Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultiva...Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultivar has not yet been reported.Meanwhile,various genome editing technologies such as transfection of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)ribonucleoprotein complexes into cells are in the limelight.Utilizing the Cas9-expressing genotype possessing a reference genome can simplify the verification of an off-target effect,resolve the economic cost of Cas9 endonuclease preparation,and avoid the complex assembly process together with single-guide RNA(sgRNA)in the transfection approach.Thus,this study was designed to generate Cas9-expressing‘Heinz 1706’lines by establishing an Agrobacterium tumefaciens-mediated transformation(ATMT)procedure.Here,we report a rapid and reproducible transformation procedure for‘Heinz 1706’by finetuning various factors:A.tumefaciens strain,pre-culture and co-culture durations,a proper combination of phytohormones at each step,supplementation of acetosyringone,and shooting/rooting method.Particularly,through eluding subculture and simultaneously inducing shoot elongation and rooting from leaf cluster,we achieved a short duration of three months for recovering the transgenic plants expressing Cas9.The presence of the Cas9 gene and its stable expression were confirmed by PCR and qRT-PCR analyses,and the Cas9 gene integrated into the T_(0) plant genome was stably transmitted to T_(1) progeny.Therefore,we anticipate that our procedure appears to ease the conventional ATMT in‘Heinz 1706’,and the created Cas9-expressing‘Heinz 1706’lines are ultimately useful in gene editing via unilateral transfection of sgRNA into the protoplasts.展开更多
In the coming decades,plant agriculture must produce the food needed to sustain the world＇s burgeoning population.This challenge is made more daunting in light of a changing climate,scarcity of inputs such as water a...In the coming decades,plant agriculture must produce the food needed to sustain the world＇s burgeoning population.This challenge is made more daunting in light of a changing climate,scarcity of inputs such as water and fertilizer,and the need to grow more crops on increasingly marginal land.To achieve food security,we will need to fully exploit plant genetics to develop new crop varieties that produce higher yields of healthier food.展开更多
Breakthroughs in the generation of programmable sequence-specific nucleases (SSNs), such as zinc finger nucleases (ZFNs),TAL effector nucleases (TALENs) and the RNA-directed nuclease CRISPR-associated protein 9 (Cas9)...Breakthroughs in the generation of programmable sequence-specific nucleases (SSNs), such as zinc finger nucleases (ZFNs),TAL effector nucleases (TALENs) and the RNA-directed nuclease CRISPR-associated protein 9 (Cas9), have greatly increased the ease of plant genome engineering (Voytas, 2013; Malzahn et al.,2017). Programmable SSNs introduce a DNA double-strand break展开更多
The major type of human liver cancer is hepatocellular carcinoma(HCC), and there are currently many risk factors that contribute to this deadly disease. The majority of HCC occurrences are associated with chronic hepa...The major type of human liver cancer is hepatocellular carcinoma(HCC), and there are currently many risk factors that contribute to this deadly disease. The majority of HCC occurrences are associated with chronic hepatitis viral infection, and hepatitis B viral(HBV) infection is currently a major health problem in Eastern Asia. Elucidating the genetic mechanisms associated with HBV-induced HCC has been difficult due to the heterogeneity and genetic complexity associated with this disease. A repertoire of animal models has been broadly used to study the pathophysiology and to develop potential treatment regimens for HBVassociated HCC. The use of these animal models has provided valuable genetic information and has been an important contributor to uncovering the factors involved in liver malignant transformation, invasion and metastasis. Recently, transposon-based mouse models are becoming more widely used in liver cancer research to interrogate the genome by forward genetics and also used to validate genes rapidly in a reverse genetic manner. Importantly, these transposon-based rapid reverse genetic mouse models could become crucial in testing potential therapeutic agents before proceeding to clinical trials in human. Therefore, this review will cover the use of transposon-based mouse models toaddress the problems of liver cancer, especially HBVassociated HCC occurrences in Asia.展开更多
CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes（Sp Cas9） cleaves double-stranded DNA targeted by a chimeric single-guide RNA（sg ...CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes（Sp Cas9） cleaves double-stranded DNA targeted by a chimeric single-guide RNA（sg RNA）. For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sg RNAs under the control of Ca MV 35 S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19 20 bp of sg RNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an Sp Cas9 expressing cassette. Twostep cloning procedures：（1） annealing two target-specific oligonucleotides with overhangs specific to the Aar I restriction enzyme site of the binary vector; and（2） ligating the annealed oligonucleotides into the two Aar I sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTMsystem and unique Eco RI/Xho I sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.展开更多
Cloven hoof animals have been a major source of nutritious animal protein for humans for at least 10 000 years. Over this time many were domesticated into livestock and some were put under selection for traits of impo...Cloven hoof animals have been a major source of nutritious animal protein for humans for at least 10 000 years. Over this time many were domesticated into livestock and some were put under selection for traits of importance or cultural preference. Accompanying domestication, diseases were transmitted from livestock to humans as a result of living in close quarters.展开更多
Plants interact with a complex network of microorganisms,forming a dynamic holobiont that is crucial for their health,growth,and adaptation.This interconnected system is deeply influenced by environmental factors,whic...Plants interact with a complex network of microorganisms,forming a dynamic holobiont that is crucial for their health,growth,and adaptation.This interconnected system is deeply influenced by environmental factors,which modulate the relationships within the plant microbiome.Key environmental drivers such as light,temperature,and moisture can alter the balance of these interactions,impacting plant immunity,resilience,and overall fitness.The traditional disease triangle model,which emphasizes plant-pathogen-environment interactions,is enhanced by incorporating the role of the microbiome,revealing how microbial communities contribute to disease outcomes.This review highlights the importance of shifting focus from studying plants in isolation to embracing an integrated approach that accounts for the intricate interactions between plants,microbes,and their surrounding environments.Comprehending these interactions is pivotal as we explore new approaches,including advanced sequencing technologies and microbiome engineering,to optimize plant-microbe relationships for improved crop resilience.These insights are vital for developing sustainable agricultural practices to address the impacts of climate change and other environmental challenges.展开更多
The ability to design and synthesize the genome of a living organism marks one of the most significant achievements in synthetic biology. The first synthetic genomes were those of bacteria （Gibson et al., 2010; Hutch...The ability to design and synthesize the genome of a living organism marks one of the most significant achievements in synthetic biology. The first synthetic genomes were those of bacteria （Gibson et al., 2010; Hutchison et al., 2016）, but an international consortium of scientists is now undertaking the ambitious task of designing and synthesizing the entire genome of Saccharornayces cerevisiae, a eukaryotic single-celled yeast （Dymond et al., 2011; Kannan and Gibson, 2017）. One feature of the redesigned yeast genome, called Saccharomyces cerevisiae 2.0 or Sc2.0, is the ability to in-duce novel genetic variation.展开更多
Precision genetics and breeding have the potential to meet the agricultural needs and goals of the world in the 21 st century.These needs include increasing the efficiency of production of animals and improving their ...Precision genetics and breeding have the potential to meet the agricultural needs and goals of the world in the 21 st century.These needs include increasing the efficiency of production of animals and improving their products with minimal impact on the environment.The USA is the major innovator in genomic science and the acknowledged leader in formulating policies to regulate genetic applications in medicine and agriculture.However,governments worldwide have been exceedingly reluctant to support the introduction of genetically modified(GM)animals into agriculture.Regulatory policies have stagnated due to legal guidelines that could not anticipate the needs and solutions that are evident today.This must change if we are to maintain planetary integrity.I propose a new,market-based regulatory model for GM livestock that has both a strong scientific foundation and has worked for 10000 years.The model is similar to that for information technology in which specific algorithms drive computer and cell phone applications.Genome engineers write genetic algorithms that drive the traits in biological organisms.Accordingly,GM products should be viewed in terms of their use and public benefit rather than by limitations to the genetic programing coming from a few highly vocal groups.Genetic algorithms(Genapps)of the 21st century will include not only introduction of synthetic genes,but also complete natural and synthetic biochemical pathways to produce agricultural products that are maximally efficient,healthy to humans and animals,and sustainable in an era of changing climates while avoiding environmental degradation.展开更多
Ecological interactions between flowers and pollinators are all about timing. Flower opening/closing and scent emissions are largely synchronized with pollinator activity, and a circadian clock regulates these rhythms...Ecological interactions between flowers and pollinators are all about timing. Flower opening/closing and scent emissions are largely synchronized with pollinator activity, and a circadian clock regulates these rhythms. However, whether the circadian clock increases a plant＇s reproductive success by regulating these floral rhythms remains untested. Flowers of Nicotiana attenuata, a wild tobacco, diurnally and rhythmically open, emit scent and move vertically through a 140° arc to interact with nocturnal hawkmoths. We tethered flowers to evaluate the importanceof flower positions for Manduca flower position dramatically sexta-mediated pollinations; nfluenced pollination. We examined the pollination success of phase-shifted flowers, silenced in circadian clock genes, NaZTL, NaLHY, and NaTOCI, by RNAi. Circadian rhythms in N. attenuata flowers are responsible for altered seed set from outcrossed pollen.展开更多
The anticancer effect of chemotherapy has been historically attributed to directly inducing proliferating cancer cell death.1 Accumulating evidence suggests that chemotherapy also engages the immune system.Some chemot...The anticancer effect of chemotherapy has been historically attributed to directly inducing proliferating cancer cell death.1 Accumulating evidence suggests that chemotherapy also engages the immune system.Some chemotherapies can induce immunogenic cell death,leading to tumor antigen uptake and presentation by dendritic cells to activate tumor-reactive T cells.2 A new study by Wang et al.adds another dimension to this paradigm by demonstrating that chemotherapy pretreatment of cancer cells can directly activate virtual memory(VM)CD8^(+)T cells to mediate tumor cytotoxicity in an antigen-independent manner.展开更多
A grand challenge facing society is climate change caused mainly by rising CO_(2) concentration in Earth’s atmosphere.Terrestrial plants are linchpins in global carbon cycling,with a unique capability of capturing CO...A grand challenge facing society is climate change caused mainly by rising CO_(2) concentration in Earth’s atmosphere.Terrestrial plants are linchpins in global carbon cycling,with a unique capability of capturing CO_(2) via photosynthesis and translocating captured carbon to stems,roots,and soils for long-term storage.However,many researchers postulate that existing land plants cannot meet the ambitious requirement for CO_(2) removal to mitigate climate change in the future due to low photosynthetic efficiency,limited carbon allocation for long-term storage,and low suitability for the bioeconomy.To address these limitations,there is an urgent need for genetic improvement of existing plants or construction of novel plant systems through biosystems design(or biodesign).Here,we summarize validated biological parts(e.g.,protein-encoding genes and noncoding RNAs)for biological engineering of carbon dioxide removal(CDR)traits in terrestrial plants to accelerate land-based decarbonization in bioenergy plantations and agricultural settings and promote a vibrant bioeconomy.Specifically,we first summarize the framework of plant-based CDR(e.g.,CO_(2) capture,translocation,storage,and conversion to value-added products).Then,we highlight some representative biological parts,with experimental evidence,in this framework.Finally,we discuss challenges and strategies for the identification and curation of biological parts for CDR engineering in plants.展开更多
The flagellin-sensing mechanism is one of the most extensively studied topics in plant defense systems.This widespread interest arises from the ability of flagellin to trigger robust and extensive responses,establishi...The flagellin-sensing mechanism is one of the most extensively studied topics in plant defense systems.This widespread interest arises from the ability of flagellin to trigger robust and extensive responses,establishing it as a cornerstone for research into other defense mechanisms.Plants recognize bacterial flagellin epitopes through plasma-membrane-localized pattern-recognition receptors,initiating pattern-triggered immunity as the frontline defense against bacterial pathogens.In this review,we comprehensively summarize flagellin-sensing mechanisms and signal transduction pathways in plants.We compare the flagellin-sensing mechanisms of plants and mammals,focusing on epitope processing and recognition.We present detailed downstream signaling events,from receptor complex formation to transcriptional reprogramming.Furthermore,we highlight the evolutionary arms race between plants and bacteria and incorporate emerging insights into how flagellin-triggered responses are modulated by receptor networking,phytocytokines,and environmental factors.These findings suggest that flagellin-mediated immune responses are highly dynamic and context dependent.By synthesizing current knowledge and recent discoveries,this review provides updated perspectives on plant–microbe interactions and aims to inspire future research in plant immunity.展开更多
User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR- associated protein 9 （d...User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR- associated protein 9 （dCasg） fused with four tandem repeats of the transcriptional activator VP16 0/1=64） could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCasg-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 （designated CRISPR-Act2.0） yielded stronger transcrip- tional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator- likeeffector activation （mTALE-Act） system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.展开更多
The circadian clock is known to increase plant growth and fitness, and is thought to prepare plants for photosynthesis at dawn and dusk; whether this happens in nature was unknown. We transformed the native tobacco, N...The circadian clock is known to increase plant growth and fitness, and is thought to prepare plants for photosynthesis at dawn and dusk; whether this happens in nature was unknown. We transformed the native tobacco, Nicotiana attenuata to silence two core clock components, NaLHY （irLHY） and NaTOC1 （irTOC1）. We characterized growth and light- and dark-adapted photosynthetic rates （Ac） throughout a 24 h clay in empty vector-transformed （EV）, irLHY, and irTOC1 plants in the field, and in NaPhyA-and NaPhyB1-silenced plants in the glasshouse. The growth rates of irLHY plants were lower than those of EV plants in the field. While irLHY plants reduced Ac earlier at dusk, no differences between irLHY and EV plants were observed at dawn in the field, irLHY, but not EV plants, responded to light in the night by rapidly increasing Ac. Under controlled conditions, EV plants rapidly increased Ac in the day compared to dark-adapted plants at night; irLHY plants lost these time-dependent responses. The role of NaLHY in gating photosynthesis is independent of the light-depen- dent reactions and red light perceived by NaPhyA, but not NaPhyB1. In summary, the circadian clock allows plants not to respond photosynthetically to light at night by anticipating and gating red light-mediated in native tobacco.展开更多
Phytochromes mainly function in photoau- totrophic organisms to adjust growth in response to fluctuating light signals. The different isoforms of plant phytochromes often display both conserved and divergent roles, pr...Phytochromes mainly function in photoau- totrophic organisms to adjust growth in response to fluctuating light signals. The different isoforms of plant phytochromes often display both conserved and divergent roles, presumably to fine-tune plant responses to environmental signals and optimize fitness. Here we describe the distinct, yet partially redundant, roles of phytochromes NaPHYA, NaPHYB1 and NaPHYB2 in a wild tobacco species, Nicotiana attenuata using RNAi-silenced phytochrome lines. Consistent with results reported from other species, silencing the expression of NaPHYA or NaPHYB2 in N. attenuata had mild or no influence on plant develop- ment as long as NaPHYBI was functional; whereas silencing the expression of NaPHYB1 alone strongly altered flowering time and leaf morphology. Thecontribution of NaPHYB2 became significant only in the absence of NaPHYB1; plants silenced for both NaPHYB1 and NaPHYB2 largely skipped the rosette- stage of growth to rapidly produce long, slender stalks that bore flowers early： hallmarks of the shade- avoidance responses. The phenotyping of phyto- chrome-silenced lines, combined with sequence and transcript accumulation independent functional analysis, suggest the diversification of the phytochromes, and a dominant role of NaPHYB1 and NaPHYB2 in N. attenuata＇s vegetative and reproductive development.展开更多
Plants use sophisticated mechanisms of gene expression to control senescence in response to environmental stress or aging.ORE1(Arabidopsis thaliana NAC092)is a master regulator of senescence that belongs to the plant-...Plants use sophisticated mechanisms of gene expression to control senescence in response to environmental stress or aging.ORE1(Arabidopsis thaliana NAC092)is a master regulator of senescence that belongs to the plant-specific NAC transcription factor protein family.ORE1 has been reported to bind to multiple DNA targets to orchestrate leaf senescence,yet the mechanistic basis for recognition of the cognate gene sequence remains unclear.Here,we report the crystal structure of the ORE1-NAC domain alone and its DNA-binding form.The structure of DNA-bound ORE1-NAC revealed the molecular basis for nucleobase recognition and phosphate backbone interactions.We showthat local versatility in the DNA-binding site,in combination with domain flexibility of the ORE-NAC homodimer,is crucial for the maintenance of binding to intrinsically flexible DNA.Our results provide a platformfor understanding other plant-specific NAC protein-DNA interactions as well as insight into the structural basis of NAC regulators in plants of agronomic and scientific importance.展开更多
Nicotiana attenuata flowers, diurnally open,emit scents and move vertically to interact with nocturnal hawkmoth and day-active hummingbird pollinators. To examine the fitness consequences of these floral rhythms, we c...Nicotiana attenuata flowers, diurnally open,emit scents and move vertically to interact with nocturnal hawkmoth and day-active hummingbird pollinators. To examine the fitness consequences of these floral rhythms, we conducted pollination trials in the plant’s native habitat with phase-shifted flowers of plants silenced in circadian clock genes. The results revealed that some pollination benefits observed under glasshouse conditions were not reproduced under natural field conditions. Floral arrhythmicity increased pollination success by hummingbirds, while reducing those by hawkmoths in the field. Thus, floral circadian rhythms may influence a plant’s fitness by filtering pollinators leading to altered seed set from outcrossed pollen.展开更多
Dear Editor, In the past few years, the use of sequence-specific nucle- ases for efficient targeted mutagenesis has provided plant biologists with a powerful new approach for understanding gene function and developin...Dear Editor, In the past few years, the use of sequence-specific nucle- ases for efficient targeted mutagenesis has provided plant biologists with a powerful new approach for understanding gene function and developing new traits. These nucleases create DNA double-strand breaks at chromosomal targeted sites that are primarily repaired by the non-homologous end joining （NHEJ） or homologous recombination （HR） pathways. NHEJ is o^en imprecise and can introduce mutations at tar- get sites resulting in the loss of gene function. In contrast, HR uses a homologous DNA template for repair and can be employed to create site-specific sequence modifications or targeted insertions （Moynahan and Jasin, 2010）.展开更多
Global demand for food and bioenergy production has increased rapidly,while the area of arable land has been declining for decades due to damage caused by erosion,pollution,sea level rise,urban development,soil salini...Global demand for food and bioenergy production has increased rapidly,while the area of arable land has been declining for decades due to damage caused by erosion,pollution,sea level rise,urban development,soil salinization,and water scarcity driven by global climate change.In order to overcome this conflict,there is an urgent need to adapt conventional agriculture to water-limited and hotter conditions with plant crop systems that display higher water-use efficiency(WUE).Crassulacean acid metabolism(CAM)species have substantially higher WUE than species performing C3 or C4 photosynthesis.CAM plants are derived from C3 photosynthesis ancestors.However,it is extremely unlikely that the C3 or C4 crop plants would evolve rapidly into CAM photosynthesis without human intervention.Currently,there is growing interest in improving WUE through transferring CAM into C3 crops.However,engineering a major metabolic plant pathway,like CAM,is challenging and requires a comprehensive deep understanding of the enzymatic reactions and regulatory networks in both C3 and CAM photosynthesis,as well as overcoming physiometabolic limitations such as diurnal stomatal regulation.Recent advances in CAM evolutionary genomics research,genome editing,and synthetic biology have increased the likelihood of successful acceleration of C3-to-CAM progression.Here,we first summarize the systems biology-level understanding of the molecular processes in the CAM pathway.Then,we review the principles of CAM engineering in an evolutionary context.Lastly,we discuss the technical approaches to accelerate the C3-to-CAM transition in plants using synthetic biology toolboxes.展开更多
文摘Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultivar has not yet been reported.Meanwhile,various genome editing technologies such as transfection of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)ribonucleoprotein complexes into cells are in the limelight.Utilizing the Cas9-expressing genotype possessing a reference genome can simplify the verification of an off-target effect,resolve the economic cost of Cas9 endonuclease preparation,and avoid the complex assembly process together with single-guide RNA(sgRNA)in the transfection approach.Thus,this study was designed to generate Cas9-expressing‘Heinz 1706’lines by establishing an Agrobacterium tumefaciens-mediated transformation(ATMT)procedure.Here,we report a rapid and reproducible transformation procedure for‘Heinz 1706’by finetuning various factors:A.tumefaciens strain,pre-culture and co-culture durations,a proper combination of phytohormones at each step,supplementation of acetosyringone,and shooting/rooting method.Particularly,through eluding subculture and simultaneously inducing shoot elongation and rooting from leaf cluster,we achieved a short duration of three months for recovering the transgenic plants expressing Cas9.The presence of the Cas9 gene and its stable expression were confirmed by PCR and qRT-PCR analyses,and the Cas9 gene integrated into the T_(0) plant genome was stably transmitted to T_(1) progeny.Therefore,we anticipate that our procedure appears to ease the conventional ATMT in‘Heinz 1706’,and the created Cas9-expressing‘Heinz 1706’lines are ultimately useful in gene editing via unilateral transfection of sgRNA into the protoplasts.
基金supported in part by grants from the National Science Foundation(Nos.IOS-1444511and IOS-1339209)
文摘In the coming decades,plant agriculture must produce the food needed to sustain the world＇s burgeoning population.This challenge is made more daunting in light of a changing climate,scarcity of inputs such as water and fertilizer,and the need to grow more crops on increasingly marginal land.To achieve food security,we will need to fully exploit plant genetics to develop new crop varieties that produce higher yields of healthier food.
基金supported by a Collaborative Funding Grant from North Carolina Biotechnology Center and Syngenta Biotechnology (2016-CFG-8003)startup funds provided by East Carolina University and University of Maryland to Y.Q.a grant from the National Science Foundation (IOS-1339209)
文摘Breakthroughs in the generation of programmable sequence-specific nucleases (SSNs), such as zinc finger nucleases (ZFNs),TAL effector nucleases (TALENs) and the RNA-directed nuclease CRISPR-associated protein 9 (Cas9), have greatly increased the ease of plant genome engineering (Voytas, 2013; Malzahn et al.,2017). Programmable SSNs introduce a DNA double-strand break
基金Supported by Health Medical Research Fund No.11122171,the Food and Health Bureau,and the Hong Kong SAR Governmentthe Department of Applied Biology and Chemical Technology,The Hong Kong Polytechnic University,Hong Kong SAR(1-ZVAG,G-YBAY,G-UA94 and 1-ZE19)the NIH IMVTP grant No.T32 AI083196-04 to Tschida BR
文摘The major type of human liver cancer is hepatocellular carcinoma(HCC), and there are currently many risk factors that contribute to this deadly disease. The majority of HCC occurrences are associated with chronic hepatitis viral infection, and hepatitis B viral(HBV) infection is currently a major health problem in Eastern Asia. Elucidating the genetic mechanisms associated with HBV-induced HCC has been difficult due to the heterogeneity and genetic complexity associated with this disease. A repertoire of animal models has been broadly used to study the pathophysiology and to develop potential treatment regimens for HBVassociated HCC. The use of these animal models has provided valuable genetic information and has been an important contributor to uncovering the factors involved in liver malignant transformation, invasion and metastasis. Recently, transposon-based mouse models are becoming more widely used in liver cancer research to interrogate the genome by forward genetics and also used to validate genes rapidly in a reverse genetic manner. Importantly, these transposon-based rapid reverse genetic mouse models could become crucial in testing potential therapeutic agents before proceeding to clinical trials in human. Therefore, this review will cover the use of transposon-based mouse models toaddress the problems of liver cancer, especially HBVassociated HCC occurrences in Asia.
基金supported by Institute for Basic Science (IBS-R021-D1)
文摘CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes（Sp Cas9） cleaves double-stranded DNA targeted by a chimeric single-guide RNA（sg RNA）. For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sg RNAs under the control of Ca MV 35 S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19 20 bp of sg RNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an Sp Cas9 expressing cassette. Twostep cloning procedures：（1） annealing two target-specific oligonucleotides with overhangs specific to the Aar I restriction enzyme site of the binary vector; and（2） ligating the annealed oligonucleotides into the two Aar I sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTMsystem and unique Eco RI/Xho I sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.
文摘Cloven hoof animals have been a major source of nutritious animal protein for humans for at least 10 000 years. Over this time many were domesticated into livestock and some were put under selection for traits of importance or cultural preference. Accompanying domestication, diseases were transmitted from livestock to humans as a result of living in close quarters.
基金supported by a grant from the Institute for Basic Science(IBS-R021-D1-2024-a00)the National Research Foundation of Korea(NRF)grant funded by the Korea government(Ho-Seok Lee).
文摘Plants interact with a complex network of microorganisms,forming a dynamic holobiont that is crucial for their health,growth,and adaptation.This interconnected system is deeply influenced by environmental factors,which modulate the relationships within the plant microbiome.Key environmental drivers such as light,temperature,and moisture can alter the balance of these interactions,impacting plant immunity,resilience,and overall fitness.The traditional disease triangle model,which emphasizes plant-pathogen-environment interactions,is enhanced by incorporating the role of the microbiome,revealing how microbial communities contribute to disease outcomes.This review highlights the importance of shifting focus from studying plants in isolation to embracing an integrated approach that accounts for the intricate interactions between plants,microbes,and their surrounding environments.Comprehending these interactions is pivotal as we explore new approaches,including advanced sequencing technologies and microbiome engineering,to optimize plant-microbe relationships for improved crop resilience.These insights are vital for developing sustainable agricultural practices to address the impacts of climate change and other environmental challenges.
文摘The ability to design and synthesize the genome of a living organism marks one of the most significant achievements in synthetic biology. The first synthetic genomes were those of bacteria （Gibson et al., 2010; Hutchison et al., 2016）, but an international consortium of scientists is now undertaking the ambitious task of designing and synthesizing the entire genome of Saccharornayces cerevisiae, a eukaryotic single-celled yeast （Dymond et al., 2011; Kannan and Gibson, 2017）. One feature of the redesigned yeast genome, called Saccharomyces cerevisiae 2.0 or Sc2.0, is the ability to in-duce novel genetic variation.
文摘Precision genetics and breeding have the potential to meet the agricultural needs and goals of the world in the 21 st century.These needs include increasing the efficiency of production of animals and improving their products with minimal impact on the environment.The USA is the major innovator in genomic science and the acknowledged leader in formulating policies to regulate genetic applications in medicine and agriculture.However,governments worldwide have been exceedingly reluctant to support the introduction of genetically modified(GM)animals into agriculture.Regulatory policies have stagnated due to legal guidelines that could not anticipate the needs and solutions that are evident today.This must change if we are to maintain planetary integrity.I propose a new,market-based regulatory model for GM livestock that has both a strong scientific foundation and has worked for 10000 years.The model is similar to that for information technology in which specific algorithms drive computer and cell phone applications.Genome engineers write genetic algorithms that drive the traits in biological organisms.Accordingly,GM products should be viewed in terms of their use and public benefit rather than by limitations to the genetic programing coming from a few highly vocal groups.Genetic algorithms(Genapps)of the 21st century will include not only introduction of synthetic genes,but also complete natural and synthetic biochemical pathways to produce agricultural products that are maximally efficient,healthy to humans and animals,and sustainable in an era of changing climates while avoiding environmental degradation.
基金supported by European Research Council advanced grant Clockwork Green (No. 293926) to I.T.B.the Global Research Lab program (2012055546) from the National Research Foundation of Korea+1 种基金Institute for Basic Science (IBS-R021-D1)the Max Planck Society
文摘Ecological interactions between flowers and pollinators are all about timing. Flower opening/closing and scent emissions are largely synchronized with pollinator activity, and a circadian clock regulates these rhythms. However, whether the circadian clock increases a plant＇s reproductive success by regulating these floral rhythms remains untested. Flowers of Nicotiana attenuata, a wild tobacco, diurnally and rhythmically open, emit scent and move vertically through a 140° arc to interact with nocturnal hawkmoths. We tethered flowers to evaluate the importanceof flower positions for Manduca flower position dramatically sexta-mediated pollinations; nfluenced pollination. We examined the pollination success of phase-shifted flowers, silenced in circadian clock genes, NaZTL, NaLHY, and NaTOCI, by RNAi. Circadian rhythms in N. attenuata flowers are responsible for altered seed set from outcrossed pollen.
文摘The anticancer effect of chemotherapy has been historically attributed to directly inducing proliferating cancer cell death.1 Accumulating evidence suggests that chemotherapy also engages the immune system.Some chemotherapies can induce immunogenic cell death,leading to tumor antigen uptake and presentation by dendritic cells to activate tumor-reactive T cells.2 A new study by Wang et al.adds another dimension to this paradigm by demonstrating that chemotherapy pretreatment of cancer cells can directly activate virtual memory(VM)CD8^(+)T cells to mediate tumor cytotoxicity in an antigen-independent manner.
基金supported by the Center for Bioenergy Innovation,a U.S.Department of Energy(DOE)Bioenergy Research Center supported by the Biological and Environmental Research(BER)programthe Laboratory Directed Research and Development program of Oak Ridge National Laboratory.DL acknowledges financial support through the National Science Foundation(NSF)under Award Number 1833402.
文摘A grand challenge facing society is climate change caused mainly by rising CO_(2) concentration in Earth’s atmosphere.Terrestrial plants are linchpins in global carbon cycling,with a unique capability of capturing CO_(2) via photosynthesis and translocating captured carbon to stems,roots,and soils for long-term storage.However,many researchers postulate that existing land plants cannot meet the ambitious requirement for CO_(2) removal to mitigate climate change in the future due to low photosynthetic efficiency,limited carbon allocation for long-term storage,and low suitability for the bioeconomy.To address these limitations,there is an urgent need for genetic improvement of existing plants or construction of novel plant systems through biosystems design(or biodesign).Here,we summarize validated biological parts(e.g.,protein-encoding genes and noncoding RNAs)for biological engineering of carbon dioxide removal(CDR)traits in terrestrial plants to accelerate land-based decarbonization in bioenergy plantations and agricultural settings and promote a vibrant bioeconomy.Specifically,we first summarize the framework of plant-based CDR(e.g.,CO_(2) capture,translocation,storage,and conversion to value-added products).Then,we highlight some representative biological parts,with experimental evidence,in this framework.Finally,we discuss challenges and strategies for the identification and curation of biological parts for CDR engineering in plants.
基金supported by grants from the Institute for Basic Science(IBS-R021-D1-2025-a00)the National Research Foundation of Korea(RS-2024-00338015)to H.-S.L.and by the Startup Fund from Duke Kunshan University to E.Y.K.D.-H.Lsupported by a postdoctoral fellowship from the National Research Foundation of Korea(NRF-2021R1A6A3A03039464).No conflict of interest is declared.
文摘The flagellin-sensing mechanism is one of the most extensively studied topics in plant defense systems.This widespread interest arises from the ability of flagellin to trigger robust and extensive responses,establishing it as a cornerstone for research into other defense mechanisms.Plants recognize bacterial flagellin epitopes through plasma-membrane-localized pattern-recognition receptors,initiating pattern-triggered immunity as the frontline defense against bacterial pathogens.In this review,we comprehensively summarize flagellin-sensing mechanisms and signal transduction pathways in plants.We compare the flagellin-sensing mechanisms of plants and mammals,focusing on epitope processing and recognition.We present detailed downstream signaling events,from receptor complex formation to transcriptional reprogramming.Furthermore,we highlight the evolutionary arms race between plants and bacteria and incorporate emerging insights into how flagellin-triggered responses are modulated by receptor networking,phytocytokines,and environmental factors.These findings suggest that flagellin-mediated immune responses are highly dynamic and context dependent.By synthesizing current knowledge and recent discoveries,this review provides updated perspectives on plant–microbe interactions and aims to inspire future research in plant immunity.
基金This work was supported by startup funds from East Carolina University and University of Maryland-College Park and a Collaborative Funding grant from North Carolina Biotechnology Center and Syngenta Biotechnology （2016-CFG-8003） to Y.Q. This work was also supported by grants, including the Sichuan Youth Science and Technology Foundation （2017JQ0005）, the National Science Foundation of China （31771486）, and the Fundamental Research Funds for the Central Universities （ZYGX2016J119） to Y.Z.
文摘User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR- associated protein 9 （dCasg） fused with four tandem repeats of the transcriptional activator VP16 0/1=64） could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCasg-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 （designated CRISPR-Act2.0） yielded stronger transcrip- tional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator- likeeffector activation （mTALE-Act） system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.
基金supported by European Research Council advanced grant Clockwork Green(No. 293926) to I.T.B.the Global Research Lab program(2012055546) from the National Research Foundation of Korea+1 种基金Human Frontier Science Program(RGP0002/2012)the Max Planck Society
文摘The circadian clock is known to increase plant growth and fitness, and is thought to prepare plants for photosynthesis at dawn and dusk; whether this happens in nature was unknown. We transformed the native tobacco, Nicotiana attenuata to silence two core clock components, NaLHY （irLHY） and NaTOC1 （irTOC1）. We characterized growth and light- and dark-adapted photosynthetic rates （Ac） throughout a 24 h clay in empty vector-transformed （EV）, irLHY, and irTOC1 plants in the field, and in NaPhyA-and NaPhyB1-silenced plants in the glasshouse. The growth rates of irLHY plants were lower than those of EV plants in the field. While irLHY plants reduced Ac earlier at dusk, no differences between irLHY and EV plants were observed at dawn in the field, irLHY, but not EV plants, responded to light in the night by rapidly increasing Ac. Under controlled conditions, EV plants rapidly increased Ac in the day compared to dark-adapted plants at night; irLHY plants lost these time-dependent responses. The role of NaLHY in gating photosynthesis is independent of the light-depen- dent reactions and red light perceived by NaPhyA, but not NaPhyB1. In summary, the circadian clock allows plants not to respond photosynthetically to light at night by anticipating and gating red light-mediated in native tobacco.
基金supported by European Research Council advanced grant Clockwork Green (293926) to ITBthe Global Research Lab program (2012055546) from the National Research Foundation of Korea+2 种基金Institute for Basic Science (IBS-R021-D1)Human Frontier Science Program (RGP0002/2012)the Max Planck Society
文摘Phytochromes mainly function in photoau- totrophic organisms to adjust growth in response to fluctuating light signals. The different isoforms of plant phytochromes often display both conserved and divergent roles, presumably to fine-tune plant responses to environmental signals and optimize fitness. Here we describe the distinct, yet partially redundant, roles of phytochromes NaPHYA, NaPHYB1 and NaPHYB2 in a wild tobacco species, Nicotiana attenuata using RNAi-silenced phytochrome lines. Consistent with results reported from other species, silencing the expression of NaPHYA or NaPHYB2 in N. attenuata had mild or no influence on plant develop- ment as long as NaPHYBI was functional; whereas silencing the expression of NaPHYB1 alone strongly altered flowering time and leaf morphology. Thecontribution of NaPHYB2 became significant only in the absence of NaPHYB1; plants silenced for both NaPHYB1 and NaPHYB2 largely skipped the rosette- stage of growth to rapidly produce long, slender stalks that bore flowers early： hallmarks of the shade- avoidance responses. The phenotyping of phyto- chrome-silenced lines, combined with sequence and transcript accumulation independent functional analysis, suggest the diversification of the phytochromes, and a dominant role of NaPHYB1 and NaPHYB2 in N. attenuata＇s vegetative and reproductive development.
基金supported by the Korean Ministry of Science,Information/Communication Technology and Future Planning(NRF-2021R1A4A1031754,NRF-2019M3E5D6066058,and NRF-2022R1F1A1068269)I.C.was funded by BK21 Plus(4120200313623)of the Ministry of Education,Korea.
文摘Plants use sophisticated mechanisms of gene expression to control senescence in response to environmental stress or aging.ORE1(Arabidopsis thaliana NAC092)is a master regulator of senescence that belongs to the plant-specific NAC transcription factor protein family.ORE1 has been reported to bind to multiple DNA targets to orchestrate leaf senescence,yet the mechanistic basis for recognition of the cognate gene sequence remains unclear.Here,we report the crystal structure of the ORE1-NAC domain alone and its DNA-binding form.The structure of DNA-bound ORE1-NAC revealed the molecular basis for nucleobase recognition and phosphate backbone interactions.We showthat local versatility in the DNA-binding site,in combination with domain flexibility of the ORE-NAC homodimer,is crucial for the maintenance of binding to intrinsically flexible DNA.Our results provide a platformfor understanding other plant-specific NAC protein-DNA interactions as well as insight into the structural basis of NAC regulators in plants of agronomic and scientific importance.
基金supported by European Research Council advanced grant Clockwork Green(No.293926)to I.T.B.the Global Research Lab program(2012055546)from South Koreathe Max Planck Society
文摘Nicotiana attenuata flowers, diurnally open,emit scents and move vertically to interact with nocturnal hawkmoth and day-active hummingbird pollinators. To examine the fitness consequences of these floral rhythms, we conducted pollination trials in the plant’s native habitat with phase-shifted flowers of plants silenced in circadian clock genes. The results revealed that some pollination benefits observed under glasshouse conditions were not reproduced under natural field conditions. Floral arrhythmicity increased pollination success by hummingbirds, while reducing those by hawkmoths in the field. Thus, floral circadian rhythms may influence a plant’s fitness by filtering pollinators leading to altered seed set from outcrossed pollen.
文摘Dear Editor, In the past few years, the use of sequence-specific nucle- ases for efficient targeted mutagenesis has provided plant biologists with a powerful new approach for understanding gene function and developing new traits. These nucleases create DNA double-strand breaks at chromosomal targeted sites that are primarily repaired by the non-homologous end joining （NHEJ） or homologous recombination （HR） pathways. NHEJ is o^en imprecise and can introduce mutations at tar- get sites resulting in the loss of gene function. In contrast, HR uses a homologous DNA template for repair and can be employed to create site-specific sequence modifications or targeted insertions （Moynahan and Jasin, 2010）.
基金This work was supported by the Center for Bioenergy Innovation(CBI),a U.S.Department of Energy Bioenergy Research Center supported by the Office of Science Biological and Environmental Research(BER)The writing of this manuscript was also supported by the Department of Energy(Office of Science,Genomic Science Program)under award number DE-SC0008834+3 种基金SDL acknowledges support from the Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education(NRF-2019R1I1A1A01061727)DL acknowledges financial support from the National Science Foundation(NSF)under Award Number 1833402KM acknowledges support from start-up funding provided by the University of California,DavisPMS acknowledges support from the Department of Energy(DE-AC02-05CH11231).
文摘Global demand for food and bioenergy production has increased rapidly,while the area of arable land has been declining for decades due to damage caused by erosion,pollution,sea level rise,urban development,soil salinization,and water scarcity driven by global climate change.In order to overcome this conflict,there is an urgent need to adapt conventional agriculture to water-limited and hotter conditions with plant crop systems that display higher water-use efficiency(WUE).Crassulacean acid metabolism(CAM)species have substantially higher WUE than species performing C3 or C4 photosynthesis.CAM plants are derived from C3 photosynthesis ancestors.However,it is extremely unlikely that the C3 or C4 crop plants would evolve rapidly into CAM photosynthesis without human intervention.Currently,there is growing interest in improving WUE through transferring CAM into C3 crops.However,engineering a major metabolic plant pathway,like CAM,is challenging and requires a comprehensive deep understanding of the enzymatic reactions and regulatory networks in both C3 and CAM photosynthesis,as well as overcoming physiometabolic limitations such as diurnal stomatal regulation.Recent advances in CAM evolutionary genomics research,genome editing,and synthetic biology have increased the likelihood of successful acceleration of C3-to-CAM progression.Here,we first summarize the systems biology-level understanding of the molecular processes in the CAM pathway.Then,we review the principles of CAM engineering in an evolutionary context.Lastly,we discuss the technical approaches to accelerate the C3-to-CAM transition in plants using synthetic biology toolboxes.
