Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we ...show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (≤3 mismatches) in the entire soybean reference genome. Targeted deep sequencing analyses show that mutations are successfully induced in FAD2 paralogues in soybean and AOC in wild tobacco. Unlike SpCas9, Cpf1 mainly induces various nucleotide deletions at target sites. No significant mutations are detected at potential off-target sites in the soybean genome. These results demonstrate that Cpf1-crRNA complex is an effective DNA-free genome-editing tool for plant genome editing.
The
Mildew Locus O (
) gene is vital for plant defense responses against fungal pathogens like powdery mildew, a significant threat to greenhouse pepper crops. Recent advancements in genome editing, ...particularly using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, have unlocked unprecedented opportunities for modifying disease-resistant genes and improving crop characteristics. However, the application of CRISPR technology in pepper cultivars has been limited, and the regeneration process remains challenging. This study addresses these limitations by investigating the feasibility of using the validated
genetic scissors system in six commercial hot pepper cultivars. We assessed the gene-editing efficiency of the previously reported high-efficiency Cas9/
single-guide RNA (sgRNA)1-ribonucleoprotein (RNP) and the low-efficiency Cas9/
sgRNA2-RNP systems by extending their application from the bell pepper 'Dempsey' and the hot pepper 'CM334' to six commercial hot pepper cultivars. Across the six cultivars,
sgRNA1 demonstrated an editing efficiency ranging from 6.3 to 17.7%, whereas
sgRNA2 exhibited no editing efficiency, highlighting the superior efficacy of sgRNA1. These findings indicate the potential of utilizing the verified Cas9/
sgRNA1-RNP system to achieve efficient gene editing at the
locus in different
cultivars regardless of their cultivar genotypes. This study provides an efficacious genome-editing tool for developing improved pepper cultivars with
-mediated enhanced disease resistance.
DNA-free, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) ribonucleoprotein (RNP)-based genome editing is a simple, convincing, and promising tool for ...precision crop breeding. The efficacy of designed CRISPR-based genome editing tools is a critical prerequisite for successful precision gene editing in crops.
This study demonstrates that soil-grown leaf- or callus-derived pepper protoplasts are a useful system for screening of efficient guide RNAs for CRISPR/Cas9 or CRISPR/Cas12a (Cpf1). CRISPR/Cas9 or Cpf1 were delivered as CRISPR/RNP complexes of purified endonucleases mixed with the designed single guide RNA, which can edit the target gene, CaMLO2 in two pepper cultivars with whole genome sequenced, Capsicum annuum 'CM334' and C. annuum 'Dempsey'. The designed guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) are conserved for CaMLO2 in both CM334 and Dempsey and cleave CaMLO2 in vitro. CRISPR/Cas9- or /Cpf1-RNP complexes were transfected into purely isolated protoplasts of the hot pepper CM334 and sweet pepper Dempsey by PEG-mediated delivery. Targeted deep sequencing analysis indicated that the targeted CaMLO2 gene was differentially edited in both cultivars, depending on the applied CRISPR/RNPs.
Pepper protoplast-based CRISPR guide-RNA selection is a robust method to check the efficacy of designed CRISPR tools and is a prerequisite for regenerating edited plants, which is a critical time-limiting procedure. The rapid and convincing selection of guide RNA against a target genome reduces the laborious efforts for tissue culture and facilitates effective gene editing for pepper improvement.
There is an increasing awareness that as a result of structural variation, a reference sequence representing a genome of a single individual is unable to capture all of the gene repertoire found in ...the species. A large number of genes affected by presence/absence and copy number variation suggest that it may contribute to phenotypic and agronomic trait diversity. Here we show by analysis of the Brassica oleracea pangenome that nearly 20% of genes are affected by presence/absence variation. Several genes displaying presence/absence variation are annotated with functions related to major agronomic traits, including disease resistance, flowering time, glucosinolate metabolism and vitamin biosynthesis.
Pepper (Capsicum annuum L.) holds immense global importance, as it is widely cultivated for its economic value in the food industry and its health benefits. Consequently, substantial breeding ...progress has been made in cultivar development, whole-genome analysis, and transformation techniques aimed at enhancing agricultural traits, including fruit development and capsaicin synthesis. However, research concerning the phylogenetic relationships within C. annuum remains insufficient. In this study, we characterized the plastome sequences of seven C. annuum, including five hot pepper and two bell pepper cultivars, while also elucidating their phylogenetic relationships. Furthermore, we conducted comparative analyses to gain insight into their evolutionary history. The seven plastomes displayed typical quadripartite structures and ranged from 156,821 to 156,922 bp, displaying highly conserved sequences. In contrast to prior studies, our phylogenomic analyses revealed that C. annuum species did not form a monophyletic group. Each subclade was thought to be related to a different evolutionary history, such as hybridization, domestication from wild ancestors, and artificial selection. Therefore, we were able to discern the relationships among cultivars based on their genetic profiles of plastomes. Our findings also revealed that the Korean landraces Younggo 4, 5, 10, and 11 share the most recent common ancestor with Mexican landrace CM334.
We developed a simple method for the fabrication of superhydrophobic surfaces on various substrates using spray coating. The fabrication method started with the blending of a modified hydrophobic ...siloxane binder, silica nanoparticles, and a volatile solvent by sonication. The mixture was spray-coated on various surfaces such as slide glass, paper, metal and fabric, forming a rough surface comprising silica particles dispersed in a hydrophobic binder. Surface hydrophobicity was affected by the surface energy of the binder and the degree of roughness. Therefore, we realized a superhydrophobic surface by controlling these two factors. The hydrophobicity of the siloxane binder was determined by the treatment of fluorine silane; the roughness was controlled by the amount of coated materials and sonication time. Thus, using the spray coating method, we obtained a superhydrophobic surface that was mechanically durable, thermally stable, and chemically resistant.
CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes（Sp Cas9） cleaves double-stranded DNA targeted by a chimeric ...single-guide RNA（sg RNA）. For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sg RNAs under the control of Ca MV 35 S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19 20 bp of sg RNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an Sp Cas9 expressing cassette. Twostep cloning procedures：（1） annealing two target-specific oligonucleotides with overhangs specific to the Aar I restriction enzyme site of the binary vector; and（2） ligating the annealed oligonucleotides into the two Aar I sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTMsystem and unique Eco RI/Xho I sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.
Adaptor protein (AP) complexes are the predominant coat proteins of membrane vesicles in post-Golgi trafficking of mammalian cells. Each AP complex contains a specific medium subunit, μ-adaptin, that ...selects cargo proteins bearing sequence-specific sorting motifs. Much less is known about the AP complexes and their μ subunits in plants. Because of uncertain homology, the μ-adaptins of Arabidopsis have been designated muA through muD Happel et al. (2004) Plant J 37(5):678–693. Furthermore, only muD has been assigned to a specific AP complex, AP-3, involved in Golgi-vacuolar trafficking Niihama et al. (2009) Plant Cell Physiol 50(12):2057–2068, Zwiewka et al. (2011) Cell Res 21(12):1711–1722, and Wolfenstetter et al. (2012) Plant Cell 24(1):215–232. In contrast, the μ subunit of neither the post-Golgi trafficking AP-1 complex nor the endocytic AP-2 complex has been identified. Here, we report the functional analysis of redundant AP-1 μ-adaptins AP1M1 (also known as muB1) and AP1M2 (also known as muB2). Coimmunoprecipitation revealed that both AP1M2 and its less strongly expressed isoform AP1M1 are complexed with the large subunit γ-adaptin of AP-1. In addition, AP1M2 was localized at or near the trans -Golgi network. Knockout mutations of AP1M2 impaired pollen function and arrested plant growth whereas the ap1m1 ap1m2 double mutant was nearly pollen-lethal. At the cellular level, the absence of AP1M2 entailed inhibition of multiple trafficking pathways from the trans -Golgi network to the vacuole and to the plasma membrane in interphase and to the plane of cell division in cytokinesis. Thus, AP-1 is crucial in post-Golgi trafficking in plant cells and required for cell division and plant growth.
In the current study, we sequenced the complete plastome of
Liebm. (1849), an attractive foliage plant. The total length of the plastome of
is 163,499 bp and it consists of three distinct regions: a ...large single-copy (90,092 bp), a small single-copy (21,737 bp), and a pair of inverted repeats (IRs, 25,835 bp). The overall GC content is 36.2%, and the genome contains 110 functional genes, excluding pseudogenes. These functional genes encompass 77 protein-coding genes, 29 transfer RNA genes, and four ribosomal RNA genes. Notably, both the
and
genes have been identified as pseudogenes. Phylogenetic analysis based on 14 representative plastomes from seven subfamilies indicates that Monsteroideae is monophyletic and sister to Pothoideae. Furthermore,
and
were shown to share a recent common ancestor, the finding for which is supported by a strong bootstrap value. The sequenced plastome of
can serve as a valuable resource for establishing phylogenetic relationships and enhancing species identification within the genus
In addition, it can facilitate investigations into the genetic characteristics of this plant.
In all eukaryotic cells, a membrane-trafficking system connects the post-Golgi organelles, such as the trans-Golgi network (TGN), endosomes, vacuoles, and the plasma membrane. This complex network ...plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. Unlike its roles in animal and yeast cells, the TGN has also been reported to function like early endosomal compartments in plant cells. However, the physiological roles of the TGN functions in plants are not understood. Here, we report a study of the SYP4 group (SYP41, SYP42, and SYP43), which represents the plant orthologs of the Tlg2/syntaxin16 Qa-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) that localizes on the TGN in yeast and animal cells. The SYP4 group regulates the secretory and vacuolar transport pathways in the post-Golgi network and maintains the morphology of the Golgi apparatus and TGN. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance responses to a fungal pathogen. We also reveal a plant cell-specific higher-order role of the SYP4 group in the protection of chloroplasts from salicylic acid-dependent biotic stress.
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