An ex vitro hairy root system from petioles of detached soybean leaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

Autor: Freitas-Alves NS; Bioprocess Engineering and Biotechnology Graduate Program, Federal University of Paraná-UFPR, Curitiba, PR, Brazil.; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Moreira-Pinto CE; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Arraes FBM; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Costa LSL; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; Molecular Biology Graduate Program, University of Brasília-UNB, Brasília, DF, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., de Abreu RA; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil., Moreira VJV; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; Molecular Biology Graduate Program, University of Brasília-UNB, Brasília, DF, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Lourenço-Tessutti IT; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Pinheiro DH; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Lisei-de-Sa ME; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Paes-de-Melo B; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Pereira BM; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil., Guimaraes PM; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Brasileiro ACM; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., de Almeida-Engler J; INRAE, Université Côte d'Azur, CNRS, 06903, Sophia Antipolis, ISA, France.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Soccol CR; Bioprocess Engineering and Biotechnology Graduate Program, Federal University of Paraná-UFPR, Curitiba, PR, Brazil., Morgante CV; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; Embrapa Semiarid, Petrolina, PE, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Basso MF; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil., Grossi-de-Sa MF; Bioprocess Engineering and Biotechnology Graduate Program, Federal University of Paraná-UFPR, Curitiba, PR, Brazil. fatima.grossi@embrapa.br.; Embrapa Genetic Resources and Biotechnology, PqEB Final, W5 Norte, PO Box 02372, Brasília, DF, 70770-917, Brazil. fatima.grossi@embrapa.br.; Molecular Biology Graduate Program, University of Brasília-UNB, Brasília, DF, Brazil. fatima.grossi@embrapa.br.; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil. fatima.grossi@embrapa.br.; Catholic University of Brasília, Brasília, DF, Brazil. fatima.grossi@embrapa.br.
Jazyk: angličtina
Zdroj: Planta [Planta] 2023 Dec 18; Vol. 259 (1), pp. 23. Date of Electronic Publication: 2023 Dec 18.
DOI: 10.1007/s00425-023-04286-x
Abstrakt: Main Conclusion: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.
(© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
Databáze: MEDLINE
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