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Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases
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作者 Tom Schreiber Anja Prange +7 位作者 Petra Schafer Thomas Iwen Ramona Grutzner Sylvestre Marillonnet Aurelie Lepage Marie Javelle Wyatt Paul Alain Tissier 《Molecular Plant》 SCIE CSCD 2024年第5期824-837,共14页
In plants and mammals,non-homologous end-joining is the dominant pathway to repair DNA doublestrand breaks,making it challenging to generate knock-in events.In this study,we identified two groups of exonucleases from ... In plants and mammals,non-homologous end-joining is the dominant pathway to repair DNA doublestrand breaks,making it challenging to generate knock-in events.In this study,we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana.We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems.In Arabidopsis thaliana,fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants.In addition,we demonstrated stable and heritable knock-ins in wheat in 1%of the primary transformants.Taken together,our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants. 展开更多
关键词 homology-directed repair knockin gene replacement CRISPR-Cas 50 exonuclease PLANTS
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BFP1:One of 700 Arabidopsis F-box proteins mediates degradation of JA oxidases to promote plant immunity
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作者 Claus Wasternack Bettina Hause 《Molecular Plant》 SCIE CSCD 2024年第3期375-376,共2页
A switch off in jasmonate(JA)signaling by hydroxylation upon wounding was described in 2008(Miersch et al.,2008).This initiated the cloning of CYP450 enzymes involved in hydroxylation and carboxylation of JA and its a... A switch off in jasmonate(JA)signaling by hydroxylation upon wounding was described in 2008(Miersch et al.,2008).This initiated the cloning of CYP450 enzymes involved in hydroxylation and carboxylation of JA and its amino acid conjugate JA-le,the most active form of JA compounds(reviewed by Wasternack and Feussner[2018]and Heitz et al.[2019]).Due to the central role of JA and JA-lle in plant stress responses and development(Wasternack and Hause,2013),catabolism of JA and its derivatives is an essential component in the sustainment of homeostasis of active JA compounds(Howe et al.,2018;Heitz et al.,2019).Catabolism of JA and JAIle takes place by JA-induced oxygenases(JOXs),also named JA-oxidases(JAOs),and by hydroxylation(CYP94B3/B1)followed by carboxylation(CYP94C1),respectively(Heitz et al.,2019).Another reaction performed by IAR3/ILL6 results in the deconjugation of JA-lle and 12-OH-JA-le to JA and 12-OH-JA,respectively(Heitz et al.,2019;Wasternack and Feussner,2018). 展开更多
关键词 CONJUGATE initiated MEDIA
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Tissue-specific signatures of metabolites and proteins in asparagus roots and exudates
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作者 Stefanie Doll Roxana Djalali Farahani-Kofoet +2 位作者 Rita Zrenner Andrea Henze Katja Witzel 《Horticulture Research》 SCIE 2021年第1期1044-1057,共14页
Comprehensive untargeted and targeted analysis of root exudate composition has advanced our understanding of rhizosphere processes.However,little is known about exudate spatial distribution and regulation.We studied t... Comprehensive untargeted and targeted analysis of root exudate composition has advanced our understanding of rhizosphere processes.However,little is known about exudate spatial distribution and regulation.We studied the specific metabolite signatures of asparagus root exudates,root outer(epidermis and exodermis),and root inner tissues(cortex and vasculature).The greatest differences were found between exudates and root tissues.In total,263 non-redundant metabolites were identified as significantly differentially abundant between the three root fractions,with the majority being enriched in the root exudate and/or outer tissue and annotated as‘lipids and lipid-like molecules’or‘phenylpropanoids and polyketides’.Spatial distribution was verified for three selected compounds using MALDI-TOF mass spectrometry imaging.Tissue-specific proteome analysis related root tissue-specific metabolite distributions and rhizodeposition with underlying biosynthetic pathways and transport mechanisms.The proteomes of root outer and inner tissues were spatially very distinct,in agreement with the fundamental differences between their functions and structures.According to KEGG pathway analysis,the outer tissue proteome was characterized by a high abundance of proteins related to‘lipid metabolism’,‘biosynthesis of other secondary metabolites’and‘transport and catabolism’,reflecting its main functions of providing a hydrophobic barrier,secreting secondary metabolites,and mediating water and nutrient uptake.Proteins more abundant in the inner tissue related to‘transcription’,‘translation’and‘folding,sorting and degradation’,in accord with the high activity of cortical and vasculature cell layers in growth-and development-related processes.In summary,asparagus root fractions accumulate specific metabolites.This expands our knowledge of tissue-specific plant cell function. 展开更多
关键词 function outer signature
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Hordedane diterpenoid phytoalexins restrict Fusarium graminearum infection but enhance Bipolaris sorokiniana colonization of barley roots
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作者 Yaming Liu Dario Esposto +10 位作者 Lisa K.Mahdi Andrea Porzel Pauline Stark Hidayat Hussain Anja Scherr-Henning Simon Isfort Ulschan.Bathe Ivan F.Acosta Alga Zuccaro Gerd U.Balcke Alain Tissier 《Molecular Plant》 SCIE CSCD 2024年第8期1307-1327,共21页
Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses.These include the induction of a cocktail of defense metabolites that typically re... Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses.These include the induction of a cocktail of defense metabolites that typically restrict pathogen virulence.Here,we investigate the interaction between barley roots and the fungal pathogens Bipolaris sorokiniana(Bs)and Fusarium graminearum(Fg)at the metabolite level.We identify hordedanes,a previously undescribed set of labdane-related diterpenoids with antimicrobial properties,as critical players in these interactions.Infection of barley roots by Bs and Fg elicits hordedane synthesis from a 60o-kb gene cluster.Heterologous reconstruction of the biosynthesis pathway in yeast and Nicotiana benthamiana produced several hordedanes,including one of the most functionally decorated products 19-β-hydroxy-hordetrienoic acid(19-OH-HTA).Barley mutants in the diterpene synthase genes of this cluster are unable to produce hordedanes but,unexpectedly,show reduced Bs colonization.By contrast,colonization by Fusarium graminearum,another fungal pathogen of barley and wheat,is 4-fold higher in the mutants completely lacking hordedanes.Accordingly,19-OH-HTA enhances both germination and growth of Bs,whereas it inhibits other pathogenic fungi,including Fg.Analysis of microscopy and transcriptomics data suggest that hordedanes delay the necrotrophic phase of Bs.Taken together,these results show that adapted pathogens such as Bs can subvert plant metabolic defenses to facilitate root colonization. 展开更多
关键词 BARLEY Hordeum vulgare diterpenoid phytoalexins gene cluster pathogenic fungi Bipolaris sorokininiana Fusarium graminearum
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The biosynthetic pathway of the hallucinogen mescaline and its heterologous reconstruction
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作者 Paula Berman Luis Alejandro de Haro +12 位作者 Ana-Rita Cavaco Sayantan Panda Younghui Dong Nikolay Kuzmich Gabriel Lichtenstein Yoav Peleg Hila Harat Adam Jozwiaki Jianghua Cai Uwe Heinig Sagit Meir Ilana Rogachev Asaph Aharoni 《Molecular Plant》 SCIE CSCD 2024年第7期1129-1150,共22页
Mescaline,among the earliest identified natural hallucinogens,holds great potential in psychotherapy treatment.Nonetheless,despite the existence of a postulated biosynthetic pathway for more than half a century,the sp... Mescaline,among the earliest identified natural hallucinogens,holds great potential in psychotherapy treatment.Nonetheless,despite the existence of a postulated biosynthetic pathway for more than half a century,the specific enzymes involved in this process are yet to be identified.In this study,we investigated the cactus Lophophora williamsii(Peyote),the largest known natural producer of the phenethylamine mescaline.We employed a multi-faceted approach,combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling,enzymatic assays,molecular modeling,and pathway engineering for pathway elucidation.We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway,and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates,likely modulating mescaline levels in Peyote.Finally,we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells,providing novel insights into several challenges hindering complete heterologous mescaline production.Taken together,our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline,safeguarding this valuable natural resource for future generations. 展开更多
关键词 mescaline Lophophora williamsi Peyote biosynthetic pathway de novo genome sequencing mo-lecular modeling
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Wound-Induced Shoot-to-Root Relocation of JA-Ile Precursors Coordinates Arabidopsis Growth 被引量:8
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作者 Adina Schulze Marlene Zimmer +4 位作者 Stefan Mielke Hagen Stellmach Charles WMelnyk Bettina Hause Debora Gasperini 《Molecular Plant》 SCIE CAS CSCD 2019年第10期1383-1394,共12页
Multicellular organisms rely on the movement of signaling molecules across cells,tissues,and organs to communicate among distal sites.In plants,localized leaf damage activates jasmonic acid(JA)-dependent transcription... Multicellular organisms rely on the movement of signaling molecules across cells,tissues,and organs to communicate among distal sites.In plants,localized leaf damage activates jasmonic acid(JA)-dependent transcriptional reprogramming in both harmed and unharmed tissues.Although it has been indicated that JA species can translocate from damaged into distal sites,the identity of the mobile compound(s),the tissues through which they translocate,and the effect of their relocation remain unknown.Here,we found that following shoot wounding,the relocation of endogenous jasmonates through the phloem is essential to initiate JA signaling and stunt growth in unharmed roots of Arabidopsis thaliana.By employing grafting experiments and hormone profiling,we uncovered that the hormone precursor cis-12-oxo-phytodienoic acid(OPDA)and its derivatives,but not the bioactive JA-Ile conjugate,translocate from wounded shoots into undamaged roots.Upon root relocation,the mobile precursors cooperatively regulated JA responses through their conversion into JA-Ile and JA signaling activation.Collectively,our findings demonstrate the existence of long-distance translocation of endogenous OPDA and its derivatives,which serve as mobile molecules to coordinate shoot-to-root responses,and highlight the importance of a controlled redistribution of hormone precursors among organs during plant stress acclimation. 展开更多
关键词 GRAFTING JASMONATE OPDA PHLOEM PHYTOHORMONE
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High-efficiencygenomeediting in plants mediated by a Cas9 gene containing multiple introns 被引量:3
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作者 Ramona Grützner Patrick Martin +5 位作者 Claudia Horn Samuel Mortensen Erin J.Cram Carolyn W.T.Lee-Parsons Johannes Stuttmann Sylvestre Marillonnet 《Plant Communications》 2021年第2期63-77,共15页
The recent discovery of the mode of action of the CRISPR/Cas9 systemhas provided biologists with a useful tool for generating site-specific mutations in genes of interest.In plants,site-targeted mutations are usually ... The recent discovery of the mode of action of the CRISPR/Cas9 systemhas provided biologists with a useful tool for generating site-specific mutations in genes of interest.In plants,site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome.The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs,including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA,and the sequence of the Cas9 nuclease itself.To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana,we investigated several features of its nucleotide and/or amino acid sequence,including the codon usage,the number of nuclear localization signals(NLSs),and the presence or absence of introns.We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one.However,the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence,which dramatically improved the editing efficiency of the constructs.None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype,whereas between 70%and 100%of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes.The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus. 展开更多
关键词 CRISPR Cas9 targeted mutagenesis gene targeting
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Reshaping of the Arabidopsis thaliana Proteome Landscape and Co-regulation of Proteins in Development and Immunity 被引量:1
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作者 Mona Bassal Mohammad Abukhalaf +11 位作者 Petra Majovsky Domenika Thieme Tobias Herr Mohamed Ayash Naheed Tabassum MHD Rami Al Shweiki Carsten Proksch Ali Hmedat Jorg Ziegler Justin Lee Steffen Neumann Wolfgang Hoehenwarter 《Molecular Plant》 SCIE CAS CSCD 2020年第12期1709-1732,共24页
Proteome remodeling is a fundamental adaptive response,and proteins in complexes and functionally related proteins are often co-expressed.Using a deep sampling strategy we define core proteomes of Arabidopsis thaliana... Proteome remodeling is a fundamental adaptive response,and proteins in complexes and functionally related proteins are often co-expressed.Using a deep sampling strategy we define core proteomes of Arabidopsis thaliana tissues with around 10 000 proteins per tissue,and absolutely quantify(copy numbers per cell)nearly 16 000 proteins throughout the plant lifecycle.A proteome-wide survey of global post-trans-lational modification revealed amino acid exchanges pointing to potential conservation of translational infi-delity in eukaryotes.Correlation analysis of protein abundance uncovered potentially new tissue-and age-specific roles of entire signaling modules regulating transcription in photosynthesis,seed development,and senescence and abscission.Among others,the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of cysteine-rich receptor-like kinases(CRKs)as ROS sensors in senescence.All of the components of ribo-some biogenesis factor(RBF)complexes were found to be co-expressed in a tissue-and age-specific manner,indicating functional promiscuity in the assembly of these less-studied protein complexes in Arabidopsis.Furthermore,we characterized detailed proteome remodeling in basalimmunity bytreatingAra-bidopsis seeldings with flg22.Through simultaneously monitoring phytohormone and transcript changes upon flg22 treatment,we obtained strong evidence of suppression of jasmonate(JA)and JA-isoleucine(JA-Ile)levels by deconjugation and hydroxylation by IAA-ALA RESISTANT3(IAR3)and JASMONATE-INDUCED OXYGENASE 2(JOX2),respectively,under the control of JASMONATE INSENSITIVE 1(MYC2),sug-gesting an unrecognized role of a new JA regulatory switch in pattern-triggered immunity.Taken together,the datasets generated in this study present extensive coverage of theArabidopsis proteome in various bio-logical scenarios,providing a rich resource available to the whole plant science community. 展开更多
关键词 ARABIDOPSIS deep proteomics protein co-expression plant immunity JASMONATE
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Determination of sex by jasmonate^FA 被引量:1
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作者 Claus Wasternack 《Journal of Integrative Plant Biology》 SCIE CAS CSCD 2020年第2期162-164,共3页
Jasmonic acid (JA) and some of its derivatives are important signals in plant stress responses such as biotic or abiotic stresses during alteration of the environment and are signals in several developmental processes... Jasmonic acid (JA) and some of its derivatives are important signals in plant stress responses such as biotic or abiotic stresses during alteration of the environment and are signals in several developmental processes such as trichome development, growth/defense balance, root growth, senescence, or light signaling (Wasternack and Hause 2013). 展开更多
关键词 DETERMINATION SEX JASMONATE
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Involvement of CYP347W1 in neurotoxin 3-nitropropionic acid-based chemical defense in mustard leaf beetle Phaedon cochleariae
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作者 Nanxia Fu Tobias Becker +3 位作者 Wolfgang Brandt Maritta Kunert Antje Burse Wilhelm Boland 《Insect Science》 SCIE CAS CSCD 2022年第2期453-466,共14页
Chrysomelina beetlesstore 3-nitropropionic acid in form of a pretoxin,isoxazolin-5-one glucoside-conjugated ester,to protect themselves against predators.Here we identified a cytochrome P450 monooxygenase,CYP347W1,to ... Chrysomelina beetlesstore 3-nitropropionic acid in form of a pretoxin,isoxazolin-5-one glucoside-conjugated ester,to protect themselves against predators.Here we identified a cytochrome P450 monooxygenase,CYP347W1,to be involved in the production of the 3-nitropropionic acid moiety of the isoxazolin-5-one glucoside ester.Knocking down CYP347W1 led to a significant depletion in the concentration of the isoxazolin-5-one glucoside ester and an increase in the concentration of the isoxazolin-5-one glucoside in the larval hemolymph.Enzyme assays with the heterologously expressed CYP347W1 showed freeβ-alanine was not the direct substrate.Homology modeling indicated thatβ-alanine-CoA ester can fit into CYP347W1’s active site.Furthermore,we proved that Phaedon cochleariae eggs are not able to de novo synthesize 3-NPA,although both isoxazolin-5-one glucoside and its 3-NPA-conjugated ester are present in the eggs.These results provide direct evidence for the involvement of CYP347W1 in the biosynthesis of a P.cochleariae chemical defense compound. 展开更多
关键词 chemical defense isoxazolin-5-one glucoside isoxazolin-5-one glucoside ester leaf beetles P450 3-nitropropionic acid(3-NPA)
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Defense-Related Calcium Signaling Mutants Uncovered via a Quantitative High-Throughput Screen in Arabidopsis thaliana 被引量:5
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作者 Stefanie Ranf Julia Grimmer +4 位作者 Yvonne P6schl Pascal Pecher Delphine Chinchilla Dierk Scheel Justin Lee 《Molecular Plant》 SCIE CAS CSCD 2012年第1期115-130,共16页
Calcium acts as a second messenger for signaling to a variety of stimuli including MAMPs (Microbe-Associated Molecular Patterns), such as fig22 and elf18 that are derived from bacterial flagellin and elongation fact... Calcium acts as a second messenger for signaling to a variety of stimuli including MAMPs (Microbe-Associated Molecular Patterns), such as fig22 and elf18 that are derived from bacterial flagellin and elongation factor Tu, respectively. Here, Arabidopsis thaliana mutants with changed calcium elevation (cce) in response to fig22 treatment were isolated and characterized. Besides novel mutant alleles of the fig22 receptor, FLS2 (Flagellin-Sensitive 2), and the receptor-associated kinase, BAK1 (Brassinosteroid receptor 1-Associated Kinase 1), the new cce mutants can be categorized into two main groups--those with a reduced or an enhanced calcium elevation. Moreover, cce mutants from both groups show differ- ential phenotypes to different sets of MAMPs. Thus, these mutants will facilitate the discovery of novel components in early MAMP signaling and bridge the gaps in current knowledge of calcium signaling during plant-microbe interactions. Last but not least, the screening method is optimized for speed (covering 384 plants in 3 or 10 h) and can be adapted to genetically dissect any other stimuli that induce a change in calcium levels. 展开更多
关键词 Calcium signaling/transport defense responses plant-microbe interactions signal transduction Arabidopsis.
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FAIR Computational Workflows 被引量:4
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作者 Carole Goble Sarah Cohen-Boulakia +5 位作者 Stian Soiland-Reyes Daniel Garijo Yolanda Gil Michael R.Crusoe Kristian Peters Daniel Schober 《Data Intelligence》 2020年第1期108-121,307,308,309,共17页
Computational workflows describe the complex multi-step methods that are used for data collection,data preparation,analytics,predictive modelling,and simulation that lead to new data products.They can inherently contr... Computational workflows describe the complex multi-step methods that are used for data collection,data preparation,analytics,predictive modelling,and simulation that lead to new data products.They can inherently contribute to the FAIR data principles:by processing data according to established metadata;by creating metadata themselves during the processing of data;and by tracking and recording data provenance.These properties aid data quality assessment and contribute to secondary data usage.Moreover,workflows are digital objects in their own right.This paper argues that FAIR principles for workflows need to address their specific nature in terms of their composition of executable software steps,their provenance,and their development. 展开更多
关键词 Computational workflow REPRODUCIBILITY Software FAIR data PROVENANCE
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