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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.

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.

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.

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.

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.