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
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
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
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
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
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
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
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
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
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
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
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
基金funded by grant no.031B0548 in the frame of the program"Crop plants of the future"from the Bundesministerium fur Bildung und Forschung to A.T.funded by the Investissement d’Avenir program of the French National Agency of Research for the project GENIUS(ANR-11-BTBR-0001_GENIUS).
文摘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.
基金supported by the Deutsche Forschungsgemeinschaft(DFG,German Research Foundation)grant no.400681449/GRK2498.
文摘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).
基金the German Federal Ministry of Education and Research(FKZ:01EA1706).
文摘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.
基金TI 800/7-1 and TI 800/7-2(SPP 2125 DECRyPT)from the Deutsche Forschungsgemeinschaft,Germany,to A.T.We also acknowledge support from the Cluster of Excellence on Plant Sciences(CEPLAS)the Deutsche Forschungsgemeinschaft under Germany's ExcellenceStrategy-EXC 2048/1-Project ID:390686111and the grant ZU263/11-2(SPP 2125DECRyPT)to A.Z.
文摘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.
基金fellowships from the Israel Ministry of Absorption and the Dean of the Plant Science Department in the Weizmann Institute.We thank Prof.David Nelson(University of Tennessee,USA)for the systematic naming of the cytochrome P450 enzymes characterized in this studyShmuel Regev and The Regev Nursery(Beit Elazari,Israel)for help in differentiating among Lophophora species+2 种基金Dr.Ziv Spiegelman for providing several N.benthamiana plantsXINTEZA(https://xinteza.com/)for funding this researchand the Adelis Foundation,the Leona M.and Harry B.Helmsley Charitable Trust,the Jeanne and Joseph Nissim Foundation for Life Sciences,Tom and Sondra Rykoff Family Foundation Research,Ron Sklare and the Raymond Burton Plant Genome Research Fund for supporting the A.A.laboratory。
文摘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.
文摘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.
基金supported by core funding of the IPBsupported by grant STU642-1/1(DFG/GRC,Deutsche Forschungsgemeinschaft)supported by the National Science Foundation MCB award 1516371 to C.L.-P.and E.C.
文摘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.
基金We thank the Leibniz Association for support and fundingMohammad Abukhalaf is funded by DFG grant HO 5063/2-1+1 种基金Mohamed Ayash is funded by DFG 400681449/GRK2498This work was also supported by de.NBI(FKZ 031 A 534A),a project of the BMBF(Bundesministerium fur Bildung und Forschung).
文摘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.
基金financial support through the program Interhana: Operational Program Education for Competitiveness–European Social Fund (project CZ.1.07/2.3.00/20.0165).
文摘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).
基金supported by the Max Planck Society and the China Scholarship Council(grant number 201406300098).
文摘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.
基金This work was supported by a Deutsche Forschungsgemeinschaft (DFG) grant,financed by the DFG grant,by the Swiss National Science Foundation grant
文摘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.
基金Carole Goble acknowledges funding by BioExcel2(H2020823830)IBISBA1.0(H2020730976)and EOSCLife(H2020824087)+3 种基金Daniel Schober’s work was financed by Phenomenal(H2020654241)at the initiation-phase of this effort,current work in kind contributionKristian Peters is funded by the German Network for Bioinformatics Infrastructure(de.NBI)and acknowledges BMBF funding under grant number 031L0107Stian Soiland-Reyes is funded by BioExcel2(H2020823830)Daniel Garijo,Yolanda Gil,gratefully acknowledge support from DARPA award W911NF-18-1-0027,NIH award 1R01AG059874-01,and NSF award ICER-1740683.
文摘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.