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The creation of new soybean varieties has been limited by genomic duplication and redundancy. Efficient multiplex gene editing and large chromosomal segment deletion through clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems are promising strategies for overcoming these obstacles. CRISPR/Cpf1 is a robust tool for multiplex gene editing. However, large chromosomal excision mediated by CRISPR/Cpf1 has been reported in only a few non-plant species. Here, we report on CRISPR/LbCpf1-induced large chromosomal segment deletions in soybean using multiplex gene targeting. The CRISPR/LbCpf1 system was optimized for direct repeat and guide RNA lengths in crispr RNA (crRNA) array. The editing efficiency was evaluated using LbCpf1 driven by the CaMV35S and soybean ubiquitin promoter. The optimized system exhibited editing efficiencies of up to 91.7%. Our results showed eight gene targets could be edited simultaneously in one step when a single eight-gRNA-target crRNA array was employed, with an efficiency of up to 17.1%. We successfully employed CRISPR/LbCpf1 to produce small fragments (

Background: Human Anatomy always causes major hindrances to the learning of nursing students. We designed this study to observe the effectiveness of the "two-line and six-stage" (TLSS) teaching mode based on model making in teaching Human Anatomy in nursing.Methods: One class in the nursing major in Grade 20 was randomly selected as the control group, in which the traditional teaching mode was adopted; while, another class in the same major and grade was selected as the observation group, in which the TLSS teaching mode was adopted. After the course teaching was completed, these students in both groups were compared from the perspective of the final theoretical examination and physical specimen assessment. Besides, the evaluation of the teaching effectiveness of these students from both groups was also investigated. Results: The observation group outperformed the control group in both theoretical examination and physical specimen assessment, which was specifically manifested in six evaluation items, including the novelty of teaching methods, the elimination of the difficult points of the course, the mobilization of the learning enthusiasm, the interestingness of the course teaching, its effects on clinical thinking training, and the interpretation of the knowledge. Conclusion: The TLSS teaching mode based on model making can improve the effectiveness in teaching Human Anatomy.

Plants have evolved sophisticated immune networks to restrict pathogen colonization. In response, pathogens deploy numerous virulent effectors to circumvent plant immune responses. However, the molecular mechanisms by which pathogen-derived effectors suppress plant defenses remain elusive. Here, we report that the nucleus-localized RxLR effector PsAvh110 from the pathogen Phytophthora sojae, causing soybean (Glycine max) stem and root rot, modulates the activity of a transcriptional complex to suppress plant immunity. Soybean like-heterochromatin protein 1-2 (GmLHP1-2) and plant homeodomain finger protein 6 (GmPHD6) form a transcriptional complex with transcriptional activity that positively regulates plant immunity against Phytophthora infection. To suppress plant immunity, the nuclear effector PsAvh110 disrupts the assembly of the GmLHP1-2/GmPHD6 complex via specifically binding to GmLHP1-2, thus blocking its transcriptional activity. We further show that PsAvh110 represses the expression of a subset of immune-associated genes, including BRI1-associated receptor kinase 1-3 (GmBAK1-3) and pathogenesis-related protein 1 (GmPR1), via G-rich elements in gene promoters. Importantly, PsAvh110 is a conserved effector in different Phytophthora species, suggesting that the PsAvh110 regulatory mechanism might be widely utilized in the genus to manipulate plant immunity. Thus, our study reveals a regulatory mechanism by which pathogen effectors target a transcriptional complex to reprogram transcription.

Cell polarity, as a vital intracellular microenvironment characteristic, has immense effects on numerous pathological and biological processes. Therefore, the tracking of polarity variations is highly essential to explore the role and mechanism of the polarity in pathophysiological processes. Herein, we designed and synthesized a novel rhodol-based fluorescent probe (RDS) sensitive to polarity by introducing a bis(2-hydroxyethylthio)methyl group, like a pair of hydrophilic and rotatable wings, into the rhodol skeleton. This unique design makes RDS adopt the colorless and non-fluorescent spirocyclic form in a low-polarity medium while the colored and fluorescent ring-open form is present in a high-polarity system, resulting in a positive-correlation response of fluorescence intension to polarity. Importantly, RDS was successfully applied to monitor the polarity changes in living cells including cancer cells, healthy cells and senescent healthy cells, visualizing that the polarity of cancer cells is lower than that of healthy cells in which the more senescent ones have higher polarity.

In plants, the apoplast is a critical battlefield for plant-microbe interactions. Plants secrete defense-related proteins into the apoplast to ward off the invasion of pathogens. How microbial pathogens overcome plant apoplastic immunity remains largely unknown. In this study, we reported that an atypical RxLR effector PsAvh181 secreted by Phytophthora sojae, inhibits the secretion of plant defense-related apoplastic proteins. PsAvh181 localizes to plant plasma membrane and essential for P. sojae infection. By co-immunoprecipitation assay followed by liquid chromatography-tandem mass spectrometry analyses, we identified the soybean GmSNAP-1 as a candidate host target of PsAvh181. GmSNAP-1 encodes a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, which associates with GmNSF of the SNARE complex functioning in vesicle trafficking. PsAvh181 binds to GmSNAP-1 in vivo and in vitro. PsAvh181 interferes with the interaction between GmSNAP-1 and GmNSF, and blocks the secretion of apoplastic defense-related proteins, such as pathogenesis-related protein PR-1 and apoplastic proteases. Taken together, these data show that an atypical P. sojae RxLR effector suppresses host apoplastic immunity by manipulating the host SNARE complex to interfere with host vesicle trafficking pathway., Author summary P. sojae secretes RxLR effectors to modulate host plant immunity. PsAvh181, an atypical RxLR effector of P. sojae, contributes to the full virulence of P. sojae. However, the mechanism by which PsAvh181 modulates plant immunity remains unknown. We found that PsAvh181 localizes to the plant plasma membrane to inhibit the secretion of apoplastic defense-related proteins such as GmGIP1, P69B and PR1. We screened target proteins of PsAvh181 by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry analyses. It was found that PsAvh181 binds to a soybean soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (GmSNAP-1), which is an important component of the SNAP receptor (SNARE) complex and plays a significant role in vesicle trafficking. Further investigation showed that PsAvh181 interferes the interaction between GmSNAP-1 and GmNSF, thus blocking the secretion of defense-related proteins and thereby modulating plant immunity. Our results reveal how a P. sojae effector suppresses plant immunity by inhibiting the secretion of apoplastic defense-related proteins.

Agrobacterium tumefaciens is a specialized plant pathogen that causes crown gall disease and is commonly used for Agrobacterium-mediated transformation. As a pathogen, Agrobacterium triggers plant immunity, which affects transformation. However, the signaling components and pathways in plant immunity to Agrobacterium remain elusive. We demonstrate that two Arabidopsis mitogen-activated protein kinase kinases (MAPKKs) MKK4/MKK5 and their downstream mitogen-activated protein kinases (MAPKs) MPK3/MPK6 play major roles in both Agrobacterium-triggered immunity and Agrobacterium-mediated transformation. Agrobacteria induce MPK3/MPK6 activity and the expression of plant defense response genes at a very early stage. This process is dependent on the MKK4/MKK5 function. The loss of the function of MKK4 and MKK5 or their downstream MPK3 and MPK6 abolishes plant immunity to agrobacteria and increases transformation frequency, whereas the activation of MKK4 and MKK5 enhances plant immunity and represses transformation. Global transcriptome analysis indicates that agrobacteria induce various plant defense pathways, including reactive oxygen species (ROS) production, ethylene (ET), and salicylic acid- (SA-) mediated defense responses, and that MKK4/MKK5 is essential for the induction of these pathways. The activation of MKK4 and MKK5 promotes ROS production and cell death during agrobacteria infection. Based on these results, we propose that the MKK4/5-MPK3/6 cascade is an essential signaling pathway regulating Agrobacterium-mediated transformation through the modulation of Agrobacterium-triggered plant immunity.

Background Agrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function. Results In this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58–64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg’ B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein–protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean. Conclusion A rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein–protein interactions and gene editing system evaluation.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. During infection of the host plant, Agrobacterium transfers T-DNA from its Ti plasmid into the host cell, which can then be integrated into the host genome. This unique genetic transformation capability has been employed as the dominant technology for producing genetically modified plants for both basic research and biotechnological applications. Agrobacterium has been well studied as a disease-causing agent. The Agrobacterium-mediated transformation process involves early attachment of the bacterium to the host's surface, followed by transfer of T-DNA and virulence proteins into the plant cell. Throughout this process, the host plants exhibit dynamic gene expression patterns at each infection stage or in response to Agrobacterium strains with varying pathogenic capabilities. Shifting host gene expression patterns throughout the transformation process have effects on transformation frequency, host morphology, and metabolism. Thus, gene expression profiling during the Agrobacterium infection process can be an important approach to help elucidate the interaction between Agrobacterium and plants. This review highlights recent findings on host plant differential gene expression patterns in response to A. tumefaciens or related elicitor molecules.

Cadmium (Cd) induces cell death in plant roots. Mitogen-activated protein kinase (MAPK) plays a role in the regulation of cell death induced by Cd in plant roots. In this study, MhMAPK4 was isolated from the roots of Malus hupehensis. Subcellular localization showed that the MhMAPK4 protein was located in the cell membrane and cytoplasm and is a transmembrane protein that is characterized by hydrophily. The expression of MhMAPK4 in the roots of M. hupehensis was up-regulated by Cd sulfate and Cd chloride. Phenotypic comparison under Cd stress showed that the growth of wild-type (WT) tobacco was lower than the transgenic lines overexpressing MhMAPK4. The fresh weight and the root length of WT also was lower than that of the transgenic tobacco. The net Cd

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. During infection of the host plant, Agrobacterium transfers T-DNA from its Ti plasmid into the host cell, which can then be integrated into the host genome. This unique genetic transformation capability has been employed as the dominant technology for producing genetically modified plants for both basic research and biotechnological applications. Agrobacterium has been well studied as a disease-causing agent. The Agrobacterium-mediated transformation process involves early attachment of the bacterium to the host’s surface, followed by transfer of T-DNA and virulence proteins into the plant cell. Throughout this process, the host plants exhibit dynamic gene expression patterns at each infection stage or in response to Agrobacterium strains with varying pathogenic capabilities. Shifting host gene expression patterns throughout the transformation process have effects on transformation frequency, host morphology, and metabolism. Thus, gene expression profiling during the Agrobacterium infection process can be an important approach to help elucidate the interaction between Agrobacterium and plants. This review highlights recent findings on host plant differential gene expression patterns in response to A. tumefaciens or related elicitor molecules.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. This pathogen is capable of transferring the T-DNA from its Ti plasmid to the host cell and, then, integrating it into the host genome. To date, this genetic transformation ability has been harnessed as the dominant technology to produce genetically modified plants for both basic research and crop biotechnological applications. However, little is known about the interaction between Agrobacterium tumefaciens and host plants, especially the host responses to Agrobacterium infection and its associated factors. We employed RNA-seq to follow the time course of gene expression in Arabidopsis seedlings infected with either an avirulent or a virulent Agrobacterium strain. Gene Ontology analysis indicated many biological processes were involved in the Agrobacterium-mediated transformation process, including hormone signaling, defense response, cellular biosynthesis, and nucleic acid metabolism. RNAseq and quantitative reverse transcription-polymerase chain reaction results indicated that expression of genes involved in host plant growth and development were repressed but those involved in defense response were induced by Agrobacterium tumefaciens. Further analysis of the responses of transgenic Arabidopsis lines constitutively expressing either the VirE2 or VirE3 protein suggested Vir proteins act to enhance plant defense responses in addition to their known roles facilitating T-DNA transformation.

The effects of sodium nitroprusside (SNP) on the multiplication, regeneration and rooting of Malushupehensis Rehd. var. pinyiensis Jiang in tissue culture have been investigated. The results showed that the multiplication of plantlets was promoted significantly by applying 20 μM SNP to the Murashige and Skoog (MS) medium containing 2.0 μM 6-benzylaminopurine (BA) and 1.0 μM zeatin (ZT). Multiplication of plantlets from the 1st subculture was more sensitive to SNP than that from the 4th or 7th subculture. The differentiation and regeneration of adventitious shoots from leaves or cotyledons increased significantly when 20–30 μM SNP was supplied to the medium MS containing 25 μM BA, 2.5 μM α-naphthaleneacetic acid (NAA) and 2.5 μM ZT. Adventitious shoots regeneration frequency from cotyledons was higher than that from leaves at the presence of SNP. The rooting of plantlets was promoted by SNP significantly and the best result for rooting was achieved in the half-strength MS medium containing 75 μM SNP. In addition, adventitious roots without callus distributed at the base of shoots when SNP was supplied.

Malus hupehensis Rehd. var. pinyiensis Jiang (Pingyi Tiancha, PYTC) is a botanical variety of Malus (tea crabapple) originating from China. This species is characterized as apomictic, and it is highly capable of resisting water-logging, shade, cold, and various diseases. Mitogen-activated protein kinase (MAPK) cascades have been implicated in the regulation of stress and developmental signals in plants. In this study, an MAPK gene, MhMAPK, has been isolated from a PYTC complementary DNA (cDNA) library using rapid amplification of cDNA ends. The gene encodes a 373-amino-acid protein with high-sequence similarity to other previously reported plants MAPKs. MhMAPK contains all 11 MAPK conserved sub-domains and the phosphorylation motif TEY, and when fused to the green fluorescent protein, it is found to be localized in the nucleus of epidermal cells of onion. Transcripts of MhMAPK accumulate when PYTC is treated with 20% polyethylene glycol and 200 mM NaCl. These results indicated that MhMAPK may be functional within the nucleus by phosphorylating transcriptional factors. This, in turn, allows plants to rapidly respond to the environmental signals.