Abstract
The generation and selection of novel fire blight resistant apple genotypes would greatly improve the management of this devastating disease, caused by Erwinia amylovora. Such resistant genotypes are currently developed by conventional breeding, but novel breeding technologies including cisgenesis could be an alternative approach. A cisgenic apple line C44.4.146 was regenerated using the cisgene FB_MR5 from wild apple Malus ×robusta 5 (Mr5), and the previously established method involving A. tumefaciens-mediated transformation of the fire blight susceptible cultivar ‘Gala Galaxy’ using the binary vector p9-Dao-FLPi. The line C44.4.146 was shown to carry only the cisgene FB_MR5, controlled by its native regulatory sequences and no transgenes were detected by PCR or Southern blot following heat induced recombinase-mediated elimination of the selectable markers. Although this line contains up to 452 bp of vector sequences, it still matches the original definition of cisgenesis. A single insertion of T-DNA into the genome of 'Gala Galaxy' in chromosome 16 was identified. Transcription of FB_MR5 in line C44.4.146 was similar to the transcription in classically bred descendants of Mr5. Three independent shoot inoculation experiments with a Mr5 avirulent strain of Erwinia amylovora were performed using scissors or syringe. Significantly lower disease symptoms were detected on shoots of the cisgenic line compared to those of untransformed 'Gala Galaxy'. Despite the fact that the pathogen can overcome this resistance by a single nucleotide mutation, this is, to our knowledge, the first prototype of a cisgenic apple with increased resistance to fire blight.
Highlights
Apple is one of the most important fruit crops worldwide considering its production level of 80.8 million tons per year [1]
In a second inoculation experiment when shoots were directly injected with E. amylovora using a syringe, the cisgenic line C44.4.146 showed a mean percentage of lesion length (PLL) of 41.1% ± 15.0%, which resulted to be significantly different from the mean PLL of 'Gala Galaxy' showing 73.9% ± 16.5% (Wilcoxon test p-value < 0.0001, Fig 7)
As a negative selection marker to exclude cells in which recombination did not occur, the gene dao1, encoding D-aminooxidase 1 [42], is present in the excisable cassette of p9-Dao-FLPi. This negative selection marker could not be applied, as it has recently been shown that the use of D-Ile containing medium completely hinders the formation of cisgenic shoots and transgenic shoots survived for several months on it [24]
Summary
Apple is one of the most important fruit crops worldwide considering its production level of 80.8 million tons per year [1]. The level of fire blight resistance of this line was assessed by means of two different shoot inoculation methods and compared with untransformed 'Gala Galaxy' plants Further characterization of this line involved the assessment of the number of T-DNA integrations, site of integration in the genome, and transcription level of the FB_MR5 gene in this line compared to conventionally bred genotypes carrying the FB_MR5 gene. To our knowledge, this is the first report of a cisgenic apple with increased resistance to fire blight
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