Restriction digestion of the target region-specific PCR product from lines 1, 2, 5 and 11 revealed that lines 1 and 5 were fully resistant to BsrI digestion, whereas line 11 showed partial digestion. However, line 2 showed complete digestion of the PCR product, similar to the wild type . Sequencing analysis of the target region PCR product from T0 lines 1, 2, 5 and 11 revealed the presence of different kinds of mutations in each line. Line 1 had a 1-nt deletion and line 2 had a 3-nt deletion; the 3-nt deletion in line 2 regenerated the BsrI restriction site, resulting in BsrI digestion of the PCR product in this line. In addition, these lines showed a single peak in the Sanger sequencing chromatogram, suggesting that they represent homozygous mutants for the CCD8Cas9 locus . However, lines 5 and 11 showed multiple peaks in the sequencing chromatogram, suggesting that they are either biallelic or chimeric mutants . Purified non-digested CCD8Cas9 target regionPCR fragments from lines 5 and 11 were cloned into a TA cloning vector. Sanger sequence analysis revealed that lines 5 and 11 are biallelic and contain four types of mutations: a 6-, 5- or 4-nt deletion, or an A insertion. Interestingly, in line 5, both alleles lost the BsrI restriction site whereas in line 11, one of the alleles restored the BsrI cut site after Cas9-mediated editing of the target . T0 transgenic tomato lines were grown to maturity and self-pollinated to generate T1 progeny. T1 progeny from lines 1, 2, 5 and 11 were designated with a small letter next to the line number . Genomic DNA was extracted from the T1 progeny and the target region of CCD8Cas9 was amplified using PCR primers fanking the target.
When this analysis was performed on T1 lines, results were similar to those with the T0 lines: lines 2a and 2b were completely digested, vertical racking indicating that the BsrI site was preserved; however, lines 1a, 1b, 5a, 5b, 5c and 11a gave PCR fragments that were resistant to BsrI digestion, indicating that the CRISPR-generated mutations in these lines were inherited from the T0 lines . Sanger sequencing of the non-BsrI-cut fragments showed deletion mutations of various lengths upstream of the PAM sequence, similar to T0. Lines 1a and 1b each contained the same 1-nt deletion and lines 2a and 2b each had the same 3-nt deletion. Moreover, biallelic mutations were detected in lines 5a, 5b, a 4-nt deletion was observed in line 5c, and a 6-nt deletion was found in line 11a . At least 15 plants from each of the T1 lines were examined for genotype at the target site using Sanger sequencing of target-site PCR products. All T1 plants from two T0 homozygous lines were homozygous for the same mutations. In contrast, the biallelic T0 lines of the Cas9-generated mutants were segregated in the T1 generation according to classic Mendelian genetics, and the ratios between the two mutations in a biallele were close to 1:2:1 as reported previously. The existence of the same mutations in sibling progeny suggested that the CRISPR mutation event occurred prior to meiosis in T0. Inheritance of the mutations in homozygous and biallelic T0 plants by T1 plants suggested that most, if not all, of the mutations resulting from genome editing activity are highly stable in nature and can be inherited in subsequent generations. Moreover, examination of transgenic region in some of the T1 generation plants suggest that 35% of line 2 and 25% of line 5, T1 plants were detected to be transgene free . The results indicated that CCD8Cas9 targeted mutations inherited to next generation in transgene free plants. The putative of-target sites associated with CCD8sgRNA were evaluated by CRISPR-P program using the CCD8sgRNA sequence against the tomato genome.
We analyzed three potential of-targets sites with high scores, which occurred in the intergenic and CDS regions of the tomato genome. Two plants from each line were selected from the T1 generations of CCD8Cas9-edited tomato plants. Sequencing of PCR products from these regions revealed no changes in the putative of-target sites in the CCD8Cas9–mutant lines .Previous studies on SL biosynthesis using rice dwarf mutants have reported that SLs regulate plant growth and morphological architecture. Furthermore, ccd8 a SL-deficient mutant of pea is known to exhibitincrease in shoot branching, lateral roots and overall dwarfing. We also observed similar phenotypic profile in CCD8Cas9 mutated lines such as highly branched shoots, increased lateral roots, decreased shoot heights and reduced fruit sizes as compared to the wild type plants . Although, morphologically, all CCD8Cas9 mutant lines showed highly branched shoots irrespective to the type of mutation but no significant differences were found in the root mass between CCD8Cas9 mutated and control plants . Interestingly, CCD8Cas9 mutated tomato lines produced considerable more number of fruits with reduced sizes as compared to the non-mutated wild-type plants . To analyze whether the CRISPR/Cas9-generated mutations in the CCD8 gene confer resistance to P. aegyptiaca, independent transgenic tomato plants from T1 lines representing the CCD8Cas9 knockout phenotypes, were triggered with P. aegyptiaca seeds. Randomly chosen T1 progeny of each lines, irrespective of their zygosity were transplanted into small pots containing soil infested with P. aegyptiaca seeds and grown for 3 months in a greenhouse. Two separate experiments with four replicates per treatment were conducted. To measure the resistance of the CCD8Cas9 mutated lines, we counted only fresh and viable parasite tubercles which are larger than 2 mm in diameter from each plant. The numbers of attached parasitictubercles and shoots were significantly reduced in the CCD8Cas9 mutated lines relative to the wild-type plants. However, the decrease in P. aegyptiaca in some of the line 11 mutants was less pronounced relative to the wild-type plants than that observed for lines 1, 2 and 5 .The tomato host plant produces different kinds of SLs—mainly orobanchol, didehydroorobanchol isomer 1 and 2, and the aromatic SL solanacols, including the recently identifed orobanchyl acetate, 7-hydroxyorobanchol isomers 1 and 2, and 7-oxoorobanchol. However, Orobanche preferentially utilizes orobanchol as the most active germination stimulant , whereas solanacol and 7-oxoorobanchol are weak stimulants.
To explore the connection between SL biosynthesis in the CCD8Cas9 mutants and their resistance to P. aegyptiaca infection, we analyzed the total orobanchol content in the roots of wild-type and CCD8Cas9 mutated T1 lines by LC–MS/MS. Orobanchol levels were significantly decreased in the CCD8Cas9 mutated lines 1b, 2a, and 11b compared to the wild type, whereas orobanchol was not detectable in lines 1a, 2b, 5a, or 5c . This is consistent with line 5 showing the highest resistance to P. aegyptiaca. In addition, although CCD8 was modified in line 11a, and the plant exhibited the typical dwarfing and shoot-branching phenotypes of reduced SL, its orobanchol content was higher than in the other modified lines. The higher orobanchol content was consistent with its lower resistance to P. aegyptiaca. To assess the higher orobanchol content in line 11a, we analyzed the DNA mutations and resulting amino acid sequences in all CCD8Cas9 mutated lines that were sampled for LC–MS/MS analysis after PCR products ligated to the TA cloning vector . The type of DNA mutation and the amino acid sequence in line 11a demonstrated that only 2 amino acids, His- 243 and Pro-244, were deleted due to Cas9 editing in the target CCD8 gene, while the rest of the coding sequence was similar to the wild-type protein .Carotenoid biosynthetic pathway derivatives all trans β- carotenoid leads to production of SL. Since CCD8 catalyze a key step in SL biosynthesis from carotenoids; hence, we are interested to discover whether CCD8Cas9 mutation affects carotenoid content and its upstream biosynthetic pathway. A simplified scheme of the correlation between carotenoid and SL biosynthesis pathways and furidone target site is illustrated in Fig. 6a. First, rolling benches to explore whether CCD8Cas9 mutation affect the carotenoid content, the content and type of carotenoid present in the root of wild type and CCD8Cas9 mutated lines were analyzed by HPLC method. Interestingly, CCD8Cas9 mutation altered the profile of differenttypes of carotenoids and its derivative, such as total carotenoids, lutein; β-carotene were substantially altered from the wild type . To further gain insight into the above results, we analyzed the expression of prominent gene Phytoene desaturase-1 and Lycopene cyclase 1-β , involved in the carotenoid biosynthetic pathway which acts upstream of CCD8. Results obtained using quantitative real-time PCR demonstrated that expression of PDS1, LCY-β and CCD8 was upregulated in CCD8Cas9 edited T1 lines as compared to the wild type .These results demonstrate that a decrease in SL content in the root of CCD8Cas9 mutants, affect the carotenoid profile by modulating expression of the gene involved in carotenoid pathway.Biotic stresses induced great economic challenges for farming and food production worldwide. Broomrapes that affect the roots of many economically important agriculture crops throughout the semiarid regions of the world especially the Mediterranean and Middle East, are regarded as some of the most serious pests in vegetable and feld crops. Efective means to control parasitic weeds are scarce and lack of novel sources of resistance limits our ability to manage newly developed, more virulent broomrape races. Therefore, an innovative solution to the problem is greatly needed.Recent work utilized the power of CRISPR/Cas9 to engineer the rice plant architecture through genomic editing of OsCCD7 gene, having decreased SL and reduced Striga hermonthica germination. Utilizing similar CRISPR/Cas9 genome-editing strategy, we have developed non-transgenic tomato mutant plants with no “foreign-DNA” that exhibits resistance to P. aegyptiaca. We designed a CCD8sgRNA construct to target the second exon of the tomato CCD8 gene to disrupt SL biosynthesis.
Several independent T0 transgenic tomato lines—1, 2, 5 and 11—were generated, of which lines 1 and 2 were homozygous mutants whereas lines 5 and 11 were biallelic in nature. In general, T0 mutants presented somatic mutations; to avoid this, T0 plants were self-pollinated to generate T1 homozygous plants. In the T1 generation, we found homozygous deletions and insertions in the target gene that were biallelic in the T0 plants without any new mutation detected. T1 lines 1, 2, 5 and 11 were selected for further analysis because of their stable genetic modification. Te different types of mutations observed in the different lines may be due to the differential activity of Cas9, depending on the transgene insertion site, as shown previously in tomato. In addition, Sanger sequencing of potential of-target sites with mismatches of less than 4nt with CCD8sgRNA did not identify any mutations. Te CRISPR/ Cas9 system has emerged as a powerful gene-editing tool and has been successful in more than 20 crop species to date. The heritability of the mutated genes and the generation of transgene-free plants are of major concern when using the CRISPR/Cas9 system. To follow heritability, the gene PDS1 was used to demonstrate the inheritance of mutations induced in Solanum lycopersicum. Tose authors showed that the CRISPR/Cas9 can efficiently induce heritable mutations in tomato plants from the T0 to T2 generation, and that homozygous and biallelic mutants are generated, in the first generation. In our study, we also showed that the CCD8Cas9 mutations induced in T0 lines are inherited by the T1 generation. Previous reports on the morphology of tomato SlCCD8 knock down plants by gene silencing have shown an increase in shoot branching, altered lateral adventitious root growth and decrease in plant height. Similarly another studies on SL bio-synthetic gene SlCCD7, demonstrates the SlCCD7 anti-sense tomato lines also display increased branching, reduced SL content and significantly decreased germination rate of O. ramosa, however no changes in carotenoid content in the roots were observed. Our results were partially consistent with the previous studies, we observed similar phenomenon in the CCD8Cas9 tomato plants. Mutated-plants displayed a dwarf phenotype, an increased number of shoot branches, and an increased number of adventitious roots compared to the wild-type plants. In contrast to the previous report, here we discovered that CCD8Cas9 mutants have altered carotenoid content and differential expression of genes involved in carotenoid bio-synthetic pathway. An explanation for the contradictory results could be due to the differences between mechanisms of siRNA and CRISPR/ Cas9 systems. We hypothesize that absence of CCD8 gene followed by mutation in tomato plants using CRISPR system, will not restore feedback regulation. However, a small percentage of functional CCD8 gene that could escape gene-silencing system will provide feedback regulation and restore the carotenoid level upstream in the carotenoid pathway.