RESUMO
Plant pathogens secrete proteins called effectors that target host cellular processes to promote disease. Recently, structural genomics has identified several families of fungal effectors that share a similar three-dimensional structure despite remarkably variable amino-acid sequences and surface properties. To explore the selective forces that underlie the sequence variability of structurally-analogous effectors, we focused on MAX effectors, a structural family of effectors that are major determinants of virulence in the rice blast fungus Pyricularia oryzae. Using structure-informed gene annotation, we identified 58 to 78 MAX effector genes per genome in a set of 120 isolates representing seven host-associated lineages. The expression of MAX effector genes was primarily restricted to the early biotrophic phase of infection and strongly influenced by the host plant. Pangenome analyses of MAX effectors demonstrated extensive presence/absence polymorphism and identified gene loss events possibly involved in host range adaptation. However, gene knock-in experiments did not reveal a strong effect on virulence phenotypes suggesting that other evolutionary mechanisms are the main drivers of MAX effector losses. MAX effectors displayed high levels of standing variation and high rates of non-synonymous substitutions, pointing to widespread positive selection shaping the molecular diversity of MAX effectors. The combination of these analyses with structural data revealed that positive selection acts mostly on residues located in particular structural elements and at specific positions. By providing a comprehensive catalog of amino acid polymorphism, and by identifying the structural determinants of the sequence diversity, our work will inform future studies aimed at elucidating the function and mode of action of MAX effectors.
Assuntos
Aminoácidos , Ascomicetos , Virulência/genética , Sequência de Aminoácidos , Ascomicetos/genéticaRESUMO
Resistance to potyviruses in plants has been largely provided by the selection of natural variant alleles of eukaryotic translation initiation factors (eIF) 4E in many crops. However, the sources of such variability for breeding can be limited for certain crop species, while new virus isolates continue to emerge. Different methods of mutagenesis have been applied to inactivate the eIF4E genes to generate virus resistance, but with limited success due to the physiological importance of translation factors and their redundancy. Here, we employed genome editing approaches at the base level to induce non-synonymous mutations in the eIF4E1 gene and create genetic diversity in cherry tomato (Solanum lycopersicum var. cerasiforme). We sequentially edited the genomic sequences coding for two regions of eIF4E1 protein, located around the cap-binding pocket and known to be important for susceptibility to potyviruses. We show that the editing of only one of the two regions, by gene knock-in and base editing, respectively, is not sufficient to provide resistance. However, combining amino acid mutations in both regions resulted in resistance to multiple potyviruses without affecting the functionality in translation initiation. Meanwhile, we report that extensive base editing in exonic region can alter RNA splicing pattern, resulting in gene knockout. Altogether our work demonstrates that precision editing allows to design plant factors based on the knowledge on evolutionarily selected alleles and enlarge the gene pool to potentially provide advantageous phenotypes such as pathogen resistance.
Assuntos
Potyvirus , Solanum lycopersicum , Edição de Genes , Solanum lycopersicum/genética , Fator de Iniciação 4E em Eucariotos/genética , Potyvirus/genética , Proteínas de Plantas/genética , Melhoramento Vegetal , Mutação , Doenças das Plantas/genéticaRESUMO
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.
Assuntos
Bryopsida , Bryopsida/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Mutagênese Sítio-DirigidaRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Malus/genética , Pyrus/genética , Quimerismo , Citidina Desaminase/genética , Marcação de Genes , Genoma de Planta , Fenótipo , Plantas Geneticamente Modificadas , Projetos de PesquisaRESUMO
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.
Assuntos
Bryopsida/genética , Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Solanum lycopersicum/genética , Solanum tuberosum/genética , Substituição de Aminoácidos/genética , Sistemas CRISPR-Cas/genética , Produtos Agrícolas/genética , Plantas Geneticamente Modificadas/genética , Streptococcus pyogenes/enzimologiaRESUMO
KEY MESSAGE: The StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base-editing strategies, leading to plants with impaired amylose biosynthesis. Genome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant-breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knock out the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss of function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.
Assuntos
Edição de Genes , Solanum tuberosum/genética , Sintase do Amido/genética , Alelos , Sistemas CRISPR-Cas , TetraploidiaRESUMO
Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through Agrobacterium-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, especially in vegetatively propagated plants. In this study, we targeted the acetolactate synthase (ALS) gene in tomato and potato by a CBE using Agrobacterium-mediated transformation. We successfully and efficiently edited the targeted cytidine bases, leading to chlorsulfuron-resistant plants with precise base edition efficiency up to 71% in tomato. More importantly, we produced 12.9% and 10% edited but transgene-free plants in the first generation in tomato and potato, respectively. Such an approach is expected to decrease deleterious effects due to the random integration of transgene(s) into the host genome. Our successful approach opens up new perspectives for genome engineering by the co-edition of the ALS with other gene(s), leading to transgene-free plants harboring new traits of interest.
Assuntos
Agrobacterium/fisiologia , Sistemas CRISPR-Cas , Citidina/genética , Edição de Genes , Técnicas de Transferência de Genes , Solanum lycopersicum/genética , Solanum tuberosum/genética , Marcação de Genes , Genes de Plantas , Técnicas de Genotipagem , Análise de Sequência de DNA , Transformação GenéticaRESUMO
Botrytis cinerea is the causing agent of the grey mold disease in more than 200 crop species. While signaling pathways leading to the basal resistance against this fungus are well described, the role of the import of sugars into host cells remains to be investigated. In Arabidopsis thaliana, apoplastic hexose retrieval is mediated by the activity of sugar transport proteins (STPs). Expression analysis of the 14 STP genes revealed that only STP13 was induced in leaves challenged with B. cinerea. STP13-modified plants were produced and assayed for their resistance to B. cinerea and glucose transport activity. We report that STP13-deficient plants exhibited an enhanced susceptibility and a reduced rate of glucose uptake. Conversely, plants with a high constitutive level of STP13 protein displayed an improved capacity to absorb glucose and an enhanced resistance phenotype. The correlation between STP13 transcripts, protein accumulation, glucose uptake rate and resistance level indicates that STP13 contributes to the basal resistance to B. cinerea by limiting symptom development and points out the importance of the host intracellular sugar uptake in this process. We postulate that STP13 would participate in the active resorption of hexoses to support the increased energy demand to trigger plant defense reactions and to deprive the fungus by changing sugar fluxes toward host cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Botrytis/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Glucose/metabolismo , Simportadores/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Transporte Biológico , Botrytis/imunologia , Resistência à Doença , Predisposição Genética para Doença , Mutação , Doenças das Plantas/genética , Simportadores/genéticaRESUMO
Clubroot caused by the protist Plasmodiophora brassicae is 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 AT5G47260 and AT5G47280 cooperate in controlling broad-spectrum quantitative partial resistance to the root pathogen P. brassicae in Arabidopsis and 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-At5.2 QTL are widespread across Arabidopsis accessions 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.
Assuntos
Arabidopsis , Resistência à Doença , Doenças das Plantas , Arabidopsis/genética , Arabidopsis/imunologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Resistência à Doença/genética , Proteínas NLR/genética , Proteínas NLR/metabolismo , Metilação de DNA , Plasmodioforídeos/fisiologia , Locos de Características Quantitativas/genética , Proteínas de Arabidopsis/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , AlelosRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci.
RESUMO
Since its discovery and first applications for genome editing in plants, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology has revolutionized plant research and precision crop breeding. Although the classical CRISPR-Cas9 system is a highly efficient tool for disruptive targeted mutagenesis, this system is mostly inefficient for the introduction of precise and predictable nucleotide substitutions. Recently, Prime Editing technology has been developed, allowing the simultaneous generation of nucleotide transitions and transversions but also short defined indels. In this study, we report on the successful use of Prime Editing in two plants of interest: the plant model Physcomitrium patens and the tetraploid and highly heterozygous potato (Solanum tuberosum). In both cases editing rates were lower than with other CRISPR-Cas9 based techniques, but we were able to successfully introduce nucleotide transversions into targeted genes, a unique feature of Prime Editing. Additionally, the analysis of potential off-target mutation sites in P. patens suggested very high targeting fidelity in this organism. The present work paves the way for the use Prime Editing in Physcomitrium patens and potato, however highlighting the limitations that need to be overcome for more efficient precision plant breeding.
Assuntos
Solanum tuberosum , Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma de Planta , Melhoramento Vegetal , Solanum tuberosum/genética , TetraploidiaRESUMO
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.
Assuntos
Edição de Genes , Solanum tuberosum , Sistemas CRISPR-Cas/genética , Plantas , RNA Guia de Cinetoplastídeos/genética , Solanum tuberosum/genética , TecnologiaRESUMO
Since 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.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Resistência à Doença/genética , Edição de Genes , Melhoramento Vegetal/métodos , Doenças das Plantas/genética , Técnicas de Silenciamento de Genes , Genoma de Planta/genética , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/microbiologiaRESUMO
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 knock-out 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.
Assuntos
Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Mutação INDEL , Solanum tuberosum/genética , Staphylococcus aureus/enzimologia , Sistemas CRISPR-Cas , Mutação da Fase de Leitura , Edição de Genes/métodos , Genoma de Planta , Plasmídeos/genética , Staphylococcus aureus/genéticaRESUMO
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.
Assuntos
Arabidopsis/metabolismo , Hexoses/metabolismo , Interações Hospedeiro-Patógeno , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Botrytis/patogenicidade , Respiração Celular , Glicólise , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismoRESUMO
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 Botrytis cinerea possesses its own functional sucrolytic machinery and hexose uptake system, and does not rely on the host apoplastic invertases.