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2. Genome Editing Tools for Potato Improvement

Author

Ravet, Karl, Sevestre, François, Chauvin, Laura, Chauvin, Jean-Eric, Lairy-Joly, Gisèle, Katz, Andrew, Devaux, Pierre, Szydlowski, Nicolas, Gallois, Jean-Luc, Pearce, Stephen, Veillet, Florian, Zhao, Kaijun, editor, Mishra, Rukmini, editor, and Joshi, Raj Kumar, editor

Published
2022
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3. The α/β hydrolase domain-containing protein 1 (ABHD1) acts as a lysolipid lipase and is involved in lipid droplet formation

Author

Torres-Romero, Ismael, primary, Légeret, Bertrand, additional, Huleux, Marie, additional, Sorigue, Damien, additional, Damm, Alicia, additional, Cuiné, Stéphan, additional, Veillet, Florian, additional, Blot, Carla, additional, Brugière, Sabine, additional, Couté, Yohann, additional, Garneau, Matthew G., additional, Kotapati, Hari Kiran, additional, Xin, Yi, additional, Xu, Jian, additional, Bates, Philip D., additional, Thiam, Abdou Rachid, additional, Beisson, Fred, additional, and Li-Beisson, Yonghua, additional

Published
2023
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8. An iterative gene‐editing strategy broadens eIF4E1 genetic diversity in Solanum lycopersicum and generates resistance to multiple potyvirus isolates

Author

Kuroiwa, Kyoka, primary, Danilo, Benoit, additional, Perrot, Laura, additional, Thenault, Christina, additional, Veillet, Florian, additional, Delacote, Fabien, additional, Duchateau, Philippe, additional, Nogué, Fabien, additional, Mazier, Marianne, additional, and Gallois, Jean‐Luc, additional

Published
2023
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12. Gene Editing in Potato Using Technology

Author

Chauvin, Laura, Sevestre, François, Lukan, Tjaša, Nogué, Fabien, Gallois, Jean-Luc, Chauvin, Jean-Eric, Veillet, Florian, Institut de Génétique, Environnement et Protection des Plantes (IGEPP), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-INSTITUT AGRO Agrocampus Ouest, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Miniaturisation pour la Synthèse, l’Analyse et la Protéomique - UAR 3290 (MSAP), Department of Biotechnology and Systems Biology [Ljubljana, Slovenia], National Institute of Biology [Ljubljana] (NIB), Institut Jean-Pierre Bourgin (IJPB), AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Rennes (UR)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-INSTITUT AGRO Agrocampus Ouest, Génétique et Amélioration des Fruits et Légumes (GAFL), and Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
[SDV.GEN.GPL]Life Sciences [q-bio]/Genetics/Plants genetics, Single-guide RNA, Agrobacterium tumefaciens, Protoplasts, [SDV]Life Sciences [q-bio], fungi, food and beverages, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, CRISPR-Cas9, Gene editing, HRM analysis, Potato, Plant regeneration
Abstract

International audience; Genome editing in the cultivated potato (Solanum tuberosum), a vegetatively propagated and highly heterozygous species, constitutes a promising trail to directly improve traits into elite cultivars. With the recent and successful development of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system in eukaryotic cells, the plant science community has gained access to a powerful, inexpensive, and easy-to-use toolbox to target and inactivate/modify specific genes. The specificity and versatility of the CRISPR-Cas9 system rely on a variable 20 bp spacer sequence at the 5' end of a single-guide RNA (sgRNA), which directs the SpCas9 (Streptococcus pyogenes) nuclease to cut the target DNA at a precise locus with no or low off-target events. Using this system, we and other teams were able to knock out specific genes in potato through the error-prone non-homologous end-joining (NHEJ) DNA repair mechanism. In this chapter, we describe strategies to design and clone spacer sequences into CRISPR-SpCas9 plasmids. We show how these constructs can be used for Agrobacterium-mediated stable transformation or transient transfection of protoplasts, and we describe the optimization of these two delivery methods, as well as of the plant regeneration processes. Finally, the molecular screening and characterization of edited potato plants are also described, mainly relying on PCR-based methods such as high-resolution melt (HRM) analysis.

Published
2021

14. A blue-print for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

Author

Guyon-Debast, Anouchka, Alboresi, Alessandro, Terret, Zoé, Charlot, Florence, Berthier, Floriane, Vendrell-Mir, Pol, Casacuberta, Josep M., Veillet, Florian, Morosinotto, Tomas, Gallois, Jean-Luc, Nogue, Fabien, Institut Jean-Pierre Bourgin (IJPB), AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Universita degli Studi di Padova, Génétique et Amélioration des Fruits et Légumes (GAFL), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Spanish National Research Council (CSIC), Institut de Génétique, Environnement et Protection des Plantes (IGEPP), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), French National Research Agency French National Research Agency (ANR) [ANR11-BTBR-0001-GENIUS], LabEx Saclay Plant Sciences-SPS [ANR-10-LABX-0040-SPS], ANR-11-BTBR-0001,GENIUS,Ingénierie cellulaire : amélioration et innovation technologiques pour les plantes d'une agriculture(2011), University of Padova [Padova, Italy], Centre for Research in Agricultural Genomics (CRAG), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-INSTITUT AGRO Agrocampus Ouest, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), ANR-10-LABX-0040,SPS,Saclay Plant Sciences(2010), Agence Nationale de la Recherche (France), Institute of Plant Sciences Paris-Saclay, Università degli Studi di Padova = University of Padua (Unipd), and Université de Rennes (UR)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-INSTITUT AGRO Agrocampus Ouest

Subjects
Gene Editing, Physcomitrella patens, Adenine deaminase, Physcomitrium patens, APRT, Base editing, CRISPR, Cas9, Cytosine deaminase, Research, cytosine deaminase, [SDV]Life Sciences [q-bio], food and beverages, Bryopsida, Methods, Mutagenesis, Site-Directed, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
Abstract

CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation., CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens., Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens., These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing., The work, including study design, data collection, analysis and interpretation and manuscript writing, was supported by the French National Research Agency (ANR11-BTBR-0001-GENIUS). The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).

Published
2021

15. Toward transgene-free genome editing in poplar plants using Agrobacterium-mediated delivery of a CRISPR/Cas9 cytidine base editor

Author

Pilate, Gilles, Veillet, Florian, Touzet, Orlane, Déjardin, Annabelle, Déjardin, Annabelle, Biologie intégrée pour la valorisation de la diversité des Arbres et de la Forêt (BioForA), Office National des Forêts (ONF)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Génétique, Environnement et Protection des Plantes (IGEPP), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Office national des forêts (ONF)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Rennes (UR)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-INSTITUT AGRO Agrocampus Ouest, and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)

Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], fungi, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, food and beverages, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BV] Life Sciences [q-bio]/Vegetal Biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BIO] Life Sciences [q-bio]/Biotechnology
Abstract

International audience; We present here the first evidence of the precise targeting of point mutations in the genome of a forest tree species using a cytidine base editor (CBE). This was done using the classical Agrobacterium cocultivation method routinely used on our model hybrid poplar clone (INRA 717-1B4) for more than 30 years. Our ultimate goal is to produce transgene-free edited poplar plants. Indeed, in perennial species with long generation time, such as trees, it is virtually impossible to get rid of alien copies introduced into the plant genome during the cocultivation step. Therefore, using a strategy already shown to be successful in tomato and potato (Veillet et al., 2019), we targeted the endogenous poplar acetolactate synthase (ALS) gene by a CBE through Agrobacterium tumefaciens cocultivation. Using an optimized procedure, we were able to regenerate at high yield chlorsulfuron-resistant plants. Interestingly, a small number of these herbicide-resistant plants do not show evidence of T-DNA integration. Molecular analyses are under way to more accurately characterize these plants. Our most recent experiments aim to evaluate on this poplar model system the co-edition of ALS with another gene.

Published
2021

16. Improvement of crispr/CASg knock-out and base editing techniques in Apple and Pear

Author

Malabarba, Jaiana, Vergne, Emilie, Dousset, Nicolas, Moizan, Julie, Veillet, Florian, Chevreau, Elisabeth, Institut de Recherche en Horticulture et Semences (IRHS), Université d'Angers (UA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-INSTITUT AGRO Agrocampus Ouest, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Institut de Génétique, Environnement et Protection des Plantes (IGEPP), Université de Rennes (UR)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-INSTITUT AGRO Agrocampus Ouest, Centre for Research in Agriculture Genomics (CRAG), Université d'Angers (UA)-AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Rennes 1 (UR1), and Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-AGROCAMPUS OUEST

Subjects
[SDV.GEN.GPL]Life Sciences [q-bio]/Genetics/Plants genetics, Genomics-informed breeding, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], New breeding tools, [SDV.SA.HORT]Life Sciences [q-bio]/Agricultural sciences/Horticulture
Abstract

International audience; CRISPR/Cas9 has become the golden technique for gene knock-out in plants. In addition to gene knock-out through the generation of INDELsmutations, base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Our previous results with CRISPR/Cas9 in apple indicated the frequent production of phenotypic and edition chimeras, after edition of theeasily scorable gene phytoene desaturase (PDS). Therefore our rst goal was to determine if adding an adventitious regeneration step from leaves of the T0 plants, would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89 % werehomogeneous albino. Furthermore, the target zone sequences of twelve of these regenerated lines (T1) were studied and compared to the T0 sequences. The results showed that 99% of the T1 alleles were predicted to producing a truncated target protein and that 67% of T1 plants had less heterogeneous editing proles than the T0. This indicates that a regeneration step can efficiently reduce the initial chimerism. The second objective was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: PDS (conferring albino and dwarfphenotype by impaired chlorophyll and gibberellin synthesis) and acetolactate synthase (ALS) (conferring resistance to chlorsulfuron). The two guideRNAs (PDS+ALS) under MdU3 and MdU6 promoters, respectively, were coupled in the pDenCas9_PmCDA1_UGI vector which has a cytidine deaminase fused to a nickase Cas9. Using this cytidine-base editor, we precisely induced DNA substitutions in the target genes, leading to discrete variation in theamino-acid sequence and generating a loss-of-function allele. The successful application of base editing in the apple and pear creates new possibilities for genome engineering to explore desirable agronomic traits in these species.

Published
2020

17. A blueprint for gene function analysis through Base Editing in the model plantPhyscomitrium (Physcomitrella) patens

Author

Guyon‐Debast, Anouchka, primary, Alboresi, Alessandro, additional, Terret, Zoé, additional, Charlot, Florence, additional, Berthier, Floriane, additional, Vendrell‐Mir, Pol, additional, Casacuberta, Josep M., additional, Veillet, Florian, additional, Morosinotto, Tomas, additional, Gallois, Jean‐Luc, additional, and Nogué, Fabien, additional

Published
2021
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18. Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops

Author

Veillet, Florian, Perrot, Laura, Guyon-Debast, Anouchka, Kermarrec, Marie-Paule, Chauvin, Laura, Chauvin, Jean-Eric, Gallois, Jean-Luc, Mazier, Marianne, Nogué, Fabien, Institut de Génétique, Environnement et Protection des Plantes (IGEPP), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Génétique et Amélioration des Fruits et Légumes (GAFL), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Jean-Pierre Bourgin (IJPB), AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ANR-10-LABX-0040-SPS, Agence Nationale de la Recherche, ANR-11-BTBR-0001,GENIUS,Ingénierie cellulaire : amélioration et innovation technologiques pour les plantes d'une agriculture(2011), ANR-17-EURE-0007,SPS-GSR,Ecole Universitaire de Recherche de Sciences des Plantes de Paris-Saclay(2017), Université de Rennes (UR)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-INSTITUT AGRO Agrocampus Ouest, and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)

Subjects
SpCas9-NG, Physcomitrella patens, base editing, physcomitrella patens, tomato, lcsh:Chemistry, [SDV.GEN.GPL]Life Sciences [q-bio]/Genetics/Plants genetics, [SDV.BV.AP]Life Sciences [q-bio]/Vegetal Biology/Plant breeding, lcsh:Biology (General), lcsh:QD1-999, potato, CBE, xCas9, CRISPR-Cas9, lcsh:QH301-705.5, alternative PAM
Abstract

Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.

Published
2020

25. The molecular dialogue between Arabidopsis thaliana and the necrotrophic fungus Botrytis cinerea leads to major changes in host carbon metabolism

Author

Veillet, Florian, Gaillard, Cécile, Lemonnier, Pauline, Coutos-Thévenot, Pierre, La Camera, Sylvain, Sucres & Echanges Végétaux-Environnement (SEVE), Ecologie et biologie des interactions (EBI), and Université de Poitiers-Centre National de la Recherche Scientifique (CNRS)-Université de Poitiers-Centre National de la Recherche Scientifique (CNRS)

Subjects
Monosaccharide Transport Proteins, Arabidopsis Proteins, [SDV]Life Sciences [q-bio], Cell Respiration, fungi, lcsh:R, Arabidopsis, food and beverages, lcsh:Medicine, Article, Host-Pathogen Interactions, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, lcsh:Q, Botrytis, lcsh:Science, Glycolysis, ComputingMilieux_MISCELLANEOUS, Hexoses
Abstract

Photoassimilates play crucial roles during plant-pathogen interactions, as colonizing pathogens rely on the supply of sugars from hosts. The competition for sugar acquisition at the plant-pathogen interface involves different strategies from both partners which are critical for the outcome of the interaction. Here, we dissect individual mechanisms of sugar uptake during the interaction of Arabidopsis thaliana with the necrotrophic fungus Botrytis cinerea using millicell culture insert, that enables molecular communication without physical contact. We demonstrate that B. cinerea is able to actively absorb glucose and fructose with equal capacities. Challenged Arabidopsis cells compete for extracellular monosaccharides through transcriptional reprogramming of host sugar transporter genes and activation of a complex sugar uptake system which displays differential specificity and affinity for hexoses. We provide evidence that the molecular dialogue between Arabidopsis cells and B. cinerea triggers major changes in host metabolism, including apoplastic sucrose degradation and consumption of carbohydrates and oxygen, suggesting an enhanced activity of the glycolysis and the cellular respiration. We conclude that beside a role in sugar deprivation of the pathogen by competing for sugar availability in the apoplast, the enhanced uptake of hexoses also contributes to sustain the increased activity of respiratory metabolism to fuel plant defences.

Published
2017

26. Two adjacent NLR genes conferring quantitative resistance to clubroot disease in Arabidopsisare regulated by a stably inherited epiallelic variation

Author

Gravot, Antoine, Liégard, Benjamin, Quadrana, Leandro, Veillet, Florian, Aigu, Yoann, Bargain, Tristan, Bénéjam, Juliette, Lariagon, Christine, Lemoine, Jocelyne, Colot, Vincent, Manzanares-Dauleux, Maria J., and Jubault, Mélanie

Abstract

Clubroot caused by the protist Plasmodiophora brassicaeis a major disease affecting cultivated Brassicaceae. Using a combination of quantitative trait locus (QTL) fine mapping, CRISPR-Cas9 validation, and extensive analyses of DNA sequence and methylation patterns, we revealed that the two adjacent neighboring NLR(nucleotide-binding and leucine-rich repeat) genes AT5G47260and AT5G47280cooperate in controlling broad-spectrum quantitative partial resistance to the root pathogen P. brassicaein Arabidopsisand that they are epigenetically regulated. The variation in DNA methylation is not associated with any nucleotide variation or any transposable element presence/absence variants and is stably inherited. Variations in DNA methylation at the Pb-At5.2QTL are widespread across Arabidopsisaccessions and correlate negatively with variations in expression of the two genes. Our study demonstrates that natural, stable, and transgenerationally inherited epigenetic variations can play an important role in shaping resistance to plant pathogens by modulating the expression of immune receptors.

Published
2024
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28. New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing.

Author

Malabarba, Jaiana, Chevreau, Elisabeth, Dousset, Nicolas, Veillet, Florian, Moizan, Julie, and Vergne, Emilie

Subjects
APPLES, CRISPRS, PEARS, ACETOLACTATE synthase, CYTIDINE deaminase, HERBICIDE resistance, PHENOTYPES
Abstract

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for "regenerated T0" lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants. [ABSTRACT FROM AUTHOR]

Published
2021
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29. Gene Editing in Potato Using CRISPR-Cas9 Technology.

Author

Chauvin L, Sevestre F, Lukan T, Nogué F, Gallois JL, Chauvin JE, and Veillet F

Subjects
CRISPR-Cas Systems genetics, Plants, RNA, Guide, CRISPR-Cas Systems genetics, Technology, Gene Editing, Solanum tuberosum genetics
Abstract

Genome editing in the cultivated potato (Solanum tuberosum), a vegetatively propagated and highly heterozygous species, constitutes a promising trail to directly improve traits into elite cultivars. With the recent and successful development of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system in eukaryotic cells, the plant science community has gained access to a powerful, inexpensive, and easy-to-use toolbox to target and inactivate/modify specific genes. The specificity and versatility of the CRISPR-Cas9 system rely on a variable 20 bp spacer sequence at the 5' end of a single-guide RNA (sgRNA), which directs the SpCas9 (Streptococcus pyogenes) nuclease to cut the target DNA at a precise locus with no or low off-target events. Using this system, we and other teams were able to knock out specific genes in potato through the error-prone non-homologous end-joining (NHEJ) DNA repair mechanism. In this chapter, we describe strategies to design and clone spacer sequences into CRISPR-SpCas9 plasmids. We show how these constructs can be used for Agrobacterium-mediated stable transformation or transient transfection of protoplasts, and we describe the optimization of these two delivery methods, as well as of the plant regeneration processes. Finally, the molecular screening and characterization of edited potato plants are also described, mainly relying on PCR-based methods such as high-resolution melt (HRM) analysis., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Published
2021
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30. Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops.

Author

Veillet F, Perrot L, Guyon-Debast A, Kermarrec MP, Chauvin L, Chauvin JE, Gallois JL, Mazier M, and Nogué F

Subjects
Amino Acid Substitution genetics, CRISPR-Cas Systems genetics, Crops, Agricultural genetics, Plants, Genetically Modified genetics, Streptococcus pyogenes enzymology, Bryopsida genetics, CRISPR-Associated Protein 9 genetics, Gene Editing methods, Solanum lycopersicum genetics, Solanum tuberosum genetics
Abstract

Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops ( Solanum lycopersicum and Solanum tuberosum ) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.

Published
2020
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31. The molecular dialogue between Arabidopsis thaliana and the necrotrophic fungus Botrytis cinerea leads to major changes in host carbon metabolism.

Author

Veillet F, Gaillard C, Lemonnier P, Coutos-Thévenot P, and La Camera S

Subjects
Arabidopsis genetics, Arabidopsis microbiology, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Botrytis pathogenicity, Cell Respiration, Glycolysis, Monosaccharide Transport Proteins genetics, Monosaccharide Transport Proteins metabolism, Arabidopsis metabolism, Hexoses metabolism, Host-Pathogen Interactions
Abstract

Photoassimilates play crucial roles during plant-pathogen interactions, as colonizing pathogens rely on the supply of sugars from hosts. The competition for sugar acquisition at the plant-pathogen interface involves different strategies from both partners which are critical for the outcome of the interaction. Here, we dissect individual mechanisms of sugar uptake during the interaction of Arabidopsis thaliana with the necrotrophic fungus Botrytis cinerea using millicell culture insert, that enables molecular communication without physical contact. We demonstrate that B. cinerea is able to actively absorb glucose and fructose with equal capacities. Challenged Arabidopsis cells compete for extracellular monosaccharides through transcriptional reprogramming of host sugar transporter genes and activation of a complex sugar uptake system which displays differential specificity and affinity for hexoses. We provide evidence that the molecular dialogue between Arabidopsis cells and B. cinerea triggers major changes in host metabolism, including apoplastic sucrose degradation and consumption of carbohydrates and oxygen, suggesting an enhanced activity of the glycolysis and the cellular respiration. We conclude that beside a role in sugar deprivation of the pathogen by competing for sugar availability in the apoplast, the enhanced uptake of hexoses also contributes to sustain the increased activity of respiratory metabolism to fuel plant defences.

Published
2017
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