Abstract
Broomrapes (Phelipanche aegyptiaca and Orobanche spp.) are obligate plant parasites that cause extreme damage to crop plants. The parasite seeds have strict requirements for germination, involving preconditioning and exposure to specific chemicals strigolactones [SLs] exuded by the host roots. SLs are plant hormones derived from plant carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase 8 (CCD8). Having no effective means to control parasitic weeds in most crops, and with CRISPR/Cas9 being an effective gene-editing tool, here we demonstrate that CRISPR/Cas9-mediated mutagenesis of the CCD8 gene can be used to develop host resistance to the parasitic weed P. aegyptiaca. Cas9/single guide (sg) RNA constructs were targeted to the second exon of CCD8 in tomato (Solanum lycopersicum L.) plants. Several CCD8Cas9 mutated tomato lines with variable insertions or deletions in CCD8 were obtained with no identified off-targets. Genotype analysis of T1 plants showed that the introduced CCD8 mutations are inherited. Compared to control tomato plants, the CCD8Cas9 mutant had morphological changes that included dwarfing, excessive shoot branching and adventitious root formation. In addition, SL-deficient CCD8Cas9 mutants showed a significant reduction in parasite infestation compared to non-mutated tomato plants. In the CCD8Cas9 mutated lines, orobanchol (SL) content was significantly reduced but total carotenoids level and expression of genes related to carotenoid biosynthesis were increased, as compared to control plants. Taking into account, the impact of plant parasitic weeds on agriculture and difficulty to constitute efficient control methods, the current study offers insights into the development of a new, efficient method that could be combined with various collections of resistant tomato rootstocks.
Highlights
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/ Cas9) system has emerged as a powerful genome-engineering technology with success in diverse organisms[1]
SLs occur in all green lineages of the plant kingdom and its synthesis start with the all trans β-carotene, a carotenoid molecule which produce 9-cis-β-carotene by the activity of Dwarf 27 (D27), after that Carotenoid Cleavage Dioxygenases 7 (CCD7) convert it into 9 cis β–apo 10′-carotenal and Carotenoid Cleavage Dioxygenases 8 (CCD8) leads to the production of carlactone, cytochrome P450 enzymes, More Axillary Growth 1 (MAX1) convert it into various SLs17
We report the development of tomato (Solanum lycopersicum L.) plants that are resistant to the parasitic weed P. aegyptiaca upon mutation of the SL-biosynthesis gene Carotenoid Cleavage Dioxygenase 8 (CCD8) using CRISPR/Cas[9]
Summary
CRISPR/Cas[9] to create host resistance in tomato plants against parasitic weeds, we chose to disrupt the SL-biosynthesis gene CCD8 (Solyc08g066650) in tomato. Orobanchol levels were significantly decreased in the CCD8Cas[9] mutated lines 1b, 2a, and 11b compared to the wild type, whereas orobanchol was not detectable in lines 1a, 2b, 5a, or 5c (Fig. 5a and Supplementary Table S3) This is consistent with line 5 showing the highest resistance to P. aegyptiaca. Results obtained using quantitative real-time PCR demonstrated that expression of PDS1, LCY-β and CCD8 was upregulated in CCD8Cas[9] edited T1 lines as compared to the wild type (Fig. 6c).These results demonstrate that a decrease in SL content in the root of CCD8Cas[9] mutants, affect the carotenoid profile by modulating expression of the gene involved in carotenoid pathway
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
