CRISPR/Cas9-mediated generation of fls2 mutant in Nicotiana benthamiana for investigating the flagellin recognition spectrum of diverse FLS2 receptors.
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ABSTRACT:
SUBMITTER: Wu L
PROVIDER: S-EPMC9491447 | biostudies-literature | 2022 Oct
REPOSITORIES: biostudies-literature
Publications
CRISPR/Cas9-mediated generation of fls2 mutant in Nicotiana benthamiana for investigating the flagellin recognition spectrum of diverse FLS2 receptors.
Wu Ling L Xiao Hongju H Zhao Lijuan L Cheng Qiang Q
Plant biotechnology journal 20220811 10
Similar Datasets
Project description:Microbe-associated molecular patterns (MAMPs) are molecules, or domains within molecules, that are conserved across microbial taxa and can be recognized by a plant or animal immune system. Although MAMP receptors have evolved to recognize conserved epitopes, the MAMPs in some microbial species or strains have diverged sufficiently to render them unrecognizable by some host immune systems. In this study, we carried out in vitro evolution of the Arabidopsis thaliana flagellin receptor FLAGELLIN-SENSING 2 (FLS2) to isolate derivatives that recognize one or more flagellin peptides from bacteria for which the wild-type Arabidopsis FLS2 confers little or no response. A targeted approach generated amino acid variation at FLS2 residues in a region previously implicated in flagellin recognition. The primary screen tested for elevated response to the canonical flagellin peptide from Pseudomonas aeruginosa, flg22. From this pool, we then identified five alleles of FLS2 that confer modest (quantitatively partial) recognition of an Erwinia amylovora flagellin peptide. Use of this Erwinia-based flagellin peptide to stimulate Arabidopsis plants expressing the resulting FLS2 alleles did not lead to a detectable reduction of virulent P. syringae pv. tomato growth. However, combination of two identified mutations into a single allele further increased FLS2-mediated responses to the E. amylovora flagellin peptide. These studies demonstrate the potential to raise the sensitivity of MAMP receptors toward particular targets.
| S-EPMC4894583 | biostudies-literature
Project description:Provided data came from a detailed study on Nicotiana benthamiana 16c plants where we use Tobacco Rattle Virus (TRV) as a molecular switch to change the chromatin state of a reporter gene (P35S::GFP) from an actively transcribed to a transcriptionally silenced state. Our approach enables us to interrogate different chromatin states of the same locus with the same set of CRISPR/Cas9 genome editing reagents and systematically describe the effect of chromatin state on the frequency and type of mutations induced at various Cas9 targets in a huge set of independently edited cells.
2022-05-04 | E-MTAB-10256 | biostudies-arrayexpress
Project description:The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant ΔXTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and ΔXTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.
| S-EPMC9433777 | biostudies-literature
Project description:BackgroundCRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS).ResultsIn this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger.ConclusionsThe tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.
| S-EPMC8943966 | biostudies-literature
Project description:Key messageTobacco etch virus accumulation declined in Nicotiana benthamiana eEF1Bγ gene-edited lines, suggesting that eEF1Bγ may be a host factor for this virus. Viruses use host factors to replicate and move from cell to cell. Therefore, the editing of genes encoding viral host factors that are not essential for plant survival enables the rapid development of plants with durable virus resistance. Eukaryotic initiation factors, such as eIF4E and eIF4G, function as host factors for viral infection, and loss-of-function mutations of these factors lead to virus resistance. Broadening the spectrum of host factor targets would help expand resources for engineering virus resistance. In this study, we tested whether editing the eukaryotic translation elongation factor gene eEF1Bγ would produce virus-resistant plants. Accordingly, we targeted the four eEF1Bγ genes in Nicotiana benthamiana for editing using virus-induced gene editing (VIGE) with Tobacco rattle virus (TRV). Although we attempted to obtain plants edited for all four eEF1Bγ homologs, we failed to identify such plants. Instead, we obtained plants with three of the four homologs knocked out, harboring 1-bp insertion/deletions resulting in premature stop codons. These eEF1Bγ-edited plants did not exhibit resistance to Potato virus X (PVX), Tobacco mosaic virus (TMV), or Tomato bushy stunt virus (TBSV) but showed reduced accumulation of Tobacco etch virus (TEV) compared to wild-type plants. These findings demonstrate the feasibility of conferring resistance in plants through gene editing of eEF1Bγ, underscoring the importance of exploring diverse host factor targets for comprehensive virus resistance.
| S-EPMC11846736 | biostudies-literature
Project description:BACKGROUND: Roles of microRNAs (miRNAs) and short interfering RNAs (siRNAs) in biotic stress responses, e.g., viral infection, have been demonstrated in plants by many studies. Tomato yellow leaf curl China virus (TYLCCNV) is a monopartite begomovirus that can systemically infect Solanaceae plants, and induces leaf curling, yellowing and enation symptoms when co-inoculated with a betasatellite (TYLCCNB). The released genome sequence of Nicotiana benthamiana provides an opportunity to identify miRNAs and siRNAs responsive to begomovirus-associated betasatellite in N. benthamiana. RESULTS: miRNAs were identified in three small RNA libraries generated using RNA isolated from N. benthamiana plants systemically infected with TYLCCNV (Y10A) alone, co-infected with Y10A and its betasatellite TYLCCNB (Y10?) or a TYLCCNB mutant (Y10m?) that contains a mutated ?C1, the sole betasatellite-encoded protein. A total of 196 conserved miRNAs from 38 families and 197 novel miRNAs from 160 families were identified. Northern blot analysis confirmed that expression of species-specific miRNAs was much lower than that of conserved miRNAs. Several conserved and novel miRNAs were found to be responsive to co-infection of Y10A and Y10? but not to co-infection of Y10A and Y10m?, suggesting that these miRNAs might play a role unique to interaction between Y10? and N. benthamiana. Additionally, we identified miRNAs that can trigger the production of phased secondary siRNAs (phasiRNAs). CONCLUSIONS: Identification of miRNAs with differential expression profiles in N. benthamiana co-infected with Y10A and Y10? and co-infected with Y10A and Y10m? indicates that these miRNAs are betasatellite-responsive. Our result also suggested a potential role of miRNA-mediated production of phasiRNAs in interaction between begomovirus and N. benthamiana.
| S-EPMC4008317 | biostudies-literature
Project description:DWARF14 (D14) is an ɑ/β-hydrolase and receptor for the plant hormone strigolactone (SL) in angiosperms. Upon SL perception, D14 works with MORE AXILLARY GROWTH2 (MAX2) to trigger polyubiquitination and degradation of DWARF53(D53)-type proteins in the SUPPRESSOR OF MAX2 1-LIKE (SMXL) family. We used CRISPR-Cas9 to generate knockout alleles of the two homoeologous D14 genes in the Nicotiana benthamiana genome. The Nbd14a,b double mutant had several phenotypes that are consistent with the loss of SL perception in other plants, including increased axillary bud outgrowth, reduced height, shortened petioles, and smaller leaves. A ratiometric fluorescent reporter system was used to monitor degradation of SMXL7 from Arabidopsis thaliana (AtSMXL7) after transient expression in N. benthamiana and treatment with the strigolactone analog GR24. AtSMXL7 was degraded after treatment with GR245DS, which has the stereochemical configuration of natural SLs, as well as its enantiomer GR24 ent-5DS. In Nbd14a,b leaves, AtSMXL7 abundance was unaffected by rac-GR24 or either GR24 stereoisomer. Transient coexpression of AtD14 with the AtSMXL7 reporter in Nbd14a,b restored the degradation response to rac-GR24, but required an active catalytic triad. We used this platform to evaluate the ability of several AtD14 mutants that had not been characterized in plants to target AtSMXL7 for degradation.
| S-EPMC8948499 | biostudies-literature
Project description:Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.
| S-EPMC7606464 | biostudies-literature
Project description: Not available
| S-EPMC10955485 | biostudies-literature
Project description:Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N-linked glycans, including the presence of β-1,2-xylose and core α-1,3-fucose residues in plants, can affect the activity, potency and immunogenicity of plant-derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N-glycosylation machinery to allow the synthesis of complex N-glycans lacking β-1,2-xylose and core α-1,3-fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant-specific α-1,3-fucosyltransferase and β-1,2-xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry-based N-glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64-binding affinity of 2G12 glycovariants produced in wild-type N. benthamiana, the newly generated FX-KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco-engineered antibody performed as well as its CHO-produced counterpart.
| S-EPMC6335070 | biostudies-literature