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

Abstract

Summary 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, MdDIPM4 knockout has been produced in two Malus × 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 in susceptibility was observed compared to control plants. Sequencing of five 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 the expression cassettes for CRISPR/Cas9, the marker gene and the 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.

Candidate gene mapping identifies genomic variations in the fire blight susceptibility genes HIPM and DIPM across the Malus germplasm

“…Similarly, the strong association (p < 0.001) of SNP marker S14_14033636 on the DIPM4b gene with PLLL and PTD indicates its potential use in marker-assisted selection to decrease fire blight susceptibility. The latter finding is consistent with previous work in which knocking out the MdDIPM4 gene via CRISPR/Cas9 led to a 50% reduction in fire blight symptoms on notoriously susceptible 'Gala' and 'Golden Delicious' cultivars 40 . These results also highlight utilization of candidate-gene based association studies to rapidly identify molecular markers linked to traits of interest for progeny and parent selection in breeding programs 59 .…”

“…Natural genetic variation in these genes can be tapped to develop cultivars with decreased fire blight susceptibility, a major goal of apple germplasm improvement efforts worldwide. The susceptibility genes, HIPMs and DIPMs, have been identified to regulate susceptibility and improved tolerance against fire blight in commercial apple cultivars 35,38,40,58 . We have found a great number of genomic variations in the 9 HIPM and DIPM genes across 145 diverse Malus accessions.…”

New Plant Breeding Techniques in Citrus for the Improvement of Important Agronomic Traits. A Review

“…Recombination/excision systems have been shown to effectively produce SMG-free apple (Kost et al, 2015;Krens et al, 2015), apricot (Petri et al, 2012), and citrus (Zou et al, 2013). Recently, Pompili et al (2020) reported the use, in apple, of a CRISPR/Cas9-FLP/FRT gene editing system aimed at the production of regenerated plants with low content of residues of exogenous DNA.…”

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

“…In this study we applied Agrobacterium -mediated transformation on two economically important fruit trees, grapevine and apple, and evaluated different constructs for the complete elimination of exogenous DNA via site-specific removal mechanisms. We designed vectors carrying the CRISPR/Cas9 system to knock-out the genes VviMLO7 and MdDIPM1 and MdDIPM4 which have a role in the susceptibility to powdery mildew in grapevine 11 and fire blight in apple 12 , respectively. Two mechanisms were tested for the removal of T-DNA.…”

“…The Flp/FRT system has been extensively used to remove undesired transgenes from transgenic apple 13 , 14 and grapevine 15 . Recently in Pompili et al 12 it has been successfully applied to remove T-DNA cassette in apple. This latter work can be considered as a starting point for our study which basically consists in the optimization of vectors for T-DNA free genome editing.…”