Abstract
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.
Abstract
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.
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Data availability
All data generated in this study are included in this manuscript and its supplementary information.
Abbreviations
- dpi:
-
Days post-inoculation
- NLS:
-
Nuclear localization signal
- PPN:
-
Plant parasitic nematodes
- RKNs:
-
Root-knot nematodes
- eGFP:
-
Enhanced green fluorescent protein
- GmPR10 :
-
Glycine max pathogenesis-related protein 10 (Glyma.17G030400)
- GmGST :
-
Glycine max glutathione S-transferase (Glyma.18G190300)
- GmExpA :
-
Glycine max expansin A (Glyma.06G021700)
- GmExpA2 :
-
Glycine max expansin A2 (Glyma.04G021600)
- GmExpLB :
-
Glycine max expansin-like B (Glyma.01G204800)
- AdEXLB8 :
-
Arachis duranensis expansin-like B8 (Aradu.MR104)
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Acknowledgements
The authors thank INCT-PlantStress Biotech, CAPES-COFECUB Project, EMBRAPA, UCB, CNPq, CAPES, and FAP-DF for the financial support.
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ITLT and CMP are grateful to CAPES/Cofecub project Sv.922/18 for the financial support for the research and student exchange program between institutions.
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MFG-S supervised this research and provided financial support. NSF-A, CEM-P, and FBMA planned and performed all experiments. FBMA and LSLC designed the CRISPR/dCas9 constructs. MEL-S provided the inoculum for the bioassays with nematodes. NSF-A, CEM-P, FBMA, LSLC, ITL-T, VJVM, and DHP contributed to the hairy root transformation and bioassays analyses. MFG-S, CVM, BP-M, BMP, PMG, and ACMB contributed to the experiment conceptualization. NSF-A wrote the manuscript. MFG-S, CVM, CRS, and MFB amended the manuscript.
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Freitas-Alves, N.S., Moreira-Pinto, C.E., Arraes, F.B.M. et al. 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. Planta 259, 23 (2024). https://doi.org/10.1007/s00425-023-04286-x
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DOI: https://doi.org/10.1007/s00425-023-04286-x