RESUMO
The movement of potyviruses, the largest genus of single-stranded, positive-sense RNA viruses responsible for serious diseases in crops, is very complex. As potyviruses developed strategies to hijack the host secretory pathway and plasmodesmata (PD) for their transport, the goal of this study was to identify membrane and/or PD-proteins that interact with the 6K2 protein, a potyviral protein involved in replication and cell-to-cell movement of turnip mosaic virus (TuMV). Using split-ubiquitin membrane yeast two-hybrid assays, we screened an Arabidopsis cDNA library for interactors of TuMV6K2. We isolated AtHVA22a (Hordeum vulgare abscisic acid responsive gene 22), which belongs to a multigenic family of transmembrane proteins, homologous to Receptor expression-enhancing protein (Reep)/Deleted in polyposis (DP1)/Yop1 family proteins in animal and yeast. HVA22/DP1/Yop1 family genes are widely distributed in eukaryotes, but the role of HVA22 proteins in plants is still not well known, although proteomics analysis of PD fractions purified from Arabidopsis suspension cells showed that AtHVA22a is highly enriched in a PD proteome. We confirmed the interaction between TuMV6K2 and AtHVA22a in yeast, as well as in planta by using bimolecular fluorescence complementation and showed that TuMV6K2/AtHVA22a interaction occurs at the level of the viral replication compartment during TuMV infection. Finally, we showed that the propagation of TuMV is increased when AtHVA22a is overexpressed in planta but slowed down upon mutagenesis of AtHVA22a by CRISPR-Cas9. Altogether, our results indicate that AtHVA22a plays an agonistic effect on TuMV propagation and that the C-terminal tail of the protein is important in this process.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Potyvirus , Potyvirus/patogenicidade , Potyvirus/fisiologia , Arabidopsis/virologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Doenças das Plantas/virologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , Replicação Viral , Nicotiana/virologia , Nicotiana/genéticaRESUMO
Successful subversion of translation initiation factors eIF4E determines the infection success of potyviruses, the largest group of viruses affecting plants. In the natural variability of many plant species, resistance to potyvirus infection is provided by polymorphisms at eIF4E that renders them inadequate for virus hijacking but still functional in translation initiation. In crops where such natural resistance alleles are limited, the genetic inactivation of eIF4E has been proposed for the engineering of potyvirus resistance. However, recent findings indicate that knockout eIF4E alleles may be deleterious for plant health and could jeopardize resistance efficiency in comparison to functional resistance proteins. Here, we explored the cause of these adverse effects by studying the role of the Arabidopsis eIF4E1, whose inactivation was previously reported as conferring resistance to the potyvirus clover yellow vein virus (ClYVV) while also promoting susceptibility to another potyvirus turnip mosaic virus (TuMV). We report that eIF4E1 is required to maintain global plant translation and to restrict TuMV accumulation during infection, and its absence is associated with a favoured virus multiplication over host translation. Furthermore, our findings show that, in the absence of eIF4E1, infection with TuMV results in the production of a truncated eIFiso4G1 protein. Finally, we demonstrate a role for eIFiso4G1 in TuMV accumulation and in supporting plant fitness during infection. These findings suggest that eIF4E1 counteracts the hijacking of the plant translational apparatus during TuMV infection and underscore the importance of preserving the functionality of translation initiation factors eIF4E when implementing potyvirus resistance strategies.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Potyvirus , Arabidopsis/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Potyvirus/fisiologia , Plantas Geneticamente Modificadas/metabolismo , Doenças das Plantas/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismoRESUMO
Resistance to viruses is an important aspect of plant breeding. One way to achieve it is to select genetic resistances based on the susceptibility factors hijacked by the virus to infect the plants. Here, we recount work done on genes encoding translation initiation factors eIF4E, some of the most successful targets for obtaining resistance to potyviruses, starting from their characterization 20 years ago. With examples from different plant species, pepper, tomato, tobacco and arabidopsis, we present the basis of this type of resistances and their characteristics, highlighting the role of gene redundancy among 4E factors, their specificity for the virus and the need for the plant of a trade-off between resistance and development. Finally, we show how the new genome editing techniques could be used in plant breeding to develop eIF4E-based resistances in crops, mimicking the functional alleles that have been selected during evolution in many crops.
Assuntos
Resistência à Doença , Fator de Iniciação 4E em Eucariotos , Doenças das Plantas , Plantas , Potyvirus , Alelos , Fator de Iniciação 4E em Eucariotos/genética , Melhoramento Vegetal , Potyvirus/genética , Plantas/virologia , Doenças das Plantas/virologiaRESUMO
The wealth of variability amongst genes controlling immunity against potyviruses in pepper (Capsicum spp.) has been instrumental in understanding plant-virus co-evolution and major determinants of plant resistance durability. Characterization of the eukaryotic initiation factor 4E1 (eIF4E1), involved in mRNA translation, as the basis of potyvirus resistance in pepper initiated a large body of work that showed that recessive resistance to potyviruses and other single-stranded positive-sense RNA viruses resulted from mutations in eukaryotic initiation factors in many plant crop species. Combining mutations in different eIF4Es in the same pepper genotype had complex effects on the breadth of the resistance spectrum and on resistance durability, revealing a trade-off between these two traits. In addition, combining eIF4E1 mutations with a quantitatively resistant genetic background had a strong positive effect on resistance durability. Analysing the evolutionary forces imposed by pepper genotypes onto virus populations allowed identifying three key factors improving plant resistance durability: the complexity of mutational pathways involved in virus adaptation to the plant resistance, the decrease of competitivity induced by these mutations on the virus and the intensity of genetic drift imposed by plant genotypes on the virus during its infection cycle.
Assuntos
Potyvirus , Potyvirus/genética , Potyvirus/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Mutação , Plantas , GenótipoRESUMO
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
The development of recessive resistance by loss of susceptibility is a consistent strategy to combat and limit damages caused by plant viruses. Susceptibility genes can be turned into resistances, a feat that can either be selected among the plant's natural diversity or engineered by biotechnology. Here, we summarize the current knowledge on the phosphoglycerate kinases (PGK), which have emerged as a new class of susceptibility factors to single-stranded positive RNA viruses, including potyviruses. PGKs are metabolic enzymes involved in glycolysis and the carbon reduction cycle, encoded by small multigene families in plants. To fulfil their role in the chloroplast and in the cytosol, PGKs genes encode differentially addressed proteins. Here, we assess the diversity and homology of chloroplastic and cytosolic PGKs sequences in several crops and review the current knowledge on their redundancies during plant development, taking Arabidopsis as a model. We also show how PGKs have been shown to be involved in susceptibility-and resistance-to viruses. Based on this knowledge, and drawing from the experience with the well-characterized translation initiation factors eIF4E, we discuss how PGKs genes, in light of their subcellular localization, function in metabolism, and susceptibility to viruses, could be turned into efficient genetic resistances using genome editing techniques.
Assuntos
Arabidopsis , Potyvirus , Arabidopsis/genética , Cloroplastos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Potyvirus/genética , Potyvirus/metabolismoRESUMO
The host susceptibility factors are important targets to develop genetic resistances in crops. Genome editing tools offer exciting prospects to develop resistances based on these susceptibility factors, directly in the cultivar of choice. Translation initiation factors 4E have long been known to be a susceptibility factor to the main genus of Potyviridae, potyviruses, but the inactivation of the eIF4E2 gene has only recently been shown to provide resistance to some isolates of pepper veinal mottle virus (PVMV) in big-fruit tomato plants. Here, using CRISPR-Cas9-NG, we show how eIF4E2 can be targeted and inactivated in cherry tomato plants. Three independent knockout alleles caused by indel in the first exon of eIF4E2, resulted in the complete absence of the eIF4E2 protein. All three lines displayed a narrow resistance spectrum to potyvirus, similar to the one described earlier for an eIF4E2 EMS mutant of M82, a big-fruit tomato cultivar; the plants were fully resistant to PVMV-Ca31, partially to PVMV-IC and were fully susceptible to two isolates of PVY assayed: N605 and LYE84. These results show how easily a resistance based on eIF4E2 can be transferred across tomato cultivar, but also confirm that gene redundancy can narrow the resistances based on eIF4E knockout.
Assuntos
Capsicum , Potyvirus , Solanum lycopersicum , Capsicum/genética , Solanum lycopersicum/genética , Doenças das Plantas/genéticaRESUMO
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
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
The translation initiation factors 4E are a small family of major susceptibility factors to potyviruses. It has been suggested that knocking out these genes could provide genetic resistance in crops when natural resistance alleles, which encode functional eIF4E proteins, are not available. Here, using the well-characterized Arabidopsis thaliana-potyvirus pathosystem, we evaluate the resistance spectrum of plants knocked out for eIF4E1, the susceptibility factor to clover yellow vein virus (ClYVV). We show that besides resistance to ClYVV, the eIF4E1 loss of function is associated with hypersusceptibility to turnip mosaic virus (TuMV), a potyvirus known to rely on the paralog host factor eIFiso4E. On TuMV infection, plants knocked out for eIF4E1 display striking developmental defects such as early senescence and primordia development stoppage. This phenotype is coupled with a strong TuMV overaccumulation throughout the plant, while remarkably the levels of the viral target eIFiso4E remain uninfluenced. Our data suggest that this hypersusceptibility cannot be explained by virus evolution leading to a gain of TuMV aggressiveness. Furthermore, we report that a functional eIF4E1 resistance allele engineered by CRISPR/Cas9 base-editing technology successfully circumvents the increase of TuMV susceptibility conditioned by eIF4E1 disruption. These findings in Arabidopsis add to several previous findings in crops suggesting that resistance based on knocking out eIF4E factors should be avoided in plant breeding, as it could also expose the plant to the severe threat of potyviruses able to recruit alternative eIF4E copies. At the same time, it provides a simple model that can help understanding of the homeostasis among eIF4E proteins in the plant cell and what makes them available to potyviruses.
Assuntos
Arabidopsis/genética , Resistência à Doença/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Doenças das Plantas/imunologia , Potyvirus/patogenicidade , Alelos , Arabidopsis/imunologia , Arabidopsis/virologia , Fator de Iniciação 4E em Eucariotos/genética , Técnicas de Inativação de Genes , Mutação com Perda de Função , Modelos Biológicos , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente ModificadasRESUMO
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 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
Tomato spotted wilt virus (TSWV; genus Orthotospovirus, family Tospoviridae) has a huge impact on a large range of plants worldwide. In this study, we determined the sequence of the large (L) RNA segment that encodes the RNA-dependent RNA polymerase (RdRp) from a TSWV isolate (LYE51) collected in the south of France. Analysis of the phylogenetic relationships of TSWV-LYE51 with other TSWV isolates shows that it is closely related to other European isolates. A 3D model of TSWV-LYE51 RdRp was built by homology with the RdRp structure of the La Crosse virus (genus Orthobunyavirus, family Peribunyaviridae). Finally, an analysis of positive and negative selection was carried out on 30 TSWV full-length RNA L sequences and compared with the phylogeny and the protein structure data. We showed that the seven codons that are under positive selection are distributed all along the RdRp gene. By contrast, the codons associated with negative selection are especially concentrated in three highly constrained domains: the endonuclease in charge of the cap-snatching mechanism, the thumb domain and the mid domain. Those three domains could constitute good candidates to look for host targets on which genetic resistance by loss of susceptibility could be developed.
Assuntos
Adaptação Fisiológica/genética , Evolução Molecular , Modelos Moleculares , Domínios Proteicos/genética , RNA Polimerase Dependente de RNA/genética , Homologia Estrutural de Proteína , Tospovirus/enzimologia , Códon/genética , França , Genoma Viral/genética , Solanum lycopersicum/virologia , Filogenia , Doenças das Plantas/virologia , RNA Viral/genética , Sequenciamento Completo do GenomaRESUMO
Translation initiation factors 4E (eIF4E) are the main source of resistance to potyvirus. We systematically assessed tomato single and double knock-out (KO) mutants of members of the eIF4E-coding gene family for resistance to Pepper veinal mottle virus (PVMV), a major constraint to tomato production. We show that the KO mutant of eIF4E2 has partial resistance to PVMV isolate IC, with plants harboring weak symptoms and low virus loads at the systemic level. The causal effect of eIF4E2 loss-of-function on resistance was confirmed on a progeny segregating for the KO mutation. The eIF4E2 KO mutant was resistant to six of the eight PVMV isolates tested and no resistance to other potyviruses was observed. This is the first evidence that mutation of eIF4E2 is in itself conferring resistance to a potyvirus and 3D protein modelling suggests that the eIF4E2 gene could be converted into a functional resistance allele.
Assuntos
Resistência à Doença/genética , Fator de Iniciação 4E em Eucariotos/genética , Potyvirus/patogenicidade , Solanum lycopersicum/genética , Capsicum/genética , Capsicum/virologia , Interações Hospedeiro-Patógeno , Mutação com Perda de Função , Solanum lycopersicum/virologia , Família Multigênica , Doenças das Plantas/virologia , Potyvirus/isolamento & purificação , Carga ViralRESUMO
Many recessive resistances against potyviruses are mediated by eukaryotic translation initiation factor 4E (eIF4E). In tobacco, the va resistance gene commonly used to control Potato virus Y (PVY) corresponds to a large deletion affecting the eIF4E-1 gene on chromosome 21. Here, we compared the resistance durability conferred by various types of mutations affecting eIF4E-1 (deletions of various sizes, frameshift or nonsense mutations). The 'large deletion' genotypes displayed the broadest and most durable resistance, whereas frameshift and nonsense mutants displayed a less durable resistance, with rapid and frequent apparition of resistance-breaking variants. In addition, genetic and transcriptomic analyses revealed that resistance durability is strongly impacted by a complex genetic locus on chromosome 14, which contains three other eIF4E genes. One of these, eIF4E-3, is rearranged as a hybrid gene between eIF4E-2 and eIF4E-3 (eIF4E-2-3 ) in the genotypes showing the most durable resistance, while eIF4E-2 is differentially expressed between the tested varieties. RNA-seq and quantitative reverse transcriptase-polymerase chain reaction experiments demonstrated that eIF4E-2 expression level is positively correlated with resistance durability. These results suggest that besides the nature of the mutation affecting eIF4E-1, three factors linked with a complex locus may potentially impact va durability: loss of an integral eIF4E-3, presence of eIF4E-2-3 and overexpression of eIF4E-2. This latter gene might act as a decoy in a non-productive virus-plant interaction, limiting the ability of PVY to evolve towards resistance breaking. Taken together, these results show that va resistance durability can in large part be explained by complex redundancy effects in the eIF4E gene family.
Assuntos
Resistência à Doença , Fator de Iniciação 4E em Eucariotos/genética , Genes de Plantas , Loci Gênicos , Nicotiana/imunologia , Nicotiana/virologia , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Potyvirus/fisiologia , Substituição de Aminoácidos/genética , Cromossomos de Plantas/genética , Ecótipo , Dosagem de Genes , Regulação da Expressão Gênica de Plantas , Genótipo , Modelos Biológicos , Mutação/genética , Fenótipo , Filogenia , Doenças das Plantas/genética , Deleção de Sequência , Nicotiana/genéticaRESUMO
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
In many crop species, natural variation in eIF4E proteins confers resistance to potyviruses. Gene editing offers new opportunities to transfer genetic resistance to crops that seem to lack natural eIF4E alleles. However, because eIF4E are physiologically important proteins, any introduced modification for virus resistance must not bring adverse phenotype effects. In this study, we assessed the role of amino acid substitutions encoded by a Pisum sativum eIF4E virus-resistance allele (W69L, T80D S81D, S84A, G114R and N176K) by introducing them independently into the Arabidopsis thaliana eIF4E1 gene, a susceptibility factor to the Clover yellow vein virus (ClYVV). Results show that most mutations were sufficient to prevent ClYVV accumulation in plants without affecting plant growth. In addition, two of these engineered resistance alleles can be combined with a loss-of-function eIFiso4E to expand the resistance spectrum to other potyviruses. Finally, we use CRISPR-nCas9-cytidine deaminase technology to convert the Arabidopsis eIF4E1 susceptibility allele into a resistance allele by introducing the N176K mutation with a single-point mutation through C-to-G base editing to generate resistant plants. This study shows how combining knowledge on pathogen susceptibility factors with precise genome-editing technologies offers a feasible solution for engineering transgene-free genetic resistance in plants, even across species barriers.
Assuntos
Sistemas CRISPR-Cas , Resistência à Doença/genética , Fator de Iniciação 4E em Eucariotos/genética , Edição de Genes , Pisum sativum/genética , Doenças das Plantas/genética , Potyvirus/patogenicidade , Alelos , Arabidopsis/genética , Arabidopsis/virologia , Pisum sativum/virologia , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Plantas Geneticamente ModificadasRESUMO
In view of major economic problems caused by viruses, the development of genetically resistant crops is critical for breeders but remains limited by the evolution of resistance-breaking virus mutants. During the plant breeding process, the introgression of traits from Crop Wild Relatives results in a dramatic change of the genetic background that can alter the resistance efficiency or durability. Here, we conducted a meta-analysis on 19 Quantitative Trait Locus (QTL) studies of resistance to viruses in plants. Frequent epistatic effects between resistance genes indicate that a large part of the resistance phenotype, conferred by a given QTL, depends on the genetic background. We next reviewed the different resistance mechanisms in plants to survey at which stage the genetic background could impact resistance or durability. We propose that the genetic background may impair effector-triggered dominant resistances at several stages by tinkering the NB-LRR (Nucleotide Binding-Leucine-Rich Repeats) response pathway. In contrast, effects on recessive resistances by loss-of-susceptibility-such as eIF4E-based resistances-are more likely to rely on gene redundancy among the multigene family of host susceptibility factors. Finally, we show how the genetic background is likely to shape the evolution of resistance-breaking isolates and propose how to take this into account in order to breed plants with increased resistance durability to viruses.