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Agrobacterium-Mediated Capsicum annuum Gene Editing in Two Cultivars, Hot Pepper CM334 and Bell Pepper Dempsey.

ABSTRACT: Peppers (Capsicum annuum L.) are the most widespread and cultivated species of Solanaceae in subtropical and temperate countries. These vegetables are economically attractive worldwide. Although whole-genome sequences of peppers and genome-editing tools are currently available, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three Agrobacterium tumefaciens strains-AGL1, EHA101, and GV3101-to investigate which Agrobacterium strain could be used for pepper transformation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. Agrobacterium tumefaciens GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the C. annuumMLO gene (CaMLO2). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to A. tumefaciens-mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.

SUBMITTER: Park SI

PROVIDER: S-EPMC8070316 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Endornaviruses include viruses that infect fungi, oomycetes, and plants. The genome of plant endornaviruses consists of linear ssRNA ranging in size from approximately 13-18 kb and lacking capsid protein and cell-to-cell movement capability. Although, plant endornaviruses have not been shown to cause detectable changes in the plant phenotype, they have been associated with alterations of the host physiology. Except for the association of cytoplasmic vesicles with infections by Vicia faba endornavirus, effects on the plant cell ultrastructure caused by endornaviruses have not been reported. Bell pepper endornavirus (BPEV) has been identified in several pepper (Capsicum spp.) species. We conducted ultrastructural analyses of cells from two near-isogenic lines of the bell pepper (C. annuum) cv. Marengo, one infected with BPEV and the other BPEV-free, and found cellular alterations associated with BPEV-infections. Some cells of plants infected with BPEV exhibited alterations of organelles and other cell components. Affected cells were located mainly in the mesophyll and phloem tissues. Altered organelles included mitochondrion, chloroplast, and nucleus. The mitochondria from BPEV-infected plants exhibited low number of cristae and electron-lucent regions. Chloroplasts contained plastoglobules and small vesicles in the surrounding cytoplasm. Translucent regions in thylakoids were observed, as well as hypertrophy of the chloroplast structure. Many membranous vesicles were observed in the stroma along the envelope. The nuclei revealed a dilation of the nuclear envelope with vesicles and perinuclear areas. The organelle changes were accompanied by membranous structure rearrangements, such as paramural bodies and multivesicular bodies. These alterations were not observed in cells from plants of the BPEV-free line. Overall, the observed ultrastructural cell alterations associated with BPEV are similar to those caused by plant viruses and viroids and suggest some degree of parasitic interaction between BPEV and the plant host.

Project description:BackgroundExpansins (EXPs) facilitate non-enzymatic cell wall loosening during several phases of plant growth and development including fruit growth, internode expansion, pollen tube growth, leaf and root development, and during abiotic stress responses. In this study, the spatial and temporal expression patterns of C. annuum α- EXPANSIN (CaEXPA) genes were characterized. Additionally, fruit-specific CaEXPA expression was correlated with the rate of cell expansion during bell pepper fruit development.ResultsSpatial expression patterns revealed that CaEXPA13 was up-regulated in vegetative tissues and flowers, with the most abundant expression in mature leaves. Expression of CaEXPA4 was associated with stems and roots. CaEXPA3 was expressed abundantly in flower at anthesis suggesting a role for CaEXPA3 in flower development. Temporal expression analysis revealed that 9 out of the 21 genes were highly expressed during fruit development. Of these, expression of six genes, CaEXPA5, CaEXPA7, CaEXPA12, CaEXPA14 CaEXPA17 and CaEXPA19 were abundant 7 to 21 days after anthesis (DAA), whereas CaEXPA6 was strongly expressed between 14 and 28 DAA. Further, this study revealed that fruit growth and cell expansion occur throughout bell pepper development until ripening, with highest rates of fruit growth and cell expansion occurring between 7 and 14 DAA. The expression of CaEXPA14 and CaEXPA19 positively correlated with the rate of cell expansion, suggesting their role in post-mitotic cell expansion-mediated growth of the bell pepper fruit. In this study, a ripening specific EXP transcript, CaEXPA9 was identified, suggesting its role in cell wall disassembly during ripening.ConclusionsThis is the first genome-wide study of CaEXPA expression during fruit growth and development. Identification of fruit-specific EXPAs suggest their importance in facilitating cell expansion during growth and cell wall loosening during ripening in bell pepper. These EXPA genes could be important targets for future manipulation of fruit size and ripening characteristics.

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Agrobacterium-Mediated Capsicum annuum Gene Editing in Two Cultivars, Hot Pepper CM334 and Bell Pepper Dempsey.

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