The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein MdDIPM4. In this work, an MdDIPM4 knock-out has been produced in two Malus x domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty-seven transgenic lines were screened to identify CRISPR/Cas9-induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss-of-function mutation were inoculated with the pathogen. Highly significant reduction of susceptibility was observed compared to control plants. Sequencing of 5 potential off-target sites revealed no mutation event. Moreover, our construct contained a heat shock-inducible FLP/FRT recombination system designed specifically to remove the T-DNA harbouring expression cassettes for CRISPR/Cas9, marker gene and FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat-treated and screened by real-time PCR to quantify the exogenous DNA elimination. The T-DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9-FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DNA

Pompili, V.; Dalla Costa, L.; Piazza, S.; Pindo, M.; Malnoy, M. (2020). Reduced fire blight susceptibility in apple cultivars using a high-efficiency CRISPR/Cas9-FLP/FRT-based gene editing system. PLANT BIOTECHNOLOGY JOURNAL, 18 (3): 845-858. doi: 10.1111/pbi.13253 handle: http://hdl.handle.net/10449/56713

Reduced fire blight susceptibility in apple cultivars using a high-efficiency CRISPR/Cas9-FLP/FRT-based gene editing system

Pompili, V.
Primo
;
Dalla Costa, L.;Piazza, S.;Pindo, M.;Malnoy, M.
Ultimo
2020-01-01

Abstract

The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein MdDIPM4. In this work, an MdDIPM4 knock-out has been produced in two Malus x domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty-seven transgenic lines were screened to identify CRISPR/Cas9-induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss-of-function mutation were inoculated with the pathogen. Highly significant reduction of susceptibility was observed compared to control plants. Sequencing of 5 potential off-target sites revealed no mutation event. Moreover, our construct contained a heat shock-inducible FLP/FRT recombination system designed specifically to remove the T-DNA harbouring expression cassettes for CRISPR/Cas9, marker gene and FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat-treated and screened by real-time PCR to quantify the exogenous DNA elimination. The T-DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9-FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DNA
Malus x domestica
DIPM
FLP/FRT recombination
Fire blight
Gene editing
Settore AGR/07 - GENETICA AGRARIA
2020
Pompili, V.; Dalla Costa, L.; Piazza, S.; Pindo, M.; Malnoy, M. (2020). Reduced fire blight susceptibility in apple cultivars using a high-efficiency CRISPR/Cas9-FLP/FRT-based gene editing system. PLANT BIOTECHNOLOGY JOURNAL, 18 (3): 845-858. doi: 10.1111/pbi.13253 handle: http://hdl.handle.net/10449/56713
File in questo prodotto:
File Dimensione Formato  
2020 PBJ Pompili et al.pdf

accesso aperto

Tipologia: Versione editoriale (Publisher’s layout)
Licenza: Creative commons
Dimensione 3.46 MB
Formato Adobe PDF
3.46 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10449/56713
Citazioni
  • ???jsp.display-item.citation.pmc??? 35
  • Scopus 94
  • ???jsp.display-item.citation.isi??? 74
social impact