Mutagenesis of two homologs ATP Binding cassette protein in tomato by CRISPR/Cas9 provide resistance against the plant parasite Phelipanche aegyptiaca

Abstract

Plant parasitic weed Phelipanche aegyptiaca and Orobanche spp. are obligate plant parasites that cause heavy damage to agricultural crop plants. Germination of the parasite seeds require exposure to specific chemical known as strigolactone [SL]. The plant hormone SL is derived from plant carotenoids via cleavage by CCD7 and CCD8 enzymes and exuded by the host roots to the rhizosphere. Here, we provide evidence that CRISPR/Cas9 mediated targeted mutagenesis of two homologues ATP Binding cassette (ABC) protein in tomato (Solyc08g067610 and Solyc08g067620), significantly reduced the germination of the parasitic weed P.aegyptiaca. Constructs harboring specific single guide RNA were prepared and targeted against conserved region in the above tomato genes (Solyc08g067610 and Solyc08g067620). Selected T0-mutated tomato plants showed different type of deletions at both locuses. Furthermore, genotype analysis of T1 plants showed that the introduced mutations stably inherited to next generation with no identified off-targets. Mutated tomato lines, showed more branching, enhanced growth of axillary buds, reduced length of primary stems and significantly reduced development of the parasitic weed P. aegyptiaca as compared to wild type plants. Moreover, in ABCCas9 mutants the expression level of the genes (CCD8 and MAX1) related to SL biosynthesis were decreased, without alteration in the root extract orobanchol [SL] as compared to control plants. Development of genetic resistance based on genome editing of host key genes are novel approaches for enhancing host-parasite resistance. The current study offers insights into ABC protein homologs and mutagenesis of ABC protein that could be used in the development of efficient method to reduce parasitic weed germination.