Magnetically textured y-Fe2O3 nanoparticles in a silica gel matrix: structural and magnetic properties

International audienceThis paper is devoted to magnetic and structural properties of anisotropic g -Fe2O3 superparamagnetic particles dispersed in a transparent xerogel matrix. The effect of frozen anisotropy axes and magnetic texture, induced by a magnetic field applied during the solidification of the matrix on the in-field magnetization process, is studied by alternating gradient force magnetometry and first and second order magneto-optical effects. The changes of magnetization curves with respect to the ferrofluid solution at the same particle concentration are interpreted on the basis of an existing statistical approach extended to systems with particle size distribution, which has to be taken into account for real samples. A very good agreement between the experiment and theory was achieved for a log-normal distribution of diameters which well resembles that deduced from electron microscopy observations in different imaging modes. This structural analysis states the parameter values used in calculations and confirms the relevance of basic assumptions of the model for the specimens studied. The experimental results and the related theoretical discussion should be of use to understand magnetic properties of other magnetically textured superparamagnetic system

A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

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

Study of molecular and biochemical mechanisms of sugar transport in source/sink relationship and during the interaction between Arabidopsis thaliana and the necrotrophic fungus Botrytis cinerea

La distribution des sucres est un processus clé dans le développement de la plante et cours des interactions plantes/microorganismes.Une recherche des acteurs moléculaires impliqués dans la répartition des ressources carbonées au cours de l'interaction avec le champignon nécrotrophe B. cinerea a été réalisée. Plusieurs familles de transporteurs de sucres et d'invertases ont été ciblées, permettant d'établir une cartographie des gènes régulés transcriptionnellement lors de l'interaction. Le rôle de certains gènes candidats a été étudié par une approche de génomique fonctionnelle afin de mettre en évidence une fonction biologique de l'allocation du carbone dans la résistance de la plante aux champignons nécrotrophes. Un système d'interaction simplifié, basé sur un dialogue moléculaire sans contact physique entre une culture cellulaire d'A. thaliana et B. cinerea, a été développé. Il a permis de mesurer les flux de sucres ainsi que les activités enzymatiques et métaboliques pour chaque partenaire. Nos résultats montrent que B. cinerea entraine une forte augmentation de l'activité invertasique pariétale dans les tissus infectés, indiquant qu'une transition source/puits a lieu. Plusieurs transporteurs de sucres sont différentiellement exprimés, certains d'entre eux modulant le devenir de l'interaction. L'activité d'absorption d'hexoses et le métabolisme primaire des cellules hôtes sont fortement stimulés, démontrant l'importance de la compétition pour les sucres à l'interface plante/agent pathogène. En conclusion, l'absorption des sucres alimente le métabolisme énergétique des cellules hôtes et participe aux mécanismes de défense de la plante.During plant development and upon pathogen infection, sugar allocation is a key process in plant physiology. Cell wall invertases and sugar transporters, involved in the sink strength, likely play a major role in the metabolic plant response. Molecular actors involved in carbohydrates allocation upon B. cinerea interaction have been identified using a transcriptional approach. Some gene families of sugar transporters and invertases have been targeted, allowing the establishment of a cartography of genes regulated during the interaction. To understand the biological role of carbon allocation during the interaction between plants and necrotrophic fungi, candidate genes have been studied using a functional genomics approach.A simplified interaction system has been developped, allowing a molecular dialogue between Arabidopsis and B. cinerea cells, without any physical contact. This system enables the monitoring of radiolabelled sugar uptake rates and some enzymatic and metabolomic activities for both the host cells and the pathogen, independently.Globally, our results demonstrate that B. cinerea infection leads to the transition from a source to a sink tissue, with a strong increase in cell wall invertase activity. The expression of some sugar transporter genes is also affected, while some of them (AtSTP1 and 13) are involved in the disease development. Besides the increase in hexose uptake activity, primary metabolism is deeply affected, highlighting the competition for apoplastic sugars that takes place at the plant/pathogen interface. Sugar retrieval appears to be a key process, fuelling host cells with energy and signal molecules, contributing to the plant defense mechanisms

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

International audienceWe 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

Targeting the AtCWIN1 gene to explore the role of invertases in sucrose transport in roots and during Botrytis cinerea infection

Cell wall invertases (CWIN) cleave sucrose into glucose and fructose in the apoplast. CWINs are key regulators of carbon partitioning and source/sink relationships during growth, development and under biotic stresses. In this report, we monitored the expression/activity of Arabidopsis cell wall invertases in organs behaving as source, sink or subjected to a source/sink transition after infection with the necrotrophic fungus Botrytis cinerea. We showed that organs with different source/sink status displayed differential CWIN activities, depending on carbohydrate needs or availabilities in the surrounding environment, through a transcriptional and posttranslational regulation. Loss-of-function mutation of the Arabidopsis cell wall invertase 1 gene, AtCWIN1, showed that the corresponding protein was the main contributor to the apoplastic sucrose cleaving activity in both leaves and roots. The CWIN-deficient mutant cwin1-1 exhibited a reduced capacity to actively take up external sucrose in roots, indicating that this process is mainly dependent on the sucrolytic activity of AtCWIN1. Using T-DNA and CRISPR/Cas9 mutants impaired in hexose transport, we demonstrated that external sucrose is actively absorbed in the form of hexoses by a sugar/H+ symport system involving the coordinated activity of AtCWIN1 with several Sugar Transporter Proteins (STP) of the plasma membrane, i.e. STP1 and STP13. Part of external sucrose was imported without apoplastic cleavage into cwin1-1 seedling roots, highlighting an alternative AtCWIN1-independent pathway for the assimilation of external sucrose. Accordingly, we showed that several genes encoding sucrose transporters of the plasma membrane were expressed. We also detected transcript accumulation of vacuolar invertase (VIN)-encoding genes and high VIN activities. Upon infection, AtCWIN1 was responsible for all the Botrytis-induced apoplastic invertase activity. We detected a transcriptional activation of several AtSUC and AtVIN genes accompanied with an enhanced vacuolar invertase activity, suggesting that the AtCWIN1-independent pathway is efficient upon infection. In absence of AtCWIN1, we postulate that intracellular sucrose hydrolysis is sufficient to provide intracellular hexoses to maintain sugar homeostasis in host cells and to fuel plant defenses. Finally, we demonstrated that B. cinerea possesses its own functional sucrolytic machinery and hexose uptake system, and does not rely on the host apoplastic invertases

Precision breeding made real with CRISPR: illustration through genetic resistance to pathogens

International audienceSince its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes, the CRISPR technology has revolutionized plant research and precision crop breeding. The CRISPR toolbox holds great promise in the production of crops with genetic disease resistance to increase agriculture resilience and reduce chemical crop protection with a strong impact on the environment and public health. In this review, we provide an extensive overview on recent breakthroughs in CRISPR technology, including the newly developed prime editing system that allows precision gene editing in plants. We present how each CRISPR tool can be selected for optimal use in accordance with its specific strengths and limitations, and illustrate how the CRISPR toolbox can foster the development of genetically pathogen-resistant crops for sustainable agriculture

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

International audienceCRISPR/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

CRISPR-induced indels and base editing using the Staphylococcus aureus Cas9 in potato

Genome editing is now widely used in plant science for both basic research and molecular crop breeding. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, through its precision, high efficiency and versatility, allows for editing of many sites in plant genomes. This system has been highly successful to produce knockout mutants through the introduction of frameshift mutations due to error-prone repair pathways. Nevertheless, recent new CRISPR-based technologies such as base editing and prime editing can generate precise and on demand nucleotide conversion, allowing for fine-tuning of protein function and generating gain-of-function mutants. However, genome editing through CRISPR systems still have some drawbacks and limitations, such as the PAM restriction and the need for more diversity in CRISPR tools to mediate different simultaneous catalytic activities. In this study, we successfully used the CRISPR-Cas9 system from Staphylococcus aureus (SaCas9) for the introduction of frameshift mutations in the tetraploid genome of the cultivated potato (Solanum tuberosum). We also developed a S. aureus-cytosine base editor that mediate nucleotide conversions, allowing for precise modification of specific residues or regulatory elements in potato. Our proof-of-concept in potato expand the plant dicot CRISPR toolbox for biotechnology and precision breeding applications

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

International audienceDespite 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