, and the isotherm data fit well with the Freundlich and Langmuir isotherm models. The adsorption mechanism of Cd2+, Pb2+, and Cu2+ by the Fe3O4/LDH-AM entailed complexation and precipitation. The experimental breakthrough curves were correlated with the Thomas model. Moreover, the Fe3O4/LDH-AM displayed superior regeneration and reusability. These results suggest that the Fe3O4/LDH-AM is an efficient adsorbent for the removal of heavy metals and can be effectively employed in practical applications.

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.

Genomes and virulence difference between two physiological races of Phytophthora nicotianae.

BACKGROUND: Black shank is a severe plant disease caused by the soil-borne pathogen Phytophthora nicotianae. Two physiological races of P. nicotianae, races 0 and 1, are predominantly observed in cultivated tobacco fields around the world. Race 0 has been reported to be more aggressive, having a shorter incubation period, and causing worse root rot symptoms, while race 1 causes more severe necrosis. The molecular mechanisms underlying the difference in virulence between race 0 and 1 remain elusive. FINDINGS: We assembled and annotated the genomes of P. nicotianae races 0 and 1, which were obtained by a combination of PacBio single-molecular real-time sequencing and second-generation sequencing (both HiSeq and MiSeq platforms). Gene family analysis revealed a highly expanded ATP-binding cassette transporter gene family in P. nicotianae. Specifically, more RxLR effector genes were found in the genome of race 0 than in that of race 1. In addition, RxLR effector genes were found to be mainly distributed in gene-sparse, repeat-rich regions of the P. nicotianae genome. CONCLUSIONS: These results provide not only high quality reference genomes of P. nicotianae, but also insights into the infection mechanisms of P. nicotianae and its co-evolution with the host plant. They also reveal insights into the difference in virulence between the two physiological races.

Kinetic, isotherm and thermodynamic investigations of phosphate adsorption onto core-shell Fe3O4@LDHs composites with easy magnetic separation assistance.

In this study, three different magnetic core-shell Fe3O4@LDHs composites, Fe3O4@Zn-Al-, Fe3O4@Mg-Al-, and Fe3O4@Ni-Al-LDH were prepared via a rapid coprecipitation method for phosphate adsorptive removal. The composites were characterized by XRD, FTIR, TEM, VSM and BET analyses. Characterization results proved the successful synthesis of core-shell Fe3O4@LDHs composites with good superparamagnetisms. Batch experiments were conducted to study the adsorption efficiency of phosphate. Optimal conditions for the phosphate adsorption were obtained: 0.05 g of adsorbent, solution pH of 3, and contact time of 60 min. Proposed mechanisms for the removal of phosphate species onto Fe3O4@LDHs composites at different initial solution pH were showed. The kinetic data were described better by the pseudo-second-order kinetic equation and KASRA model. The adsorption isotherm curves showed a three-region behavior in the ARIAN model. It had a good fit with Langmuir model and the maximum adsorption capacity followed the order of Fe3O4@Zn-Al-LDH>Fe3O4@Mg-Al-LDH>Fe3O4@Ni-Al-LDH. Thermodynamic analyses indicated that the phosphate adsorption process was endothermic and spontaneous in nature. The three Fe3O4@LDHs composites could be easily separated from aqueous solution by the external magnetic field in 10s. These novel magnetic core-shell Fe3O4@LDHs adsorbents may offer a simple single step adsorption treatment option to remove phosphate from water without the requirement of pre-/post-treatment for current industrial practice.

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.

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.

A reusable naphthalimide-functionalized magnetic fluorescent nanosensor for the simultaneous determination and removal of trace Hg2+ in aqueous solution.

On the basis of high selective and sensitive interaction of 1,8-naphthalimide with Hg(2+) and the formation of stable neutral imide-Hg-imide complexes, we designed and synthesized a novel magnetic fluorescent sensor (S1) employed Fe(3)O(4) magnetic nanoparticles and 1,8-naphthalimide fluorescent sensor. Under optimum conditions, S1 exhibits the high selectivity toward Hg(2+) over other metal ions, with the detection limit of 1.03×10(-8)M. We demonstrated that a reliable fluorescence response of S1 toward Hg(2+) over a broad pH range (pH=5.0-9.0) could be reused at least four cycles. The maximum sorption capacity of S1 was about 5.6 mg g(-1). In addition, the removal of Hg(2+) in water was achieved by the aggregation-induced sedimentation (AIS) strategy. Moreover, the suspended magnetic nanoparticles could be removed by external magnetic field, and the secondary pollution was avoided. The above-mentioned results indicate that this approach may serve as a foundation of the preparation of the multifunctional magnetic fluorescent sensor for simple, rapid, and simultaneous determination and removal of trace Hg(2+) and other pollutants in environmental samples.