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Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes

Molecular Plant 2023年第3期16卷 616-631页

作者： Qian Liu Chenglu Zhao Kai Sun Yinlu Deng Zhenghe Li State Key Laboratory of Rice Biology Institute of BiotechnologyZhejang UniversityHangzhouChina Ministry of Agricutture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests Zhejiang UniversityHangzhouChina Key Laboratory of Biology of Crop Pathogens and Insects of zhejang Province Zhejang UniversityHangzhouChina

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding.However,the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science.Here,we report an efficient,non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus(TSWV),an RNA virus with a host range of over 1000 plant species.We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts.The resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base editors.In systemically infected plant tissues,the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency.Plants with heritable,bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection.Moreover,we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100%of regenerated plants and further augmented the recovery of heritable mutations.Because many plants are recalcitrant to stable transformation,the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.

关键词： genome editing CRISPR-Cas base editor RNA virus vector tomato spotted wilt virus bunyavirus delivery non-transgenic

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Disruption of three polyamine uptake transporter genes in rice by CRISPR/Cas9 gene editing confers tolerance to herbicide paraquat

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aBIOTECH 2022年第2期3卷 140-145页

作者： Yu-Shu Lyu Li-Miao Cao Wen-Qian Huang Jian-Xiang Liu Hai-Ping Lu State Key Laboratory of Plant Physiology and Biochemistry Cllge of Life SciencesZhejang UniversityHangzhou 310058China State Key Laboratory of Rice Biology Zhejang Provincial Key Laboratory of Crop Genetie ResourcesInstitute of Crop ScienceZhejiang UniversityHangzhou 310058China

Weeds are a major biotic constraint that can cause dramatic crop production losses.Herbicide technology has been widely used by farmers as the most cost-effective weed control measure,and development of new strategy to improve herbicide tolerance in plants is urgently needed.The CRISPR/Cas9-based genome editing tool has been used in diverse applications related to agricultural technology for crop improvement.Here we identified three polyamine uptake transporter(PUT)genes in rice that are homologous to the Arabidopsis AtRMV1.We successfully demonstrate that CRISPR/Cas9-targeted mutagenesis of OsPUT1/2/3 greatly improves paraquat resistance in rice without obvious yield penalty.Therefore,manipulation of these loci could be valuable for producing transgene-free rice with improved herbicide resistance in future.

关键词： Paraquat OsPUTs CRISPR/Cas9 Rice

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High-resolution anatomical and spatial transcriptome analyses reveal two types of meristematic cell pools within the secondary vascular tissue of poplar stem

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Molecular Plant 2023年第5期16卷 809-828页

作者： Juan Du Yichen Wang Wenfan Chen Mingling Xu Ruhong Zhou Huixia Shou Jun Chen State Key Laboratory of Plant Physiology and Biochemistry College of Life SciencesZhejang University866Yu Hangtang RoadHangzhouZhejiang 310058China MOE Key Laboratory of Biosystems Homeostasis&Protection College of Life SciencesZhejang University866 Yu Hangtang RoadHangzhouZhejang 310058China Institute of Quantitative Biology College of Life SciencesZhejiang UniversityHangzhouZhejiang 310058China Hangzhou Botanical Garden Taoyuanling RoadHangzhouZhejiang 310013China

The secondary vascular tissue emanating from meristems is central to understanding how vascular plants such as forest trees evolve,grow,and regulate secondary radial growth.However,the overall molecular characterization of meristem origins and developmental trajectories from primary to secondary vascular tissues in woody tree stems is technically challenging.In this study,we combined high-resolution anatomic analysis with a spatial transcriptome(ST)technique to define features of meristematic cells in a developmental gradient from primary to secondary vascular tissues in poplar stems.The tissue-specific gene expression of meristems and derived vascular tissue types were accordingly mapped to specific anatomical domains.Pseudotime analyses were used to track the origins and changes of meristems throughout the development from primary to secondary vascular tissues.Surprisingly,two types of meristematic-like cell pools within secondary vascular tissues were inferred based on high-resolution microscopy combined with ST,and the results were confirmed by in situ hybridization of,transgenic trees,and single-cell sequencing.The rectangle shape procambium-like(PCL)cells develop from procambium meristematic cells and are located within the phloem domain to produce phloem cells,whereas fusiform shape cambium zone(CZ)meristematic cells develop from fusiform metacambium meristematic cells and are located inside the CZ to produce xylem cells.The gene expression atlas and transcriptional networks spanning the primary transition to secondary vascular tissues generated in this work provide new resources for studying the regulation of meristem activities and the evolution of vascular plants.A web server(https://***/stRNAPal/)was also established to facilitate the use of ST RNA-seq data.

关键词： poplar stem primary and secondary meristem primary and secondary vascular tissue spatial transcriptome single-cell sequencing

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Structures and ion transport mechanisms of plant high-affinity potassium transporters

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Molecular Plant 2024年第3期17卷 409-422页

作者： Jiangqin Wang Yanping Luo Fan Ye Zhong Jie Ding Shao Jian Zheng Shuai Qiao Yong Wang Jiangtao Guo Wei Yang Nannan Su international Institutes of Medicine the Fourth Affiliated HospitalZhejang University School of MedicineYiwuZhejiang 322000China Department of Biophysics and Department of Neurology of the Fourth Affiliated Hospital Zhejiang University School of MedicineHangzhouZhejang 310058China State Key Laboratory of Plant Environmental Resilience College of Life SciencesZhejang UniversityHangzhouZhejang 310058China College of Life Sciences Zhejiang UniversityHangzhouZhejiang 310058China NHC and CAMS Key Laboratory of Medical Neurobiology MOE Frontier Science Center for Brain Science and Brain-machine IntegrationSchool of Brain Science and Brain MedicineZhejiang UniversityHangzhouZhejiang 310058China Nanhu Brain-computer Interface Institute Hangzhou.Zheiang 311100.China

Plant high-affinity K^(+) transporters(HKTs)mediate Na^(+) and K^(+) uptake,maintain Na^(+)/K^(+) homeostasis,and therefore play crucial roles in plant salt tolerance.In this study,we present cryoelectron microscopy structures of HKTs from two classes,classI HKT1;1 from Arabidopsis thaliana(AtHKT1;1)and class II HKT2;1 from Triticum aestivum(TaHKT2;1),in both Na^(+) -and K^(+) -bound states at 2.6-to 3.0-A resolutions.BothAtHKT1;1and TaHKT2;1function ashomodimers.Each HKT subunit consists of four tan-dem domain units(D1-D4)with a repeated K^(+) -channel-like M-P-M topology.In each subunit,D1-D4 assemble into an ion conduction pore with a pseudo-four-fold symmetry.Although both TaHKT2;1and AtHKT1;1 have only one putative Na^(+) ion bound in the selectivity filter with a similar coordination pattern,the two HKTs display different K^(+) binding modes in the filter.TaHKT2;1 has three K^(+) ions bound in the selec-tivity filter,but AtHKT1;1 has only two K^(+) ions bound in the filter,which has a narrowed external entrance due to the presence of a Ser residue in the first filter motif.These structures,along with compu-tational,mutational,and electrophysiological analyses,enable us to pinpoint key residues that are critical for the ion selectivity of HKTs.The findings provide new insights into the ion selectivity and ion transport mechanisms of plant HKTs and improve our understanding about how HKTs mediate plant salt tolerance and enhance crop growth.

关键词： HKTs structure ion selectivity mechanism salt tolerance

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Roles of scaffolding protein Gab1 in regulation of myeloid dendritic cell-mediated allergic asthma

Roles of scaffolding protein Gab1 in regulation of myeloid d...

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中国生物化学与分子生物学会第十一次会员代表大会暨201...

作者： Yun Zhang Yun Xu Dong Cen Jiaqi Xu Xue Zhang Yuehai Ke Zhejang University Zhejang University

Scaffolding proteins Gab1 is ubiquitously expressed and belongs to the GAB family(GABs),which integrates multiple signal events.This protein family has recently attracted interestthat genetic variants of human GAB1 are considered a risk factor for asthma.Our previousresearch has revealed a novel function of Gab2 in mediating mucin gene expression andGoblet cell hyperplasia(GCH),which is an important process in asthma.However,the functionof Gab1 in asthma remains unclear.In asthma exacerbated patients,we found a prominentelevated level of Gab1 in peripheral blood monouclear cells compared to that of convalescencepatients.Next,we generated conditional Gab1 deletion mice in myeloid cells(Gab1 KO mice)to determinate physiological the role of Gab1 in asthma.Lung histologyand mucus metaplasia indicated that disruption of Gab1 in dendritic cells(DCs)leads to alleviatedasthmatic inflammation.Future investigation showed that loss of Gab1results inimpaired DC migration to lymph node,while has no influences on DC maturation、antigenuptake and presentation.Mechanically,Gab1 is essentially involved in CCL19-mediatedAKT activation.Together,these finding has characterized a novel of Gab1 in mediating DCmigration and that deregulation of Gab1 signal in lungs predisposes asthmatic inflammation.

关键词： Gab1 dendritic cell allergic asthma

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Phosphate-dependent regulation of vacuolar trafficking of OsSPX-MFSs is critical for maintaining intracellular phosphate homeostasis in rice

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Molecular Plant 2023年第8期16卷 1304-1320页

作者： Runze Guo Qi Zhang Kun Qian Yinghui Ying Wenying Liao Lening Gan Chuanzao Mao Yong Wang James Whelan Huixia Shou State Key Laboratory of Piant Physiology and Biochemistry Cllege of Life SciencesZhejiang UniversityHangzhouZhejang 310058P.R.China Hainan Institute Zhejiang UniversitySanya 572025China The Provincial International Science and Technology Cooperation Base on Engineering Biology International Campus of Zhejiang UniversityHainingZhejang 314400China Zhijiang lab Hangzhou 310012China

Vacuolar storage of inorganic phosphate(Pi)is essential for Pi homeostasis in plants.The SPX-MFS family proteins have been demonstrated to be vacuolar Pi transporters in many plant species.Transcriptional regulation of the predominant transporter among rice SPX-MFSs,OsSPX-MFS3,was only moderately suppressed by Pi starvation.Thus,post-transcriptional mechanisms were hypothesized to regulate the activity of OsSPX-MFS3.In this study,we found that the tonoplast localization of OsSPX-MFSs is inhibited under Pi-depleted conditions,resulting in their retention in the pre-vacuolar compartments(PVCs).A yeast two-hybrid screen identified that two SNARE proteins,OsSYP21 and OsSYP22,interact with the MFS domain of OsSPX-MFS3.Further genetic and cytological analyses indicate that OsSYP21 and OsSYP22 facilitate trafficking of OsSPX-MFS3 from PVCs to the tonoplast.Although a homozygous frameshift mutation in OsSYP22 appeared to be lethal,tonoplast localization of OsSPX-MFS3 was significantly inhibited in transgenic plants expressing a negative-dominant form of OsSYP22(OsSYP22-ND),resulting in reduced vacuolar Pi concentrations in OsSYP22-ND plants.Under Pi-depleted conditions,the interaction between OsSYP22 and OsSPX-MFS3 was disrupted,and this process depended on the presence of the SPX domain.Deleting the SPX domains of OsSPX-MFSs resulted in their tonoplast localization under both Pi-depleted and Pi-replete conditions.Complementation of the osspx-mfs1/2/3 triple mutants with the MFS domain or the SPX domain of OsSPX-MFS3 confirmed that the MFS and SPX domains are responsive to Pi transport activity and Pi-dependent regulation,respectively.These data indicated that the SPX domains of OsSPX-MFSs sense cellular Pi(InsP)levels and,under Pi-depleted conditions,inhibit the interaction between OsSPX-MFSs and OsSYP21/22 and subsequent trafficking of OsSPX-MFSs from PVCs to the tonoplast.

关键词： fir homeostasis inhibited

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PDGFR in PDGF-BB/PDGFR Signaling Pathway Does Orchestrates Osteogenesis ina Temporal Manner

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Research 2024年第1期2023卷 33-48页

作者： Fangqian Wangg Yuxiao Ye Zengjie Zhang Wangsiyuan Teng Hangxiang Sun Xupeng Chai Xingzhi Zhou Jiayu Chen Haochen Mou Yinwang Eloy Xiaoqiang Jin Liang Chen Zhenxuan Shao Yan Wu Yue Shen An Liu Peng Lin Jianwei Wang Xiaohua Yu Zhaoming Ye Department of Orthopedic Surgery the Second Affiliated HospitalZhejang University School of MedicineHangzhou CityZhejiang ProvincePR China Orthopedics Research Institute of Zhejang University Hangzhou CityZhejiang ProvincePR China Key Laboratory of Motor System Disease Research and Precision Therapy of Zhejiang Province Hangzhou CityZhejiang ProvincePR China School of Material Science and Engineering University of New South WalesSydney 2052Australia

Platelet-derived growth factor-BB(PDGF-BB)/platelet-derived growth factor receptor-β(PDGFR-β)pathway is conventionally considered as an important pathway to promote osteogenesis;however,recent study suggested its role during osteogenesis to be controversial.Regarding the differential functions of this pathway during 3 stages of bone healing,we hypothesized that temporal inhibition of PDGF-BB/PDGFR-βpathway could shift the proliferation/differentiation balance of skeletal stem and progenitor cells,toward osteogenic lineage,which leads to improved bone regeneration.We first validated that inhibition of PDGFR-βat late stage of osteogenic induction effectively enhanced differentiation toward osteoblasts.This effect was also replicated invivo by showing accelerated bone formation when block PDGFR-βpathway at late stage of critical bone defect healing mediated using biomaterials.Further,we found that such PDGFR-βinhibitor-initiated bone healing was also effective in the absence of scaffold implantation when administrated intraperitoneally.Mechanistically,timely inhibition of PDGFR-βblocked extracellular regulated protein kinase 1/2 pathway,which shift proliferation/differentiation balance of skeletal stem and progenitor cell to osteogenic lineage by upregulating osteogenesis-related products of Smad to induce osteogenesis.This study offered updated understanding of the use of PDGFR-βpathway and provides new insight routes of action and novel therapeutic methods in the field of bone repair.

关键词： healing sized implantation

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Exact Boundary Controllability of Fifth-order KdV Equation Posed on the Periodic Domain

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Journal of Partial Differential Equations 2022年第2期35卷 163-172页

作者： YANG Shuning ZHAO Xiangqing Schol of Information and Engineering Zhejang Ocean UniversityZhoushan316022China Department of Mathmatics Suqian UniversitySuqian 223800China

In this paper,we show by Hilbert Uniqueness Method that the boundary value problem of fifth-order KdV equation{y_(t)-y_(5x)=0,(x,t)∈(0,2π)×(0,T),y(t,2π)-y(t,0)=h_(0)(t),y_(x)(t,2π)-y_(x)(t,0)=h_(1)(t),y_(2x)(t,2π)-y_(2x)(t,0)=h_(2)(t),y_(3x)(t,2π)-y_(3x)(t,0)=h_(3)(t),y_(4x)(t,2π)-y_(4x)(t,0)=h_(4)(t),(with boundary data as control inputs)is exact controllability.

关键词： Fifth-order KdV equation Hilbert Uniqueness Method exact controllability

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Structural variation(SV)-based pan-genome and GWAS reveal the impacts of SVs on the speciation and diversification of allotetraploid cottons

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Molecular Plant 2023年第4期16卷 678-693页

作者： Shangkun Jin Zegang Han Yan Hu Zhanfeng Si Fan Dai Lu He Yu Cheng Yiqjan Li Ting Zhao Lei Fang Tianzhen Zhang zhejang Provincial Engineering Center for Crop Precision Breeding Advanced Seed InstitutePlant Precision Breeding AcademyCollege of Agriculture and BiotechnologyZhejiang UniversityHangzhouChina Hainan Institute of Zhejiang university Sanya 572025China

Structural variations(SVs)have long been described as being involved in the origin,adaption,and domes-tication of species.However,the underlying genetic and genomic mechanisms are poorly understood.Here,we report a high-quality genome assembly of Gossypium barbadense acc.Tanguis,a landrace that is closely related to formation of extra-long-staple(ELS)cultivated cotton.An SV-based pan-genome(Pan-SV)was then constructed using a total of 182593 non-redundant SVs,including 2236 inversions,97398 insertions,and 82959 deletions from 11 assembled genomes of allopolyploid cotton.The utility of this Pan-sV was then demonstrated through population structure analysis and genome-wide association studies(GWASs).Using segregation mapping populations produced through crossing ELS cotton and the landrace along with an Sv-based GWAs,certain SVs responsible for speciation,domestication,and improvement in tetraploid cottons were identified.Importantly,some of the SVs presently identified as associated with the yield and fiber quality improvement had not been identified in previous SNP-based GWAS.In particular,a 9-bp insertion or deletion was found to associate with elimination of the interspecific reproductive isolation between Gossypium hirsutum and G.barbadense.Collectively,this study provides new insights into genome-wide,gene-scale SVs linked to important agronomic traits in a major crop spe-cies and highlights the importance of sVs during the speciation,domestication,and improvement of culti-vated crop species.

关键词： genome assembly structural variations SV-based pan-genome GWAS introgression QTL mapping

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Enantioselective Reductive Cross-Coupling of Aryl/Alkenyl Bromides with Benzylic Chlorides via Photoredox/Biimidazoline Nickel Dual Catalysis

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Chinese Journal of Chemistry 2022年第9期40卷 1033-1038页

作者： Tongtong Li Xiaokai Cheng Jiamin Lu Huifeng Wang Qun Fang Zhan Lu Department of Chemistry Zhejiang University 866 Yuhangtang roadHangzhouZhejiang 310058China College of Chemistry ZhengzhouUniversityZhengzhouHenan 450001China Hangzhou Innovation Center Zhejang UniversityHangzhouZhejiang 311200China

The asymmetric reductive arylation and alkenylation of benzylic chloride under photoredox/nickel dual catalysis using chiral bimidazoline(Bilm)ligand is reported to access 1,1-diaryl alkanes and aryl allylic compounds with good yield as well as stereo-and enantioselectivities.This protocol uses more commercially available and less expensive C(sp^(2))-Br as the electrophile coupling partner.A primary result using alkenyl chloride and alkyl chloride is also reported.Various functional groups are tolerated and the applications of this method are investigated by late-stage functionalization and gram-scale reaction.

关键词： Cross-coupling Photocatalysis Enantioselectivity Stereoselectivity Bimidazoline

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