Article

Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology

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Abstract

Genome editing in plants has been boosted tremendously by the development of the CRISPR/Cas9 technology. This powerful tool allows substantial improvement of plant traits in addition to those provided by classical breeding. Here we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) by utilizing Cas9/sgRNA technology to disrupt the recessive eIF4E gene function. Cas9/sgRNA constructs were targeted to the N' and C' terminus of the eIF4E gene. Small deletions and SNPs were observed in the eIF4E gene targeted sites of T1 generation transformed cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eIF4E mutant plants were selected for production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eIF4E sites exhibited immunity to Cucumber vein yellowing virus (ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous-mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in the cucumber crop, non-transgenically, not visibly affecting plant development, and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants. This article is protected by copyright. All rights reserved.

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... Jia et al. [1,26,27] used CRISPR/ Cas9 to disrupt CsLOB1 gene in grapefruit Duncan (Citrus paradisi Macf.) to produce canker-resistant citrus varieties. CRISPR/Cas9mediated genome editing was used for the production of transgenefree crop plants; cucumber rice wheat and tomato [28][29][30][31][32]. CRISPR/ Cas9 was also used to develop abiotic stress tolerant crops through the regulatory mechanism of stress/ABA-activated protein kinase2 (SAPK2)-mediated stress tolerance in rice [11,33]. ...
... Am J Biomed Sci & Res American Journal of Biomedical Science & Research Copy@ Nwawuba Stanley Udogadiwas disrupted to produce a variety of canker-resistant citrus using CRISPR/Cas9 system[27]. Finally, the eukaryotic translation initiation factor 4E (eIF4E) and eukaryotic translation initiation factor isoform 4E (eIF(iso)4E) genes which are identified to be a resistance gene in a wide range of hosts were altered to develop a virus-resistant cucumber (Cucumis sativus) and Arabidopsis plants using the CRISPR/Cas9 system[28,98]. ...
... The anti-browning mushroom (Agaricus bisporus) and waxy corn have been reported to already have passed the US biosafety regulations[11]. Additionally, transgene-free genetically modified crops plants developed using CRISPR/Cas9 system have been reported for tomato, rice, maize, wheat, and cucumber[28,30,32,81,100,101]. ...
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Global recent developments and extensive body of evidence have established the fact that crop productivity and yield has declined, and the agricultural sector suffers a huge threat as a result of abiotic and biotic factors. In an attempt to mitigate the challenge of food shortfall, poor plant yield, intolerance to abiotic and biotic stresses and poor adaptability of crops, several methods have been adopted including conventional breeding technologies but has hit a plateau in recent times. However, breakthrough in molecular biology and biotechnology has been demonstrated to provide an improved alternative to the conventional methods for crop improvements. At the present, sequence-specific genome editing technologies particularly the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Caspase 9) genome editing technology (CRISPR/Cas9) has so far shown the greatest potential in mitigating the emerging challenges in crop improvement. CRISPR/Cas9 technology have been used for specific genome modification in many crops and the progress in CRISPR/Cas9 technology in crop improvement has been outstanding including development of abiotic stress tolerant crop plants, development of disease resistant variety of crop plants, and generation of transgene free genome edited crop plants. There is an expectation that the application of CRISPR/Cas9 technology in variety of crop would revolutionize the agricultural sector in the second green revolution to ensure food and nutritional security of the teeming global population particularly among tropical regions. Therefore, this review provides knowledge on the potentials of CRISPR/Cas9 for crop improvement.
... The significant imperative restricting the productivity of cucurbits crops is various infections brought about by fungi, bacteria and viruses (Chandrasekaran et al. 2016). Traditional breeding appears to have restricted application because of nonaccessibility of tolerant gene(s) in genetic system of a cucurbits crop. ...
... Powdery mildew on watermelon, vine declines, Phytophthora blight, bacterial wilt, illnesses caused by Fusarium species, and various diseases caused by viruses, including Melon necrotic spot carmovirus and several members of the crinivirus genus (Clough and Hamm, 1995). Rotation, fumigation, minimising injury during harvest, chlorine spray or hot water treatment after harvest, sanitation, drip irrigation, culling symptomatic fruit before storage, pathogen-free seed, plastic mulch or other soil barrier, deep ploughing, adjusting soil pH, host controlling weeds and insects are some of the techniques used to manage various diseases, Plant resistance, fungicides, solarization, greenhouse climate manipulation, improved soil drainage, treated seed, planting when soil is not too cold, roguing sick plants, and correct storage conditions, including refrigeration A variety of transgenic cucurbit lines have been created and field tested by a number of commercial enterprises (Chandrasekaran et al. 2016;Azadi et al. 2011). ...
... This method has recently been used to build resistance to a variety of viruses (Baltes et al. 2015). By interrupting the activity of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene, a virus-resistant cucumber was created using CRISPR-Cas9 (Chandrasekaran et al. 2016). ...
... Host susceptibility proteins (encoded by recessive genes) provide essential assistance at different stages of the virus life cycle (Diaz-Pendon et al., 2004;Schie & Takken, 2014;Truniger & Aranda, 2009). Host susceptibility recessive genes serve as targets for the development of crop virus resistance by gene modification through genome editing technologies (Cao et al., 2020;Chandrasekaran et al., 2016;Gal-On et al., 2017). ...
... ToMV (similar to isolate 99-1 [KR537870.1]), and TMV (KU321698) mechanically according to (Chandrasekaran et al., 2016) at the two-leaf stage and were grown in a greenhouse at a constant temperature of 25°C or a nethouse with temperatures ranging between 21 and 31°C. 4.2 | Generation of SlTOM1a, SlTOM3, and SlOM1b tomato mutants using CRISPR/Cas9 technology The knockout of the SlTOM1a, SlTOM3, SlTOM1b, and SlARL8 genes by CRISPR/Cas9 was performed as in Corem et al. (2018). ...
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During tobamovirus–host coevolution, tobamoviruses developed numerous interactions with host susceptibility factors and exploited these interactions for replication and movement. The plant‐encoded TOBAMOVIRUS MULTIPLICATION (TOM) susceptibility proteins interact with the tobamovirus replicase proteins and allow the formation of the viral replication complex. Here CRISPR/Cas9‐mediated mutagenesis allowed the exploration of the roles of SlTOM1a, SlTOM1b, and SlTOM3 in systemic tobamovirus infection of tomato. Knockouts of both SlTOM1a and SlTOM3 in sltom1a/sltom3 plants resulted in an asymptomatic response to the infection with recently emerged tomato brown rugose fruit virus (ToBRFV). In addition, an accumulation of ToBRFV RNA and coat protein (CP) in sltom1a/sltom3 mutant plants was 516‐ and 25‐fold lower, respectively, than in wild‐type (WT) plants at 12 days postinoculation. In marked contrast, sltom1a/sltom3 plants were susceptible to previously known tomato viruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), indicating that SlTOM1a and SlTOM3 are not essential for systemic infection of TMV and ToMV in tomato plants. Knockout of SlTOM1b alone did not contribute to ToBRFV and ToMV resistance. However, in triple mutants sltom1a/sltom3/sltom1b, ToMV accumulation was three‐fold lower than in WT plants, with no reduction in symptoms. These results indicate that SlTOM1a and SlTOM3 are essential for the replication of ToBRFV, but not for ToMV and TMV, which are associated with additional susceptibility proteins. Additionally, we showed that SlTOM1a and SlTOM3 positively regulate the tobamovirus susceptibility gene SlARL8a3. Moreover, we found that the SlTOM family is involved in the regulation of plant development. Knockouts of both SlTOM1a and SlTOM3 resulted in an asymptomatic response to ToBRFV, but were susceptible to ToMV and TMV, and SlTOM1a and SlTOM3 positively regulate the tobamovirus susceptibility gene SlARL8a3.
... We (Leibman et al. 2011;. The eukaryotic initiation factor 4E (EIF4E) gene on chromosome 1 modified by CRISPR-cas9 conferred resistance to CMV (Chandrasekaran et al. 2016). Monogenic resistances have already been mapped and/or introduced in cucumber elite germplasm (Martín-Hernández and Picó 2021). ...
... Nevertheless, as no SNP was detected in the EiF4G sequence in this study, we assumed that this gene was not involved in resistance to CMV. More recently, an EiF4E gene was shown to be involved in cucumber CMV resistance by CRISPR-Cas9 (Chandrasekaran et al. 2016). Note that EiF4E was located in one of the minor QTLs for ZYMV on chr1 but did not have any SNP within the EiF4E sequence. ...
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The mapping and introduction of sustainable resistance to viruses in crops is a major challenge in modern breeding, especially regarding vegetables. We hence assembled a panel of cucumber elite lines and landraces from different horticultural groups for testing with six virus species. We mapped 18 quantitative trait loci (QTL) with a multiloci genome wide association studies (GWAS), some of which have already been described in the literature. We detected two resistance hotspots, one on chromosome 5 for resistance to the cucumber mosaic virus (CMV), cucumber vein yellowing virus (CVYV), cucumber green mottle mosaic virus (CGMMV) and watermelon mosaic virus (WMV), colocalizing with the RDR1 gene, and another on chromosome 6 for resistance to the zucchini yellowing mosaic virus (ZYMV) and papaya ringspot virus (PRSV) close to the putative VPS4 gene location. We observed clear structuring of resistance among horticultural groups due to plant virus coevolution and modern breeding which have impacted linkage disequilibrium (LD) in resistance QTLs. The inclusion of genetic structure in GWAS models enhanced the GWAS accuracy in this study. The dissection of resistance hotspots by local LD and haplotype construction helped gain insight into the panel’s resistance introduction history. ZYMV and CMV resistance were both introduced from different donors in the panel, resulting in multiple resistant haplotypes at same locus for ZYMV, and in multiple resistant QTLs for CMV.
... Interaction was disrupted between VPg and eIF4E by the application of CRISPR/Cas9 that mutated eIF4E in tetraploid potato genome ( Figures 5A,B). Previously, the CRISPR/Cas9 technology was used to inhibit the function of the recessive eIF4E gene, resulting in viral resistance in cucumber (Cucumis sativus L.) (Chandrasekaran et al., 2016). It has been reported that Eva1, a variant of eIF4E-1, provides resistance to PVY in S. tuberosum, S. chacoense, and S. demissum. ...
... It is constitutively active throughout the cell cycle and is particularly efficient at fixing DSBs due to the efficiency of repair proteins ku70 and ku80. The CRISPR/Cas9 approach followed by NHJE-repair has been widely utilized to generate resistance in agricultural plants, such as in rice against bacterial blight, in cucumber against cucumber vein yellowing virus, and in tomato against powdery mildew (Chandrasekaran et al., 2016;Nekrasov et al., 2017;Shibata et al., 2018;Zafar et al., 2020b). Editing all alleles in a polyploid genome simultaneously is complex and challenging. ...
Article
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Potato (Solanum tuberosum L) is an important staple food around the world, and potato virus Y (PVY) is a major constraint to potato production. The VPg of PVY interacts with the initiation factor eIF4E, that works as a susceptibility factor during infection. The interaction between eIF4E protein and VPg has been disrupted by CRISPR/Cas9. A homozygous conserved region of eIF4E of potato variety Kruda was mutated by CRISPR/Cas9. Tracking of insertion, deletion and conversion events were confirmed by Sanger sequencing with ~15% editing efficiency. Truncated and mutated eIF4E proteins unable to interact with VPg and virus was note able to exploit the host machinery for replication and systemic spreading as judged by the symptoms. Mutated eIF4E lines showed enhanced resistance to PVYO strain. DAS-ELISA and RT-PCR validated the results of phenotype produced in transgenic plants. This is the first study to use CRISPR/Cas9 in cultivated potatoes to generate immunity to PVY. CRISPR/Cas9 resistance in tetraploid lines allows the development of new resistant potato cultivars.
... The resulting eifiso4e mutants were resistant to turnip mosaic virus (TuMV) infection without other traits affected (Pyott et al., 2016). Chandrasekaran et al. (2016) demonstrated that knocking out cucumber eIF4E resulted in resistance to a wide range of viruses (Chandrasekaran et al., 2016). Also, Gomez et al. (2019) showed that simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduced cassava brown streak disease symptom severity and incidence (Gomez et al., 2019). ...
... The resulting eifiso4e mutants were resistant to turnip mosaic virus (TuMV) infection without other traits affected (Pyott et al., 2016). Chandrasekaran et al. (2016) demonstrated that knocking out cucumber eIF4E resulted in resistance to a wide range of viruses (Chandrasekaran et al., 2016). Also, Gomez et al. (2019) showed that simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduced cassava brown streak disease symptom severity and incidence (Gomez et al., 2019). ...
Article
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The cap‐binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularisation of mRNAs and their subsequent cap‐dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knock‐out mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non‐transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non‐mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male‐sterility phenotype, while crossing with wild‐type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild‐type. An RNA‐Seq analysis identified critical genes in pollen development that were down‐regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E‐specific mRNA translation initiation is a limiting factor for male gametes formation in melon.
... The pRCS binary vector containing Cas9-sgRNA was generated based on 35S:Cas9-AtU6:sgRNA-eIF4E [42]. The sgRNA-eIF4E was replaced by a sgRNA-RDR1c sequence using the appropriate primers (Table S4), as per [42]. ...
... The pRCS binary vector containing Cas9-sgRNA was generated based on 35S:Cas9-AtU6:sgRNA-eIF4E [42]. The sgRNA-eIF4E was replaced by a sgRNA-RDR1c sequence using the appropriate primers (Table S4), as per [42]. ...
Article
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RNA-dependent RNA polymerase 1 (RDR1) plays a crucial defense role against plant viruses by secondary amplification of viral double-stranded RNA in the gene-silencing pathway. In this study, it was found that melon (Cucumis melo) encodes four RDR1 genes (CmRDR1a, b, c1 and c2) similar to the CsRDR1 gene family of cucumber (C. sativus). However, in contrast to cucumber, melon harbors a truncated CmRDR1b gene. In healthy plants, CmRDR1a was expressed, whereas the expression of CmRDR1c1/c2 was not detected. CmRDR1a expression level increased 20-fold upon cucumber mosaic virus (CMV) infection and was not increased in melon plants infected with zucchini yellow mosaic virus (ZYMV), cucumber vein yellowing virus (CVYV) and cucumber green mottle mosaic virus (CGMMV). The expression of CmRDR1c1/c2 genes was induced differentially by infection with viruses from different families: high levels of ~340-, 172- and 115-fold increases were induced by CMV, CVYV and CGMMV, respectively, and relatively low-level increases by potyvirus infection (4- to 6-fold). CMV mutants lacking the viral silencing suppressor 2b protein did not cause increased CmRDR1c/c2 expression; knockout of CmRDR1c1/c2 by CRISPR/Cas9 increased susceptibility to CMV but not to ZYMV. Therefore, it is suggested that the sensitivity of melon to viruses from different families is a result of the loss of function of CmRDR1b.
... Although there was less bacterial infiltration through the stomata in this modified type, its necrotroph resistance was unaffected. Cucumber was the first species in the Curcubitaceae family to successfully report gene editing using the CRISPR-Cas9 system (Chandrasekaran et al., 2016) [5] . For the establishment of widespread viral resistance, the authors focused on the elF4E gene at two distinct loci. ...
... Although there was less bacterial infiltration through the stomata in this modified type, its necrotroph resistance was unaffected. Cucumber was the first species in the Curcubitaceae family to successfully report gene editing using the CRISPR-Cas9 system (Chandrasekaran et al., 2016) [5] . For the establishment of widespread viral resistance, the authors focused on the elF4E gene at two distinct loci. ...
Article
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The demand for food security is expanding as the world's population expands gradually. It is now urgently necessary to apply cutting-edge technologies to accelerate the pace and scale of our vegetable production. One of the primary components of the global food supply chain is vegetable crops. They have evolved into staple meals in many cultures throughout the world because of their variety, flavour profile, and nutritional value. However, due to their physiology, they are more vulnerable to harm from biotic stress and climate change. Therefore, there is an urgent need for novel kinds with higher yields, greater adaptability, and tolerance to biotic and abiotic stress. Conventional breeding is difficult and, depending on the species, might take over 20 years to generate a new competitive variety since it depends on the genetic variability already existing in the genetic pool. Here is where the variety, usability, and high efficiency of technologies like CRISPR/Cas9 may produce superior outcomes. Genome editing using CRISPR/Cas9 has the potential to profoundly alter how crop development techniques are developed in the near future.
... CRISPR/Cas9 has already been applied in cucumber, melon and watermelon (Chandrasekaran et al. 2016;Hoogvorst et al. 2019;Tian et al. 2017). However, research is still limited due to the fairly recent introduction of CRISPR/Cas9 and to the fact that cucurbits are recalcitrant plants for agrobacterium mediated transformation needed for CRISPR/CAS9 editing. ...
... However, research is still limited due to the fairly recent introduction of CRISPR/Cas9 and to the fact that cucurbits are recalcitrant plants for agrobacterium mediated transformation needed for CRISPR/CAS9 editing. In cucumber Chandrasekaran et al. in 2016 edited eIF4E (eukaryotic translation initiation factor 4E) gene which was a known S-gene (Nicaise et al. 2003;Julio et al. 2015) for potyviruses in other plant species and conferred resistance to Zucchini yellow mosaic virus, Papaya ring spot mosaic virus-W, and the Ipomovirus Cucumber vein yellowing virus. In the same way Pechar et al. in 2021 edited the melon eIF4E in order to achieve resistance to potyviruses. ...
Article
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Tomato leaf curl New Dehli virus (ToLCNDV) is a whitefly transmitted plant virus that is affecting European melon cultivation for over a decade. Since its first introduction in the Mediterranean basin the virus has been associated with significant economic losses including lower yields and cracked non-marketable fruits in Spain and other key cucurbits production areas. Since there is no chemical application against viral pathogens the focus is geared towards resistance breeding. Various QTLs associated with ToLCNDV resistance have been reported over the recent years in melon and other cucurbits. In the current review we summarize the latest advances in melon breeding for ToLCNDV resistance and present all relevant loci known so far in cucurbits. As a way forward in the future we propose an alternative to traditional resistance gene introgression breeding by exploiting the knowledge on genes that confer susceptibility to the virus in melon and other cucurbits.
... The application of CRISPIR/cas9 is tremendous and more robust with high throughput manipulation of target genes. Some of the recent advancements in CRISPR/cas9-based genome-editing for the development of biotic and abiotic stresses include powdery mildew resistance in bread wheat [63], late blight resistance in potato [12], beet severe curly virus resistance [64], turnip mosaic virus resistance in A. thaliana [65], blast resistance in rice [66], cucumber vein yellowing virus [67], drought tolerance in maize [68], potassium deficiency tolerance in rice [69] (see review by Jaganathan et al. [70]). ...
... Sustainable agricultural production to feed the projected population of 9.8 billion by 2050 posed an unprecedented challenge to plant breeders. Disease [66], [69] C. sativus [67] Z. mays [68] Highest efficiency and universality for any target organism caused by bacterial and fungal pathogens contributes to 15% yield loss and the other 3% by viral pathogens [72], altogether exacerbating the demand for better breeding technologies for disease resistance. Of interesting phytopathogenic aspect is the crosspathogenicity of most principal fungal and viral pathogens to potato, tomato, and pepper (Table 2), which urges the breeders for the development of interspecific, broad-spectrum, and durable resistance. ...
Article
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Genome-editing tools for the development of traits to tolerate abiotic and biotic adversaries are the recently devised breeding techniques revolutionizing molecular breeding by addressing the issues of rapidness and precision. To that end, disease resistance development by disrupting disease susceptibility genes (S genes) to intervene in the biological mechanism of pathogenicity has significantly improved the techniques of molecular breeding. Despite the achievements in genome-editing aimed at the intervention of the function of susceptibility determinants or gene regulatory elements, off-target effects associated with yield-related traits are still the main setbacks. The challenges are attributed to the complexity of the inheritance of traits controlled by pleiotropic genes. Therefore, a more rigorous genome-editing tool with ultra-precision and efficiency for the development of broad-spectrum and durable disease resistance applied to staple crop plants is of critical importance in molecular breeding programs. The main objective of this article is to review the most impressive progresses achieved in resistance breeding against the main diseases of three Solanaceae crops (potato, Solanum tuberosum; tomato, Solanum lycopersicum and pepper, Capsicum annuum) using genome-editing by disrupting the sequences of S genes, their promoters, or pathogen genes. In this paper, we discussed the complexity and applicability of genome-editing tools, summarized the main disease of Solanaceae crops, and compiled the recent reports on disease resistance developed by S-gene silencing and their off-target effects. Moreover, GO count and gene annotation were made for pooled S-genes from biological databases. Achievements and prospects of S-gene-based next-generation breeding technologies are also discussed.
... The immature genetic transformation system of cucumber restricts the application of CRISPR/ Cas9. Fewer studies concerning the application of CRISPR/ Cas9 in cucumbers have been reported; however, both have the same bottleneck of low mutation efficiency [19,26]. ...
... Chanderasekaran et al. [26] obtained three edited plants of the eIF4E gene with Cas9/sgRNA from 5 PCR positive cucumber strains. However, this paper did not mention the number of explants, thus failing to compare genetic transformation efficiency. ...
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BackgroundsThe narrow genetic basis of cucumber makes breeding of this species difficult. CRISPR/Cas9 system is characteristic of simple design, low cost and high efficiency, which has opened a new path for cucumber functional genetics and the development of cucumber mocular breeding. However, the immature genetic transformation system is the main limiting factor for applying this technology in cucumber.Methods and ResultsIn this study, a Histochemical β-glucuronidase (GUS) assay was used to analyze the effect of various parameters, including slight scratch of explants, pre-culture time, acetosyringone (AS) concentration, infection time in Agrobacterium solution, and co-culture period on the transformation efficiency. The results showed that the explants slightly scratched after cutting, pre-cultured for 1 day, Agrobacterium bacterial solution containing AS, and 20 min length of infection could significantly increase the GUS staining rate of explants. On this basis, two sequences with high specificity (sgRNA-1 and sgRNA-2) targeted different loci of gene CsGCN5 were designed. The corresponding vectors Cas9-sgRNA-1 and Cas9-sgRNA-2 were constructed and transformed using the above-optimized cucumber genetic transformation system, and three and two PCR positive lines were obtained from 210 and 207 explants, respectively. No sequence mutation at target loci of CsGCN5 was detected in the Cas9-sgRNA-1 transformed three PCR positive lines. However, one mutant line with targeted homozygous change was recognized from the Cas9-sgRNA-2 transformed two PCR positive lines.Conclusion In this study, 2.4‰ of total explants had directed mutation in the CsGCN5 gene. The results in the present study would be beneficial to further optimize and improve the efficiency of the genetic transformation of cucumber.
... In contrast, no new variants were found in plants carrying sgRNAs targeting the IR sequences in N. benthamiana plants. A second technique for achieving viral disease resistance entails altering plant genes that provide virus resistance qualities, segregating the CRISPR/Cas9 tool, and releasing nontransgenic mutants into the field (Chandrasekaran et al., 2016;Pyott et al., 2016;Macovei et al., 2018). Plant host factors, such as the eukaryotic translation initiation factors eIF4E, eIF(iso)4E, and eIF4G, are required by RNA viruses to maintain their life cycle (Sanfaçon, 2015). ...
... Plant host factors, such as the eukaryotic translation initiation factors eIF4E, eIF(iso)4E, and eIF4G, are required by RNA viruses to maintain their life cycle (Sanfaçon, 2015). By modifying two different sites of the host susceptibility gene eIF4E with CRISPR/Cas9, Chandrasekaran et al. (2016) were able to develop cucumber plants that were resistant to potyviruses. Homozygous eif4e mutants demonstrated protection to viruses from the Potyviridae family, such as zucchini yellow mosaic virus (ZYMV), cucumber vein yellowing virus (CVYV), and papaya ring spot mosaic virus-W (PRSV-W). ...
Article
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Abiotic stress such as cold, drought, saline-alkali stress and biotic stress including disease and insect pest are the main factors that affect plant growth and limit agricultural productivity. In recent years, with the rapid development of molecular biology, genome editing techniques have been widely used in botany and agronomy due to their characteristics of high efficiency, controllable and directional editing. Genome editing techniques have great application potential in breeding resistant varieties. These techniques have achieved remarkable results in resistance breeding of important cereal crops (such as maize, rice, wheat, etc.), vegetable and fruit crops. Among them, CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) provides a guarantee for the stability of crop yield worldwide. In this paper, the development of CRISRR/Cas and its application in different resistance breeding of important crops are reviewed, the advantages and importance of CRISRR/Cas technology in breeding are emphasized, and the possible problems are pointed out.
... Compared with its use in animal cells, CRISPR-Cas9 editing in plants remains in its infancy, yet it has already been used to modify the genes of a wide range of plant species [5,6]. CRISPR experiments in plants have developed virus-resistant cucumbers [7], citrus trees immune to disease [8] and rice lines with enhanced crop yield [9]. ...
Article
[Formula: see text] Standfirst: To feed an ever-growing population in an increasingly volatile climate, new technologies are required; is CRISPR the key to reducing food waste and creating climate change-proof crops?
... The technology was further used to disrupt the coding region of CsLOB1 resulting in no canker symptoms in Duncan grapefruit [46]. [47] conducted a research in cucumber by disrupting eIF4E (Eukaryotic translation initiation factor 4E), broad virus resistance was developed. The plants were seen immune to cucumber Vein Yellowing virus (Ipomovirus) and were also resistant against potyviruses, Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. ...
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CRISPR/Cas systems are the third-generation genome editing systems, which appeared in 2012 and quickly became a superstar in genome editing tools because of their great simplicity and usability compared to with ZFN and TALEN. CRISPR/Cas was originally identified as an effective acquired immune system in bacteria against virus infection and relies on RNA-DNA binding to achieve sequence specificity in genome editing. CRISPR/Cas9 system has become widely used in plants for characterizing gene function and crop improvement. Crops such as tomato, rice, banana and wheat are excellent model plants for biological research and are most important applied plants for genome editing. Genome editing has also been applied in plant breeding for improving fruit yield and quality, increasing stress resistance, accelerating the domestication of wild tomato, and recently customizing tomato cultivars for urban agriculture. In addition, genome editing is continuously innovating, and several new genome editing systems such as the recent prime editing, a breakthrough in precise genome editing, have recently been applied in plants. In this review, the advances in applications of CRISPR/Cas systems genome editing technology to enhance specific features in plants in order to mitigate postharvest losses and wastes are summarized.
... The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/ CRISPR-associated) system, a genome editing technology, has been applied for the genetic improvement of plant viruses resistance and has accelerated resistance breeding (Zhang et al. 2019b). In cucumbers, knocking out the eIF4E gene using the CRISPR/Cas9 system resulted in broad-spectrum resistance to members of the Potyviridae family, including cucumber vein yellowing virus, papaya ringspot virus, and zucchini yellow mosaic virus (Chandrasekaran et al. 2016). In wheat, TaeIF(iso)4e-mutant lines were obtained using CRISPR-Cas9 genome-editing technology, which revealed that the corresponding mutations can potentially confer WSSMV and WYMV resistance (Hahn et al. 2021). ...
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In China, soil-borne viruses transmitted by the root parasite Polymyxa graminis have caused significant yield loss in winter wheat for many years. At present, it is believed that two main soil-borne RNA viruses, namely wheat yellow mosaic virus (WYMV) and Chinese wheat mosaic virus (CWMV) are responsible for such losses. The molecular characteristics and infection processes of these two viruses have been intensively investigated and described substantially in detail, following the complete sequencing of their respective genomes. In this review, we highlight our recent findings on the distribution of WYMV and CWMV in China, the associated crop damage, the biological functions of WYMV and CWMV proteins as well as the viral temperature sensitivities. We also describe the characteristics of the resistance genes and discuss the novel virus–plant arms race strategies in hope of enlarging our understanding on the theme of virus-plant interactions. Finally, we compare current disease-management options and suggest the application of biotechnology-based genetic resistance to develop more cost-effective countermeasures for controlling soil-borne virus diseases in the future.
... Unlike RNA interference (binding siRNA with the mRNA of the target gene to reduce the level of gene expression), this is a precise tool to introduce targeted mutations strategically in the host genome [64]. This technique was soon applied to develop broad-spectrum antiviral [65], dwarf [48], and gynoecious cucumber lines [49]. ...
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Cucumber (Cucumis sativus L.), belonging to the gourd family (Cucurbitaceae), is one of the major vegetable crops in China. Conventional genetic breeding methods are ineffective for improving the tolerance of cucumber to various environmental stresses, diseases, and pests in the short term, but bio-engineering technologies can be applied to cucumber breeding to produce new cultivars with high yield and quality. Regeneration and genetic transformation systems are key technologies in modern cucumber breeding. Compared with regeneration systems, genetic transformation systems are not yet fully effective, and the low efficiency of genetic transformation is a bottleneck in cucumber cultivation. Here, we systematically review the key factors influencing the regeneration and genetic transformation of cucumber plants, including the selection of genotype, source of explants and forms of exogenous hormones added to the medium, the methods of transgene introduction and co-cultivation, and selection methods. In addition, we also focus on recent advances in the study of molecular mechanisms underlying important agronomic traits using genetic transformation technology, such as fruit length, fruit warts, and floral development. This review provides reference information for future research on improvements in cucumber varieties.
... Simultaneous mutation of the three homeoalleles of TaMLO conferred heritable broad-spectrum resistance to powdery mildew in hexaploid bread wheat (Wang et al., 2014). Similarly, Zhang et al. (2017) and Ipomovirus (cucumber) has also been reported (Dale et al., 2017;Jia et al., 2017;Peng et al., 2017;Wang et al., 2017;Gomez et al., 2019;Chandrasekaran et al., 2016; Table 2). ...
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The global climate change and unfavourable abiotic and biotic factors are limiting agricultural productivity and therefore intensifying the challenges for crop scientists to meet the rising demand for global food supply. The introduction of applied genetics to agriculture through plant breeding facilitated the development of hybrid varieties with improved crop productivity. However, the development of new varieties with the existing gene pools poses a challenge for crop breeders. Genetic engineering holds the potential to broaden genetic diversity by the introduction of new genes into crops. But the random insertion of foreign DNA into the plant’s nuclear genome often leads to transgene silencing. Recent advances in the field of plant breeding include the development of a new breeding technique called genome editing. Genome editing technologies have emerged as powerful tools to precisely modify the crop genomes at specific sites in the genome, which has been the longstanding goal of plant breeders. The precise modification of the target genome, the absence of foreign DNA in the genome-edited plants, and the faster and cheaper method of genome modification are the remarkable features of the genome-editing technology that have resulted in its widespread application in crop breeding in less than a decade. This review focuses on the advances in crop breeding through precision genome editing. This review includes: an overview of the different breeding approaches for crop improvement; genome editing tools and their mechanism of action and application of the most widely used genome editing technology, CRISPR/Cas9, for crop improvement especially for agronomic traits such as disease resistance, abiotic stress tolerance, herbicide tolerance, yield and quality improvement, reduction of anti-nutrients, and improved shelf life; and an update on the regulatory approval of the genome-edited crops. This review also throws a light on development of high-yielding climate-resilient crops through precision genome editing.
... CRISPR-Cas9 was implemented to mutate some of these recessive genes, among them eukaryotic translation initiation factors, i.e., eIF4E and eIF4G, have been widely explored to prevent viral infection in diverse crop species (Mushtaq et al., 2020;Hashimoto et al., 2016). In cucumber, CRISPR-Cas9mediated targeted knock-out of eIF4E exhibited complete protection against a wide range of viruses (Chandrasekaran et al., 2016). Reports on rice are also encouraging, where the knock-out of the rice gene eIF4G using CRISPR-Cas9 displayed resistance against tungro disease (Macovei et al., 2018;Kumam et al., 2022). ...
Article
Genome editing technology has rapidly evolved to knock-out genes, create targeted genetic variation, install precise insertion/deletion and single nucleotide changes, and perform large-scale alteration. The flexible and multipurpose editing technologies have started playing a substantial role in the field of plant disease management. CRISPR-Cas has reduced many limitations of earlier technologies and emerged as a versatile toolbox for genome manipulation. This review summarizes the phenomenal progress of the use of the CRISPR toolkit in the field of plant pathology. CRISPR-Cas toolbox aids in the basic studies on host-pathogen interaction, in identifying virulence genes in pathogens, deciphering resistance and susceptibility factors in host plants, and engineering host genome for developing resistance. We extensively reviewed the successful genome editing applications for host plant resistance against a wide range of biotic factors, including viruses, fungi, oomycetes, bacteria, nematodes, insect pests, and parasitic plants. Recent use of CRISPR-Cas gene drive to suppress the population of pathogens and pests has also been discussed. Furthermore, we highlight exciting new uses of the CRISPR-Cas system as diagnostic tools, which rapidly detect pathogenic microorganism. This comprehensive yet concise review discusses innumerable strategies to reduce the burden of crop protection.
... To enhance plants biotic resistance to viruses, two methods have been used: (1) CRISPR-Cas was used to target the N and C ends of eIF4E-produced nontransgenic homozygous plants in the T3 generation, which showed immunity to cucumber vein yellow virus and pumpkin mosaic virus, as well as resistance to papaya ring spot mosaic virus (PRSV-W) (Chandrasekaran, J. et al., 2016). ...
Article
Genome-editing technologies provide unprecedented opportunities for agriculture crop improvement with unprecedented precision and speed. This review examines the current state of CRISPR/Cas9 genome editing in cereal, vegetables oilseed crops medicinal plants, and fruits. Important agronomic and quality traits in agriculture crops have been achieved through genome editing. Adaptive traits to mitigate the effects of climate change, tolerance to biotic stresses, higher yields, more optimal plant architecture, improved grain quality and nutritional content, and safer products. Genome editing, on the other hand, has already transformed all crops improvement and is poised to shape future agricultural practises in tandem with other breeding innovations.
... In another example, Cas9/sub genomic RNA (sgRNA technology) was successfully utilized to develop virus-resistant cucumber by disrupting eIF4E (eukaryotic translation initiation factor 4E) gene. eIF4E sites targeted transgenic plants develop resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus (Chandrasekaran et al. 2016). Similarly, Downy mildew and Powdery mildew are both serious fungal infections that affect grapes. ...
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Climate change leads to frequent alterations in environmental factors with a reciprocal impact on crop productivity. Over the last few decades, various approaches have been used for producing more stress-tolerant and climate-flexible crops. Genetic engineering is one of the approaches used to modify multiple characters or to improve more than one agronomic trait in plants. These instances simultaneously demand simultaneous genetic manipulation of multiple genes, necessitating stacking or pyramiding of multiple genes as compared to single-gene manipulations, and the genetic engineering of plants using multiple genes is technically challenging. In the last two decades, considerable progress has been made with respect to the development and application of the methods for gene pyramiding in transgenic context. The conventional methods of gene stacking include the crossing of individual transgenic plants, co-transformation using multiple plant expression constructs, transformation with single constructs carrying multiple transgenes as well as with the constructs carrying polycistronic transgenes. These methods have been instrumental for gene stacking in several commercialized crops. The tools of targeted genome editing (ZFN, TALEN, and CRISPR) that carry out precise genetic modifications, have opened new avenues in the area of crop biotechnology for defending plants against various stresses. The present review covers the current status of biotechnological techniques used to combat biotic and abiotic stresses in crop plants and describes multiple associated challenges.
... For example, loss-of-function mutation of eIF4Es would result in broad-spectrum resistance to potyviruses, potexviruses, bymoviruses, cucumoviruses, ipomoviruses, and carmoviruses [70][71][72][73][74][75] . EXA1 has been reported as a recessive resistance gene required for the infection of viruses, bacteria, and oomycete pathogens in several model plants [44][45][46][47] . ...
Article
Plant viruses recruit multiple host factors for translation, replication, and movement in the infection process. The loss-of-function mutation of the susceptibility genes will lead to the loss of susceptibility to viruses, which is referred to as “recessive resistance”. Essential for potexvirus Accumulation 1 (EXA1) has been identified as a susceptibility gene required for potexvirus, lolavirus, and bacterial and oomycete pathogens. In this study, EXA1 knockdown in potato (StEXA1) was found to confer novel resistance to potato virus Y (PVY, potyvirus) in a strain-specific manner. It significantly compromised PVYO accumulation but not PVYN:O and PVYNTN. Further analysis revealed that StEXA1 is associated with the HC-Pro of PVY through a member of eIF4Es (StnCBP). HC-ProO and HC-ProN, two HC-Pro proteins from PVYO and PVYN, exhibited strong and weak interactions with StnCBP, respectively, due to their different spatial conformation. Moreover, the accumulation of PVYO was mainly dependent on the stress granules (SGs) induced by StEXA1 and StnCBP, whereas PVYN:O and PVYNTN could induce SGs by HC-ProN independently through an unknown mechanism. These results could explain why StEXA1 or StnCBP knockdown conferred resistance to PVYO but not to PVYN:O and PVYNTN. In summary, our results for the first time demonstrate that EXA1 can act as a susceptibility gene for PVY infection. Finally, a hypothetical model was proposed for understanding the mechanism by which StEXA1 interacts with StnCBP to facilitate PVY accumulation in potato through the SG-dependent RNA regulatory pathway.
... eIF4E, eIF4G, and their isoforms are effective host translation initiation factors, which are exploited to achieve defense against various sub-species of viruses. Chandrasekaran et al. (2016) reported the eukaryotic initiation factor, eIF4Eknockout in cucumber through CRISPR/Cas9, which exhibited complete resistance to viruses involved in papaya ringspot, zucchini yellow mosaic, and cucumber vein yellowing diseases. Recent advancements in CRISPR/Cas9 technologies resulted in increasing reports of efficient plant DNA virus resistance. ...
Article
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Leaf curl disease in a chili plant is caused mainly by Chili leaf curl virus (ChiLCV) (Family: Geminiviridae, Genus: Begomovirus). ChiLCV shows a widespread occurrence in most of the chili (Capsicum spp.) growing regions. ChiLCV has a limited host range and infects tomatoes (Solanum lycopersicum), potatoes (S. tuberosum), and amaranth (Amaranthus tricolor). The virus genome is a monopartite circular single-stranded DNA molecule of 2.7 kb and associated with α and β-satellites of 1.3 and 1.4 kb, respectively. The virus genome is encapsulated in distinct twinned icosahedral particles of around 18–30 nm in size and transmitted by Bemisia tabaci (Family: Aleyrodidae, Order: Hemiptera). Recently, bipartite begomovirus has been found to be associated with leaf curl disease. The leaf curl disease has a widespread distribution in the major equatorial regions viz., Australia, Asia, Africa, Europe, and America. Besides the PCR, qPCR, and LAMP-based detection systems, recently, localized surface-plasmon-resonance (LPSR) based optical platform is used for ChiLCV detection in a 20–40 μl of sample volume using aluminum nanoparticles. Management of ChiLCV is more challenging due to the vector-borne nature of the virus, therefore integrated disease management strategies need to be followed to contain the spread and heavy crop loss. CRISPR/Cas-mediated virus resistance has gained importance in disease management of DNA and RNA viruses due to certain advantages over the conventional approaches. Therefore, CRISPR/Cas system-mediated resistance needs to be explored in chili against ChiLCV.
... This caused overexpression of Cas9 endonuclease in Nicotiana benthamiana and conferred resistance toward TYLCV. Chandrasekaran et al. (2016) develop cucumber-resistant line against cucumber vein yellowing virus, potyviruses, zucchini yellow mosaic virus, and papaya ringspot mosaic virus-W. They have achieved this by disrupting eIF4E and generating transgene-free heterozygous eIF4E mutant plants. ...
... To enhance plants biotic resistance to viruses, two methods have been used: (1) CRISPR-Cas was used to target the N and C ends of eIF4E-produced nontransgenic homozygous plants in the T3 generation, which showed immunity to cucumber vein yellow virus and pumpkin mosaic virus, as well as resistance to papaya ring spot mosaic virus (PRSV-W) (Chandrasekaran, J. et al., 2016). ...
... SgRNAs targeting the replication proteins, intergenic regions and coat proteins significantly reduced the symptoms of bean golden mosaic virus [21]. Moreover, removal of eIF(iso)4E gene in Arabidopsis improved resistance against turnip mosaic virus [22] The translational factor, Eukaryotic translation initiation factor 4E (eif4e), which negatively regulate resistance in plants has been successfully edited in several plant RNA viruses including Potyviruses [23]. VPG, a Virus-encoded protein binds to Eukaryotic translation initiation factor 4E (eIF4E), and leads to mutations in the eIF4E, resulting in quantitative resistant phenotypic variation [23]. ...
Article
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Agricultural crop production is critically restrained by many plant pathogens and pests, particularly where a number of them exist concurrently. A use disease-resistant plant variety is one of the most effective ways to reduce the negative impacts of plant pathogens on crop production and yield. Recent advancements in novel breeding techniques offer the potential to expedite disease resistance breeding in a variety of plants. Because of its ability to make precise alterations in plant genomes and trait stacking through multiplexing, CRISPR/Cas9-based genome editing has become as the most effective technique for genetic enhancement. CRISPR/Cas9 is a fast evolving genome editing tool that has been successfully used in many species, including experimental and agricultural plants. Disease resistance is rapidly engineered by CRISPR-based genome editing tools by directly targeting specific nucleotide sites of plant pathogens. This method is highly effective, having low risk of off-target effects as compared to other genome editing techniques, and is reasonably simple to use. CRISPR-Cas technology produce incredible broad-spectrum disease resistance in crops with the assistance of CRISPR-Cas technology where specific host susceptibility genes are targeted (Susceptibility-gene approach) or DNA of plant pathogens is cleaved (bacteria, viruses, or fungi) in order to restrain their emergence. CRISPR-Cas technology is successfully being employed to enhance crop immunity and tolerance to number of plant pathogens, and thereafter leading to the Nisa et al. 2 development of disease-resistant crops. In this review, we will discuss about theCRISPR-Cas9 mediated genome editing technology and its utilization in plant disease resistance.
... Cas9/subgenomic RNA (sgRNA) technology has been used to generate recessive inactivation of eukaryotic translation initiation factor 4E (eIF4E) gene. This research suggests that eIF4E inhibition promotes resistance against Cucumber vein yellowing virus [101]. In the cucumber drought stress regulatory pathway, cucumber activating factor1 (CsATAF1) is a positive regulator that can reduce the accumulation of reactive oxygen species (ROS) [102]. ...
Article
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Cucumbers are rich in vitamins and minerals. The cucumber has recently become one of China’s main vegetable crops. More specifically, the adjustment of the Chinese agricultural industry’s structure and rapid economic development have resulted in increases in the planting area allocated to Chinese cucumber varieties and in the number of Chinese cucumber varieties. After complete sequencing of the “Chinese long” genome, the transcriptome, proteome, and metabolome were obtained. Cucumber has a small genome and short growing cycle, and these traits are conducive to the application of molecular breeding techniques for improving fruit quality. Here, we review the developments and applications of molecular markers and genetic maps for cucumber breeding and introduce the functions of gene families from the perspective of genomics, including fruit development and quality, hormone response, resistance to abiotic stress, epitomizing the development of other omics, and relationships among functions.
... CRISPR-mediated gene drive can be used to disseminate transgenes and mutations in the target populations (Fig. 4). On the other hand, as an alternative to targeting the viral genome now with the help of CRISPR, plant genes, i.e., S genes enabling the transmission and settling of the virus within the plants can be targeted (Chandrasekaran et al. 2016;Pyott et al. 2016) which provide an alternative way to control whiteflies. In a nutshell, all these discussed strategies could be utilized with the proper identification of whitefly at the species level and the fulfillment of regulatory quarantine measures that will assist in better pest management. ...
Article
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Bemisia tabaci species complex (whitefly) is one of the most dangerous pests that destroy many important crops worldwide. It causes damage to the host plant by feeding on phloem sap as well as transmitting a wide range of devastating plant viruses (especially begomoviruses) that cause severe epidemics on crops. To fend off the menace, modern genomic-based strategies have been adapted to minimize the crop losses due to this destructive pest. Genetic engineering techniques, e.g., transgenics and RNA interference (RNAi) have shown promising results in controlling B. tabaci in plants; however, these techniques often face challenges due to the concerns about GMOs in food crops. With the enhanced knowledge about B. tabaci genomics, new technologies, e.g., manipulation of microbiota or CRISPR-based genome editing have shown promising results in several insect pests and could have an instrumental role in controlling agricultural pests including whitefly. Genome editing is an eco-friendly approach that can be employed to suppress or even destroy the target species. In this review, we have discussed B. tabaci as a pest and advancement in control strategies of B. tabaci. Various potential targets for genome editing have also been discussed that could be used in gene-editing technologies for the efficient management of B. tabaci and the viruses it transmits. Finally, we also outlined the future perspective and effective use of genome editing technology in developing CRISPR-based gene drive for whitefly population modification, suppression, and eradication.
... We identified regenerated plants with loss-of-function mutations in the combinations of one, two, three and all four targeted genes in the T0 generation, indicating that CRISPR/Cas9 can be efficiently harnessed for precision combinatorial gene editing applications in tobacco. eIF-mediated potyvirus resistance has been extensively studied in many plants 12,48,53,54 , which is based on the interaction of the viral genome-linked protein (VPg) with the eukaryotic translation initiation factor eIF4E or its orthologue eIF(iso)4E to initiate viral RNA translation and subsequent virus replication 55,56 . Consequently, down regulation of the above translation initiation factors or mutations that disrupt the binding of VPg to the corresponding eIFs could result in resistance to potyviruses 57,58 . ...
Article
Full-text available
Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVYO accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVYO confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVYO in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.
... Similarly, other transporter proteins are also prominent candidates for genome editing for the enhancement of abiotic stress tolerance. These factors indicate that the CRISPR/Cas system can be harnessed prolifically for this novel purpose and will be the future of targeting minor genes of complex quantitative traits related to abiotic stresses [20]. ...
Article
Full-text available
Agriculture plays a predominant role in economy as well as it is considered as the backbone of economic system for developing countries like India. A recent advancement in the field of life sciences is gene editing. It is one of the boundless examples of techniques used to explore the understanding of the biological phenomenon. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing approach is a choice of approach due to its simplicity, ease of access, cost, and flexibility. In this review, numerous CRISPR/Cas based approaches have been discussed, also considered recent advancements and challenges to implicate those in the crop improvement programs. In this paper, CRISPR/C as approaches have been used to improve the biotic and a biotic stress tolerance, and traits related to yield and plant architecture have been discussed. This paper, highlights the challenges to implement the gene editing in plants like wheat, canola, and sugarcane. We have also discussed about multi-target editing approaches. Gene editing has potential to edit multiple targets at the same time makes it possible to take up more challenging tasks required to engineer chosen crop plants. Similarly, advances like precision gene editing, promoter bashing, and methylome editing will also be discussed. The present review also provides a list of available computational tools and servers facilitating designing of guide-RNA targets construct designs, and data analysis. The data provided here useful for the exploration of technological advances in gene editing field for the crop improvement.
... CRISPR/Cas9 system has been used for broad-spectrum resistance targeting and disrupting translation initiation like factors eIF4E gene without affecting the plant genome in cucumber. Immunity was exhibited against the family Potyviridae, mainly Cucumber vein yellowing virus (CVYV), Zucchini yellow mosaic virus (ZYMV), and Papaya ring spot mosaic virus-W (PRSV) by introducing small deletions and SNPs in recessive eIF4E gene in T1 generation of cucumber [40]. Cassava brown streak virus (CBSV) is a major constraint for Central and Eastern Africa cassava yields. ...
Article
Full-text available
Crop plants are prone to several yield-reducing biotic and abiotic stresses. The crop yield reductions due to these stresses need addressing to maintain an adequate balance between the increasing world population and food production to avoid food scarcities in the future. It is impossible to increase the area under food crops proportionately to meet the rising food demand. In such an adverse scenario overcoming the biotic and abiotic stresses through biotechnological interventions may serve as a boon to help meet the globe's food requirements. Under the current genomic era, the wide availability of genomic resources and genome editing technologies such as Transcription Activator-Like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and Clustered-Regularly Interspaced Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas) has widened the scope of overcoming these stresses for several food crops. These techniques have made gene editing more manageable and accessible with changes at the embryo level by adding or deleting DNA sequences of the target gene(s) from the genome. The CRISPR construct consists of a single guide RNA having complementarity with the nucleotide fragments of the target gene sequence, accompanied by a protospacer adjacent motif. The target sequence in the organism's genome is then cleaved by the Cas9 endonuclease for obtaining a desired trait of interest. The current review describes the components, mechanisms, and types of CRISPR/Cas techniques and how this technology has helped to functionally characterize genes associated with various biotic and abiotic stresses in a target organism. This review also summarizes the application of CRISPR/Cas technology targeting these stresses in crops through knocking down/out of associated genes.
... CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has transformed gene editing in plants. With the recent availability of full-length and draft genomes of several crops, the use of gene manipulation in crops such as wheat, maize, sorghum, barley, cucumber, rice, banana and cassava has been facilitated by applying genome sequence information to understand the gene structures as well as discover and edit genes coding for economically important traits (Chandrasekaran et al., 2016;Svitashev et al., 2016;Odipio et al., 2017;Srivastava et al., 2017;Gomez et al., 2019;Kim et al., 2019;Tripathi et al., 2020;Yan et al., 2020;Bahariah et al., 2021;Ma et al., 2021). ...
Chapter
Agriculture in the tropical world faces many challenges because of growing populations and environmental degradation. Climate change is exacerbating these problems but also extending them to a wider range of environments globally. Tropical crops may be increasingly suitable for regions much further from the equator. Many tropical crops have not had a long history of research investment that has supports the major crops from temperate regions. As genome sequencing technology has advanced rapidly in recent years it has been applied to many tropical species that have not been well researched in the past. Advances in genome sequencing can be views as providing an opportunity for our understanding of the biology and breeding of tropical crop plants to catch up with that for better-studied species. Genomics provides a platform that can be used to support germplasm analysis and conservation, climate adaptation, improvement of crop performance, and food nutritional and functional quality.
... Similarly, other transporter proteins are also prominent candidates for genome editing for the enhancement of abiotic stress tolerance. These factors indicate that the CRISPR/Cas system can be harnessed prolifically for this novel purpose and will be the future of targeting minor genes of complex quantitative traits related to abiotic stresses [20]. ...
Article
Full-text available
Agriculture plays a predominant role in economy as well as it is considered as the backbone of economic system for developing countries like India. A recent advancement in the field of life sciences is gene editing. It is one of the boundless examples of techniques used to explore the understanding of the biological phenomenon. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing approach is a choice of approach due to its simplicity, ease of access, cost, and flexibility. In this review, numerous CRISPR/Cas based approaches have been discussed, also considered recent advancements and challenges to implicate those in the crop improvement programs. In this paper, CRISPR/CAS approaches have been used to improve the biotic and abiotic stress tolerance, and traits related to yield and plant architecture have been discussed. This paper, highlights the challenges to implement the gene editing in plants like wheat, canola, and sugarcane. We have also discussed about multi-target editing approaches. Gene editing has potential to edit multiple targets at the same time makes it possible to take up more challenging tasks required to engineer chosen crop plants. Similarly, advances like precision gene editing, promoter bashing, and methylome editing will also be discussed. The present review also provides a list of available computational tools and servers facilitating designing of guide-RNA targets construct designs, and data analysis. The data provided here useful for the exploration of technological advances in gene editing field for the crop improvement.
... Tomato, citrus, and orange became resistant to bacterial disease by knocking out the SlJAZ2, CsLOB1, and CsWRKY22 genes, respectively [48][49][50]. The CRISPR/Cas9 tool was used to knock out the eif4e gene, which produces virus resistance to cucumber vein yellowing virus, papaya ring spot mosaic virus W, and zucchini yellow mosaic virus [51]. Cotton became resistant to the fungal disease Verticillium dahliae after knocking out the 14-3-3 gene with the application of CRISPR/Cas9 [52]. ...
Article
Full-text available
The long-term goal of scientists and breeders is to study the efficacy of a gene, as well as to use it in the development of human life and in the development of improved quality and varieties of crops. CRISPR/Cas9, a gene-editing tool, has already uncovered a wide range of applications in areas such as human disease diagnosis and the development of new crop varieties. This review provides basic ideas about CRISPR/Cas9 as well as its importance in the current context. CRISPR/Cas9 editing tool has more contributions to plant science than medical science. As a mature cutting-edge biotechnological technique, CRISPR/Cas9 has been applied in a variety of crop related research and development areas including disease resistance, plant development, abiotic tolerance, morphological development, secondary metabolism, and fiber formation. Lastly, some of the limitations of this system have been mentioned, and aspects of more research in the future have been suggested. Through this review, readers will better be able to understand the CRISPR/Cas9 genome editing system and will be familiar with much of the research that has occurred from the past to the present in a range of science fields.
... Similarly, the CRISPR-Cas9 genome editing tool was used to develop virus resistance in cucumber (Cucumis sativus L.). Here, genome editing was utilized to alter the function of the eIF4E gene (eukaryotic translation initiation factor 4E) of C. sativus, and the transformed plants showed resistance against Potyviruses (Zucchini yellow mosaic virus and Papaya ringspot mosaic virus) and Ipomovirus (Cucumber vein yellowing virus) (Chandrasekaran et al. 2016). Wheat dwarf virus (WDV) is a phloem-limited, insect-transmitted virus and belongs to the Geminiviridae family. ...
Article
Diseases caused by plant pathogens pose significant challenges to crop yield and quality, thus incurring a serious constraint on the global food supply. Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated nuclease) system has revolutionized the field of biology and biotechnology. New technologies derived from CRISPR-Cas enable crop genetic improvement for plant disease resistance and the development of tools for the early detection of pathogens. This review discusses canonical and advanced genome editing tools available for understanding host–pathogen interaction and developing host resistance. We categorically cite examples of the use of genome editing technology in developing disease-resistant crops. CRISPR-Cas9 tools have enabled us to apply novel strategies to confer resistance against several plant pathogens like bacteria, viruses, fungi, and nematodes. CRISPR diagnostic methods have been developed by leveraging the CRISPR-Cas system’s precise ability to bind and cleave nucleic acids. We have also discussed the different CRISPR-diagnostic methods developed for precise and fast plant disease detection.
... In Arabidopsis, point mutations in eIF(iso)4E gene were found to impart complete resistance against the turnip mosaic virus (Pyott et al., 2016). Likewise, in cucumber, eukaryotic translation initiation factor eIF(iso)4E was engineered using the CRISPR/Cas9 system to generate heritable homozygous point mutations that conferred resistance to the mutants against zucchini yellow mosaic virus, papaya ringspot mosaic virus-W, and vein yellowing virus (Chandrasekaran et al., 2016). In Nicotiana benthamiana, sgRNA/Cas9-mediated broad-spectrum immunity was ...
Article
Full-text available
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system was initially discovered as an underlying mechanism for conferring adaptive immunity to bacteria and archaea against viruses. Over the past decade, this has been repurposed as a genome-editing tool. Numerous gene editing-based crop improvement technologies involving CRISPR/Cas platforms individually or in combination with next-generation sequencing methods have been developed that have revolutionized plant genome-editing methodologies. Initially, CRISPR/Cas nucleases replaced the earlier used sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), to address the problem of associated off-targets. The adaptation of this platform led to the development of concepts such as epigenome editing, base editing, and prime editing. Epigenome editing employed epi-effectors to manipulate chromatin structure, while base editing uses base editors to engineer precise changes for trait improvement. Newer technologies such as prime editing have now been developed as a “search-and-replace” tool to engineer all possible single-base changes. Owing to the availability of these, the field of genome editing has evolved rapidly to develop crop plants with improved traits. In this review, we present the evolution of the CRISPR/Cas system into new-age methods of genome engineering across various plant species and the impact they have had on tweaking plant genomes and associated outcomes on crop improvement initiatives.
... The Xanthomonas oryzae pv oryzae-(Xoo) rice system is well studied and used as a model for studying various susceptible genes (SWEET genes) in monocots. Transcription activator-like Effectors (TALEs) secreted by Xoo strains through Type III effectors interact with EBEs (effector binding elements) in promoter regions of SWEET genes to induce expression of genes for the production of sucrose that makes [40]. Cassava brown streak virus (CBSV) is a major constraint for Central and Eastern Africa cassava yields. ...
Article
Crop plants are prone to several yield-reducing biotic and abiotic stresses. The crop yield reductions due to these stresses need addressing to maintain an adequate balance between the increasing world population and food production to avoid food scarcities in the future. It is impossible to increase the area under food crops proportionately to meet the rising food demand. In such an adverse scenario overcoming the biotic and abiotic stresses through biotechnological interventions may serve as a boon to help meet the globe’s food requirements. Under the current genomic era, the wide availability of genomic resources and genome editing technologies such as Transcription Activator-Like Efector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and Clustered-Regularly Interspaced Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas) has widened the scope of overcoming these stresses for several food crops. These techniques have made gene editing more manageable and accessible with changes at the embryo level by adding or deleting DNA sequences of the target gene(s) from the genome. The CRISPR construct consists of a single guide RNA having complementarity with the nucleotide fragments of the target gene sequence, accompanied by a protospacer adjacent motif. The target sequence in the organism’s genome is then cleaved by the Cas9 endonuclease for obtaining a desired trait of interest. The current review describes the components, mechanisms, and types of CRISPR/Cas techniques and how this technology has helped to functionally characterize genes associated with various biotic and abiotic stresses in a target organism. This review also summarizes the application of CRISPR/Cas technology targeting these stresses in crops through knocking down/out of associated genes.
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In developing nations, where arable land per capita is declining but human and animal populations are constantly expanding, the key constraint for food and nutritional security for the human population in the next years will be sustained plant productivity and crop yield(s). Apart from the genetic potential of plant species, agricultural plant output is quite variable and is impacted by a variety of physical, abiotic, and biotic factors. Transgenic technology has the potential to cope with these situations and to feed the teeming millions through crop improvement strategies. The basic objective of any transformation technique is to get the desired gene into the cell’s nucleus without compromising the cell’s capacity to live. The plant is considered to be transformed if the inserted gene is functional and the gene product is produced. The plant is called transgenic after the gene introduced is stable, inherited, and expressed in following generations. Therefore, transgenic plants are plants that have been genetically engineered with novel traits and are identified as a class of genetically modified organism (GMO). Several GM crops such as corn, cassava, soybean, canola, squash, tobacco, mustard, tomato, rice, papaya, cotton, alfalfa, sugar beet, and brinjal have been commercialized worldwide, and some are under pipeline. These crops and their products have now gained acceptance in several countries across the world. Previously, the focus of development of transgenic plants was to develop water, salinity, temperature, insect, and disease tolerance, but now the focus of this technology is enhancement of nutritional components in edible crops so as to improve people’s health. Transgenic technology is an indispensable tool for the biotechnologists and has a bright future towards sustainable development goals.KeywordsTransgenic technologyGMOGenetic modificationBiosafetyCrop improvementBt technology
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Plant diseases caused by diverse pathogens lead to serious reduction in crop yield and threaten food security worldwide. Genetic improvement of plant immunity is considered as the most effective and sustainable approach to control crop diseases. In the last decade, our understanding of plant immunity at both molecular and genomic levels has improved greatly. Combined with advances in biotechnologies, particularly CRISPR/Cas9-based genome editing, we can now rapidly identify new resistance genes and engineer disease resistance crop plants like never before. In this review, we summarize the current knowledge of plant immunity and outline existing and new strategies for disease resistance improvement in crop plants. We also discuss existing challenges in this field and suggest directions for future studies.
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Traditional breeding has successfully selected beneficial traits for food, feed, and fibre crops over the last several thousand years. The last century has seen significant technological advancements particularly in marker assisted selection and the generation of induced genetic variation, including over the last few decades, through mutation breeding, genetic modification, and genome editing. While regulatory frameworks for traditional varietal development and for genetic modification with transgenes are broadly established, those for genome editing are lacking or are still evolving in many regions. In particular, the lack of “foreign” recombinant DNA in genome edited plants and that the resulting SNPs or INDELs are indistinguishable from those seen in traditional breeding has challenged development of new legislation. Where products of genome editing and other novel breeding technologies possess no transgenes and could have been generated via traditional methods, we argue that it is logical and proportionate to apply equivalent legislative oversight that already exists for traditional breeding and novel foods. This review analyses the types and the scale of spontaneous and induced genetic variation that can be selected during traditional plant breeding activities. It provides a base line from which to judge whether genetic changes brought about by techniques of genome editing or other reverse genetic methods are indeed comparable to those routinely found using traditional methods of plant breeding.
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Plant viruses are the major pathogens that cause heavy yield loss in potato. The important viruses are potato virus X, potato virus Y and potato leaf roll virus around the world. Besides these three viruses, a novel tomato leaf curl New Delhi virus is serious in India. Conventional cum molecular breeding and transgenics approaches have been applied to develop virus resistant potato genotypes. But progress is slow in developing resistant varieties due to lack of host genes and long breeding process, and biosafety concern with transgenics. Hence, CRISPR-Cas mediated genome editing has emerged as a powerful technology to address these issues. CRISPR-Cas technology has been deployed in potato for several important traits. We highlight here CRISPR-Cas approaches of virus resistance through targeting viral genome (DNA or RNA), host factor gene and multiplexing of target genes simultaneously. Further, advancement in CRISPR-Cas research is presented in the area of DNA-free genome editing, virus-induced genome editing, and base editing. CRISPR-Cas delivery, transformation methods, and challenges in tetraploid potato and possible methods are also discussed.
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The world’s food and nutritional security is adversely affected by the plant diseases which in turn affects the economic growth and environmental sustainability at the global level. The threat in climate change combined with increasing food production demand poses growing stress on the agro-ecosystems. Worldwide, due to pests and diseases, around 20–40% of crop losses occur in agricultural productivity every year. Basic understanding on the infection process of plant pathogens and their interaction with the host during disease establishment, virulence/avirulence/effector genes in plant pathogens and resistance genes in hosts becomes mandatory for developing disease-resistant cultivars in a sustainable manner. Different management strategies, viz. host plant resistance, use of agrochemicals, cultural practices, and biocontrol agents, have been practiced for reducing the losses caused by plant pathogens. But the sustainability is a question mark because of the rapid occurrence of new pathotypes/ strains/ races of plant pathogens which are infecting the major crops of economic value. With the development of novel genome editing techniques, it is feasible to generate pathogen-resistant crops which are durable with less time. One such powerful genome editing tool is CRISPR/Cas due to its high precision, robustness, minimal off-target effects, and ability to edit multiple targets.KeywordsGenome editingCRISPR/CasPlant pathogensCereals cropsDisease resistance
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Plants subjected to phytopathogens can synthesize peptides with antimicrobial properties. In the last few decades, there has been an impressive increase in the prospection of antimicrobial peptides (AMPs) due to continuous demand for the development and manufacture of new antibiotics. In this setting, plants have attracted scientific and pharmaceutical interest and are considered promising AMPs biofactories. However, it is a great challenge to explore the diversity of actions needed to obtain a pharmaceutical product with AMPs derived from plants on a large scale. This review presents the last 5 years main findings on plants used as AMPs biofactories. Published works in this period were reviewed, and perspectives are presented on recombinant AMPs for drug production that appear in a plant‐based system, as well as products available on the market.
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Plant viruses are known to infect most economically important crops and pose a major threat to global food security. Currently, few resistant host phenotypes have been delineated, and while chemicals are used for crop protection against insect pests and bacterial or fungal diseases, these are inefficient against viral diseases. Genetic engineering emerged as a way of modifying the plant genome by introducing functional genes in plants to improve crop productivity under adverse environmental conditions. Recently, new breeding technologies, and in particular the exciting CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated proteins) technology, was shown to be a powerful alternative to engineer resistance against plant viruses, thus has great potential for reducing crop losses and improving plant productivity to directly contribute to food security. Indeed, it could circumvent the “Genetic modification” issues because it allows for genome editing without the integration of foreign DNA or RNA into the genome of the host plant, and it is simpler and more versatile than other new breeding technologies. In this review, we describe the predominant features of the major CRISPR/Cas systems and outline strategies for the delivery of CRISPR/Cas reagents to plant cells. We also provide an overview of recent advances that have engineered CRISPR/Cas‐based resistance against DNA and RNA viruses in plants through the targeted manipulation of either the viral genome or susceptibility factors of the host plant genome. Finally, we provide insight into the limitations and challenges that CRISPR/Cas technology currently faces and discuss a few alternative applications of the technology in virus research. Plant viruses pose a major threat to global food security. In this review we provide an overview of the use and limitations of CRISPR/Cas‐based technology for resistance against DNA and RNA viruses in plants.
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Genetic engineering or breeding entails deliberate modifications of the characteristics of an organism by manipulating its genetic makeup. This technique has been used to develop crops which are high yielding, resistance to pest and disease, high nutritional value, high oil content etc. Genetic engineering technique offers mankind the ability to develop crops with desirable characteristics within a very short period compared to conventional breeding methods. The use of this breeding method has resulted in producing some varieties of crops which requires farmers to invest low during production for higher income after harvest. The aim of this article is to summarise the known role genetically modified crops has played to reduce hunger and the provision of food with the required nutritional value in helping the world to become a food secured one.
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Background: The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results: In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions: These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.
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Plant viruses recruit cellular translation factors not only to translate their viral RNAs but also to regulate their replication and potentiate their local and systemic movement. Because of the virus dependence on cellular translation factors, it is perhaps not surprising that many natural plant recessive resistance genes have been mapped to mutations of translation initiation factors eIF4E and eIF4G or their isoforms, eIFiso4E and eIFiso4G. The partial functional redundancy of these isoforms allows specific mutation or knock-down of one isoform to provide virus resistance without hindering the general health of the plant. New possible targets for antiviral strategies have also been identified following the characterization of other plant translation factors (eIF4A-like helicases, eIF3, eEF1A and eEF1B) that specifically interact with viral RNAs and proteins and regulate various aspects of the infection cycle. Emerging evidence that translation repression operates as an alternative antiviral RNA silencing mechanism is also discussed. Understanding the mechanisms that control the development of natural viral resistance and the emergence of virulent isolates in response to these plant defense responses will provide the basis for the selection of new sources of resistance and for the intelligent design of engineered resistance that is broad-spectrum and durable.
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The CRISPR/Cas9 system is becoming an important genome editing tool for crop breeding. Although it has been demonstrated that target mutations can be transmitted to the next generation, their inheritance pattern has not yet been fully elucidated. Here, we describe the CRISPR/Cas9-mediated genome editing of four different rice genes with the help of online target-design tools. High-frequency mutagenesis and a large percentage of putative biallelic mutations were observed in T0 generations. Nonetheless, our results also indicate that the progeny genotypes of biallelic T0 lines are frequently difficult to predict and that the transmission of mutations largely does not conform to classical genetic laws, which suggests that the mutations in T0 transgenic rice are mainly somatic mutations. Next, we followed the inheritance pattern of T1 plants. Regardless of the presence of the CRISPR/Cas9 transgene, the mutations in T1 lines were stably transmitted to later generations, indicating a standard germline transmission pattern. Off-target effects were also evaluated, and our results indicate that with careful target selection, off-target mutations are rare in CRISPR/Cas9-mediated rice gene editing. Taken together, our results indicate the promising production of inheritable and "transgene clean" targeted genome-modified rice in the T1 generation using the CRISPR/Cas9 system.
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The ability to selectively alter genomic DNA sequences in vivo is a powerful tool for basic and applied research. The CRISPR/Cas9 system precisely mutates DNA sequences in a number of organisms. Here, the CRISPR/Cas9 system is shown to be effective in soybean by knocking-out a green fluorescent protein (GFP) transgene and modifying nine endogenous loci. Targeted DNA mutations were detected in 95% of 88 hairy-root transgenic events analyzed. Bi-allelic mutations were detected in events transformed with eight of the nine targeting vectors. Small deletions were the most common type of mutation produced, although SNPs and short insertions were also observed. Homoeologous genes were successfully targeted singly and together, demonstrating that CRISPR/Cas9 can both selectively, and generally, target members of gene families. Somatic embryo cultures were also modified to enable the production of plants with heritable mutations, with the frequency of DNA modifications increasing with culture time. A novel cloning strategy and vector system based on In-Fusion® cloning was developed to simplify the production of CRISPR/Cas9 targeting vectors, which should be applicable for targeting any gene in any organism. The CRISPR/Cas9 is a simple, efficient, and highly specific genome editing tool in soybean. Although some vectors are more efficient than others, it is possible to edit duplicated genes relatively easily. The vectors and methods developed here will be useful for the application of CRISPR/Cas9 to soybean and other plant species.
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Targeted genome editing using artificial nucleases has the potential to accelerate basic research as well as plant breeding by providing the means to modify genomes rapidly in a precise and predictable manner. Here we describe the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a recently-developed tool for the introduction of site-specific double stranded DNA breaks. We highlight the strengths and weaknesses of this technology compared with two well-established genome editing platforms: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). We summarize recent results obtained in plants using CRISPR/Cas9 technology, discuss possible applications in plant breeding and consider potential future developments. Copyright © 2014. Published by Elsevier Inc.
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Transgenic resistance to plant viruses is an important technology for control of plant virus infection, which has been demonstrated for many model systems, as well as for the most important plant viruses, in terms of the costs of crop losses to disease, and also for many other plant viruses infecting various fruits and vegetables. Different approaches have been used over the last 28 years to confer resistance, to ascertain whether particular genes or RNAs are more efficient at generating resistance, and to take advantage of advances in the biology of RNA interference to generate more efficient and environmentally safer, novel "resistance genes." The approaches used have been based on expression of various viral proteins (mostly capsid protein but also replicase proteins, movement proteins, and to a much lesser extent, other viral proteins), RNAs [sense RNAs (translatable or not), antisense RNAs, satellite RNAs, defective-interfering RNAs, hairpin RNAs, and artificial microRNAs], nonviral genes (nucleases, antiviral inhibitors, and plantibodies), and host-derived resistance genes (dominant resistance genes and recessive resistance genes), and various factors involved in host defense responses. This review examines the above range of approaches used, the viruses that were tested, and the host species that have been examined for resistance, in many cases describing differences in results that were obtained for various systems developed in the last 20 years. We hope this compilation of experiences will aid those who are seeking to use this technology to provide resistance in yet other crops, where nature has not provided such.
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The CRISPR/Cas9 system is highly efficient at generating targeted mutations in stable transgenic tomato plants, and homozygous deletions of a desired size can be created in the first generation.
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Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.
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In the past few years, the development of sequence-specific DNA nucleases has progressed rapidly and such nucleases have shown their power in generating efficient targeted mutagenesis and other genome editing applications. For zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), an engineered array of sequence-specific DNA binding domains are fused with the DNA nuclease Fok1. These nucleases have been successful in genome modifications by generating double strand breaks (DSBs), which are then repaired through non-homologous end joining (NHEJ) or homologous recombination (HR) in different species, including mouse, tobacco and rice. Recently, another breakthrough technology for genome editing, the CRISPR/Cas system, was developed. CRISPR (clustered regulatory interspaced short palindromic repeats) loci are variable short spacers separated by short repeats, which are transcribed into non-coding RNAs. The non-coding RNAs form a functional complex with CRISPR-associated (Cas) proteins and guide the complex to cleave complementary invading DNA. After the initial development of a programmable CRISPR/Cas system, it has been rapidly applied to achieve efficient genome editing in human cell lines, zebrafish and mouse. However, there is still no successful application in plants reported.
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Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.
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Two methods for the detection of Cucumber vein yellowing virus (CVYV) on infected plants were developed, based on the information provided by cDNA clones covering the 3-end of the genome of a Spanish isolate (CVYV-AILM). The sequenced portion of the CVYV-AILM genome showed a 96.6% aminoacid identity with that of a reported sequence of another CVYV isolate from Israel (Lecoq et al., 2000). The first detection method used a RNA specific probe for hybridization with nucleic acids extracted from infected plants. The probe was complementary to a portion of the CVYV genome including the C-terminal part of the NIb and most of the coat protein (CP) coding regions. The second detection method employed polyclonal antisera raised against recombinant viral CP expressed in bacteria. The specific antibodies were used to detect the presence of virus particles in plant extracts. Both procedures resulted in a highly specific detection of CVYV in plants infected with different isolates of the virus. No interference was observed with other cucurbit-infecting viruses. Sensitivities achieved were sufficient for routine diagnosis of the presence of the virus in plants.
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Plant viruses are important agricultural pathogens, and are responsible for a significant number of commercially relevant plant diseases. There are very few efficient control measures for viral diseases, but the use of genetic resistance appears to be the most promising strategy, often conferring effective protection without additional costs or labour during the growing season, and without damaging the environment. Sources of virus resistance have been identified for most crop species, and many resistant cultivars are already commercially available and of widespread cultivation; however, much remains to be learned about genetic resistance. This review article considers three main aspects that require intense investigation. First, we review the identification of sources of resistance and how plant breeders and pathologists have focused on aspects of the breeding process particularly relevant to viruses, such as germplasm screening and the dissection of resistance phenotypes. Second, we review how molecular mechanisms controlling resistance have been unravelled, looking at case studies where resistance mechanisms are now understood in detail for each stage of the infection cycle. Third, we turn to the durability of resistance in a global context, examining factors that influence durability and how this can be predicted. We conclude with a short discussion of the technological and scientific opportunities provided by recent advances in the field.
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For some plant positive-sense RNA viruses, a protein known as VPg (short for virus protein, genome linked) is covalently linked to the 5' end of the viral RNA. The VPg is an intrinsically disordered protein, and this property would confer an ability to bind several proteins. Accordingly, the potyvirus VPg interacts with many proteins, notably host factors involved in protein synthesis within viral replication factories or within the nucleus. The number of protein partners, the clustering of the various interactions centering around it, the biological importance for some of these interactions (e.g. VPg-eIF4E) and the intrinsically disordered state of the protein are all elements that support the notion that VPg is a hub protein that controls many processes leading to virus production and spread.
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The use of genetic resistance is considered to be the most effective and sustainable approach to the control of plant pathogens. Although most of the known natural resistance genes are monogenic dominant R genes that are predominant against fungi and bacteria, more and more recessive resistance genes against viruses have been cloned in the last decade. Interestingly, of the 14 natural recessive resistance genes against plant viruses that have been cloned from diverse plant species thus far, 12 encode the eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. This review is intended to summarize the current state of knowledge about eIF4E and the possible mechanisms underlying its essential role in virus infection, and to discuss recent progress and the potential of eIF4E as a target gene in the development of genetic resistance to viruses for crop improvement.
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The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance. To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2. These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato-potyvirus interactions.
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Two recessive genes (cyv1 and cyv2) are known to confer resistance against Clover yellow vein virus (ClYVV) in pea. cyv2 has recently been revealed to encode eukaryotic translation initiation factor 4E (eIF4E) and is the same allele as sbm1 and wlm against other potyviruses. Although mechanical inoculation with crude sap is rarely able to cause infection of a cyv2 pea, biolistic inoculation of the infectious ClYVV cDNA clone does. At the infection foci, the breaking virus frequently emerges, resulting in systemic infection. Here, a derived cleaved-amplified polymorphic sequence analysis showed that the breakings were associated with a single nonsynonymous mutation on the ClYVV genome, corresponding to an amino-acid substitution at position 24 (isoleucine to valine) on the P1 cistron. ClYVV with the point mutation was able to break the resistance. This is a first report demonstrating that P1 is involved in eIF4E-mediated recessive resistance.
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The characterization of natural recessive resistance genes and Arabidopsis virus-resistant mutants have implicated translation initiation factors of the eIF4E and eIF4G families as susceptibility factors required for virus infection and resistance function. To investigate further the role of translation initiation factors in virus resistance we set up a TILLING platform in tomato, cloned genes encoding for translation initiation factors eIF4E and eIF4G and screened for induced mutations that lead to virus resistance. A splicing mutant of the eukaryotic translation initiation factor, S.l_eIF4E1 G1485A, was identified and characterized with respect to cap binding activity and resistance spectrum. Molecular analysis of the transcript of the mutant form showed that both the second and the third exons were miss-spliced, leading to a truncated mRNA. The resulting truncated eIF4E1 protein is also impaired in cap-binding activity. The mutant line had no growth defect, likely because of functional redundancy with others eIF4E isoforms. When infected with different potyviruses, the mutant line was immune to two strains of Potato virus Y and Pepper mottle virus and susceptible to Tobacco each virus. Mutation analysis of translation initiation factors shows that translation initiation factors of the eIF4E family are determinants of plant susceptibility to RNA viruses and viruses have adopted strategies to use different isoforms. This work also demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. We have also developed a complete tool that can be used for both forward and reverse genetics in tomato, for both basic science and crop improvement. By opening it to the community, we hope to fulfill the expectations of both crop breeders and scientists who are using tomato as their model of study.
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SUMMARY Recent work carried out to characterize recessive mutations which render experimental hosts non-permissive to viral infection (loss-of-susceptibility mutants) seems to be converging with new data on natural recessive resistance in crop species, and also with functional analyses of virus avirulence determinants. Perhaps the most well known examples are the studies that identified the eukaryotic translation initiation factors 4E(iso) (eIF(iso)4E) and 4E(eIF4E) as the host factors required for potyvirus multiplication within experimental and natural hosts, respectively, and the potyviral genome-linked protein (VPg) as the viral factor that directly interacts with eIF4E to promote potyvirus multiplication. The purpose of this paper is to review the available information on the characterization of loss-of-susceptibility mutants in experimental hosts, natural recessive resistances and virus avirulence factors, and also to comment on possible implications for the design of new sources of sustainable virus resistance.
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About half of the approximately 200 known virus resistance genes in plants are recessively inherited, suggesting that this form of resistance is more common for viruses than for other plant pathogens. The use of such genes is therefore a very important tool in breeding programs to control plant diseases caused by pathogenic viruses. Over the last few years, the detailed analysis of many host/virus combinations has substantially advanced basic research on recessive resistance mechanisms in crop species. This type of resistance is preferentially expressed in protoplasts and inoculated leaves, influencing virus multiplication at the single-cell level as well as cell-to-cell movement. Importantly, a growing number of recessive resistance genes have been cloned from crop species, and further analysis has shown them all to encode translation initiation factors of the 4E (eIF4E) and 4G (eIF4G) families. However, not all of the loss-of-susceptibility mutants identified in collections of mutagenized hosts correspond to mutations in eIF4E and eIF4G. This, together with other supporting data, suggests that more extensive characterization of the natural variability of resistance genes may identify new host factors conferring recessive resistance. In this chapter, we discuss the recent work carried out to characterize loss-of-susceptibility and recessive resistance genes in crop and model species. We review actual and probable recessive resistance mechanisms, and bring the chapter to a close by summarizing the current state-of-the-art and offering perspectives on potential future developments.
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Protein synthesis is principally regulated at the initiation stage (rather than during elongation or termination), allowing rapid, reversible and spatial control of gene expression. Progress over recent years in determining the structures and activities of initiation factors, and in mapping their interactions in ribosomal initiation complexes, have advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studying the regulation of translation initiation by mechanisms that include the modulation of initiation factor activity (which affects almost all scanning-dependent initiation) and through sequence-specific RNA-binding proteins and microRNAs (which affect individual mRNAs).
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Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant watermelon plant introduction PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon cultivar 'New Hampshire Midget' ('NHM') showed the presence of single nucleotide polymorphisms (SNPs). Initial analysis of the identified SNPs in association studies indicated that SNPs in the eIF4E, but not eIF(iso)4E, were closely associated to the phenotype of ZYMV-resistance in 70 F(2) and 114 BC(1R) progenies. Subsequently, we focused our efforts in obtaining the entire genomic sequence of watermelon eIF4E. Three SNPs were identified between PI 595203 and NHM. One of the SNPs (A241C) was in exon 1 and the other two SNPs (C309A and T554G) were in the first intron of the gene. SNP241 which resulted in an amino acid substitution (proline to threonine) was shown to be located in the critical cap recognition and binding area, similar to that of several plant species resistance to potyviruses. Analysis of a cleaved amplified polymorphism sequence (CAPS) marker derived from this SNP in F(2) and BC(1R) populations demonstrated a cosegregation between the CAPS-2 marker and their ZYMV resistance or susceptibility phenotype. When we investigated whether such SNP mutation in the eIF4E was also conserved in several other PIs of C. lanatus var. citroides, we identified a different SNP (A171G) resulting in another amino acid substitution (D71G) from four ZYMV-resistant C. lanatus var. citroides (PI 244018, PI 482261, PI 482299, and PI 482322). Additional CAPS markers were also identified. Availability of all these CAPS markers will enable marker-aided breeding of watermelon for ZYMV resistance.
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An aphid-transmissible (AT) and two non-aphid-transmissible (NAT) isolates of zucchini yellow mosaic virus (ZYMV) were studied. The predicted amino acid sequences of the coat protein (CP) of the three virus isolates were analysed and compared. The NAT isolates differed from the AT isolate in having a Thr instead of an Ala residue at position 10 in the conserved Asp-Ala-Gly triplet in the N-terminal region of CP. Aphid transmissibility was restored in a progeny virus derived from an infectious clone of the ZYMV-NAT isolate in which Thr was changed back to Ala by site-directed mutagenesis. However this mutation did not have any effect on the multiplication rate in squash, which was significantly higher than that of the AT isolate. The involvement of this mutation in aphid transmission and virus multiplication is discussed.
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The yeast LexA interaction trap was used to screen a cDNA library from Arabidopsis thaliana in order to identify proteins that interact with the viral protein genome linked (VPg)-proteinase of turnip mosaic potyvirus. The screen allowed the isolation of four candidate cDNA clones. Clones pHC4, pHC21, and pHC40 were partially sequenced but no homologies to known proteins were found. However, the amino acid sequence deduced from the complete nucleotide sequence of pSW56 revealed that it was the eukaryotic initiation factor (iso) 4E [eIF(iso)4E]. Deletion analysis indicated that the VPg domain was involved in the interaction with the plant protein. Interaction between the viral protein and the cellular protein was confirmed by ELISA-based binding experiments. eIF(iso)4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus.
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The interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the translation eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana has previously been reported. eIF(iso)4E binds the cap structure (m7GpppN, where N is any nucleotide) of mRNAs and has an important role in the regulation in the initiation of translation. In the present study, it was shown that not only did VPg bind eIF(iso)4E but it also interacted with the eIF4E isomer of A. thalianaas well as with eIF(iso)4E of Triticum aestivum (wheat). The interaction domain on VPg was mapped to a stretch of 35 amino acids, and substitution of an aspartic acid residue found within this region completely abolished the interaction. The cap analogue m7GTP, but not GTP, inhibited VPg-eIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. The biological significance of this interaction was investigated. Brassica perviridis plants were infected with a TuMV infectious cDNA (p35Tunos) and p35TuD77N, a mutant which contained the aspartic acid substitution in the VPg domain that abolished the interaction with eIF(iso)4E. After 20 days, plants bombarded with p35Tunos showed viral symptoms, while plants bombarded with p35TuD77N remained symptomless. These results suggest that VPg-eIF(iso)4E interaction is a critical element for virus production.
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The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanis