CRISPR/Cas9 mediated mutagenesis of MORE AXILLARY GROWTH 1 in tomato confers resistance to root parasitic weed Phelipanche aegyptiaca

Root parasitic weeds infect numerous economically important crops, affecting total yield quantity and quality. A lack of an efficient control method limits our ability to manage newly developing and more virulent races of root parasitic weeds. To control the parasite induced damage in most host crops, an innovative biotechnological approach is urgently required. Strigolactones (SLs) are plant hormones derived from carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase (CCD) 7, CCD8 and More Axillary Growth 1 (MAX1) genes. SLs act as branching inhibitory hormones and strictly required for the germination of root parasitic weeds. Here, we demonstrate that CRISPR/Cas9-mediated targted editing of SL biosynthetic gene MAX1, in tomato confers resistance against root parasitic weed Phelipanche aegyptiaca. We designed sgRNA to target the third exon of MAX1 in tomato plants using the CRISPR/Cas9 system. The T0 plants were edited very efficiently at the MAX1 target site without any non-specific off-target effects. Genotype analysis of T1 plants revealed that the introduced mutations were stably passed on to the next generation. Notably, MAX1-Cas9 heterozygous and homozygous T1 plants had similar morphological changes that include excessive growth of axillary bud, reduced plant height and adventitious root formation relative to wild type. Our results demonstrated that, MAX1-Cas9 mutant lines exhibit resistance against root parasitic weed P. aegyptiaca due to reduced SL (orobanchol) level. Moreover, the expression of carotenoid biosynthetic pathway gene PDS1 and total carotenoid level was altered, as compared to wild type plants. Taking into consideration, the impact of root parasitic weeds on the agricultural economy and the obstacle to prevent and eradicate them, the current study provides new aspects into the development of an efficient control method that could be used to avoid germination of root parasitic weeds.


Results
Targeted mutagenesis of MAX1 gene in tomato using CRISPR/Cas9. To analyze the effectiveness of the genome editing approach and to generate host resistance against root parasitic weed P. aegyptiaca, we targeted the SL-biosynthesis gene MAX1 (Solyc08g062950) in the host plant tomato using CRISPR/Cas9 system. The Cas9/sgRNA vector construct was designed to target the third exon of MAX1 (position 778-797 bp in the coding region) having an XmaI restriction site located next to the protospacer adjacent motif (PAM) (Fig. 1a,b). Five transgenic lines 1, 2, 4, 5 and 8 were independently generated and presence of transgene was confirmed by kanamycin resistance and PCR amplification of pcoCas9 (Fig. 1c). To evaluate the types of mutation generated by Cas9/MAX1-sgRNA in T 0 lines 1, 2, 4, 5 and 8, the flanking region was PCR amplified and the presence of indel mutations was tested by XmaI restriction digestion. Restriction analysis suggests that line 1 contained about equal amounts of undigested and digested DNA, in contrast other lines 2, 4, 5 and 8 seem non-mutants with most of the DNA digested similarly to wild type plants (Fig. 1d). DNA sequencing analysis of the target region from T 0 line 1 shows multiple peaks possibly due to the presence of biallelic or heterozygous mutations (Fig. 2a), however in lines 2, 4, 5 and 8 no mutation was detected hence discarded. To explore the kind of mutation, existing in line 1, the MAX1 target region was PCR amplified and directly cloned into a TA cloning vector and sequenced. Analysis of the DNA sequences revealed that T 0 line 1 plant is heterozygous and contains in-frame deletion of 9 nt which causes removal of the amino acid (Lys-252, Arg-253 and Iso-254) in mutant as compared to wild-type protein after Cas9-mediated editing of the target (Fig. 2b,c).
Usually, T 0 transgenic lines are somatic in nature, hence T 0 line 1 was grown to maturity and self-pollinated to generate T 1 plants. Genomic DNA was isolated from the T 1 plants of line 1 and the target region of MAX1 was again amplified using PCR primers flanking the target. When this analysis was performed on T 1 plants of line 1, results were like those with the T 0 line 1. At least 64 plants of the T 1 lines were analyzed for genotype at the target site using sanger DNA sequencing. T 1 plants from the T 0 heterozygous line 1 were segregated according to mendelian law and the kind of mutation that existed was separated as 1:2:1 as reported previously (Table S1) 28 . The existence of T 0 mutation by T 1 plants suggested that the mutation resulting from CRISPR/Cas9 activity are highly stable in nature and inherited to the next generation without any alteration. Additionally, the presence of the transgene region (pcoCas9) was examined in the T 1 generation plants. Our results demonstrated that 35.7% (5/14) T 1 plants were detected to be transgene-free (Table S1). We have also analyzed potential off-target effects that occurred due to non-specific activity of Cas9, associated with MAX1-sgRNA in the tomato genome. At least two plants were selected from the T 1 generations of MAX1-Cas9 edited homozygous lines. Sequencing analysis of PCR products from these regions revealed no change in the potential off-target sites (Fig. S1, Table S2).

Alteration of morphological phenotype in MAX1-Cas9 mutated plants. Previous studies with
RNA silencing of MAX1 in tomato suggest that max1 mutants exhibit an increase in shoot branching, lateral roots and overall dwarfing 29 . Surprisingly, in our study, the heterozygous lines had shown more growth of axillary buds as compare to control even in T 0 generation (Fig. 3a). Consistently, we also observed similar phenotypic changes in the MAX1-Cas9 mutated homozygous T 1  heterozygous and homozygous tomato lines from T 1 generation, and mixed with P. aegyptiaca seeds (15 mg/kg soil) and grown for three months in a greenhouse with optimum control conditions. To analyze the resistance, we counted only fresh and viable parasite tubercles and shoots that are larger than 2 mm in diameter. Results obtained suggested that the total number of germinated parasite tubercles and shoots were significantly reduced in the MAX1-Cas9 mutated heterozygous and homozygous plants as compared to the wild-type plants (Fig. 5a). To further correlate resistance to P. aegyptiaca infection and SL content of the MAX1-Cas9 mutated plants, we analyzed the total orobanchol content in the root extract of wild-type and MAX1-Cas9 edited host plants. We observed a significant reduction of total orobanchol content in both heterozygous and homozygous plants as compared to the wild type. However, the decrease in orobanchol was more pronounced in homozygous as compared to heterozygous mutant plants (Fig. 5b).
MAX1-Cas9 mutation affects upstream carotenoid-biosynthesis pathway. SLs are carotenoid derivatives and carotenoids are isoprenoid pigments that differ in structure and stereometric configuration with a system of conjugated double bonds that is responsible for their colors 9,30 . In plants, SL and carotenoid biosynthesis take place mainly in the plastids 31 . It is initiated by the condensation of two molecules of geranylgeranyl diphosphate and with a series of reactions in presence of different enzymes, they produce diverse active molecules such as lycopene, lutein, β -carotene and violaxanthin 9,32 . To assess whether targeted mutagenesis of MAX1 gene induces feedback regulation and affect transcript level of SL biosynthetic gene CCD8 and MAX1, expression analysis was performed in homozygous lines using quantitative real-time PCR, however, we did not www.nature.com/scientificreports/ observe any significant difference in the expression of CCD8 and MAX1 transcript (Fig. 5c). This data suggests that mutagenesis of MAX1 does not affect expression of CCD8 or the feedback pathway. Previous studies with Petunia hybrida ATP Binding Cassette transporter explored that PDR1 act as SL transporter which has a key role in regulating the growth of axillary branches and symbiotic interactions with arbuscular mycorrhizae. Additionally, P. hybrida pdr1 mutants are defective in SL exudation from their roots, resulting in reduced germination of P. ramosa 33 . To investigate whether the resistance against root parasitic weed depends on SL exporter the expression of ABCG45, a homolog of PDR1 in tomato, was analyzed in MAX1 mutants and we did not observe any significant change in the transcript, which suggest that ABCG45 is not involved in parasitic weed resistance mechanism (Fig. 5c). Further, to explore whether defective SL biosynthesis in MAX1-Cas9 mutant affects the behavior of the upstream carotenoid-biosynthesis pathway, we analyzed the expression of phytoene desaturase-1 (PDS1), a gene involved in the biosynthesis of β-carotene 30,34 . Our results demonstrated that the expression of PDS1 is significantly upregulated in MAX1-Cas9-edited homozygous T 1 lines as compared to the wild type (Fig. 5c). To further validate the PDS1 expression data, we analyzed the total carotenoids content in the root of www.nature.com/scientificreports/ MAX1 mutated plants. Our results suggest that homozygous mutants have substantially increased content of total carotenoids along with β-carotene and lutein as compare to heterozygous and wild type plants (Fig. 5d).

Discussion
Plant-parasitic weeds exert biotic stresses and create significant constraints for farming and food production worldwide. Root parasite Phelipanche and Orobanche spp. attacks many economically important crops throughout the Mediterranean and semi-arid regions and are regarded as the most serious pests 35 . Hence, to control parasitic weeds effectively, an innovative solution is urgently needed. A major goal of plant genome editing is to improve crop yield and quality. Studies on host plant-parasitic weed interactions have explored many key host genes associated with parasitic weed resistance [36][37][38][39] .
In this study, using CRISPR/Cas9 genome-editing system, we have developed a non-transgenic MAX1 mutant of tomato that exhibits reduced orobanchol content and increased resistance to P. aegyptiaca. We designed a sgRNA to target the third exon of the tomato MAX1 gene to disrupt SL biosynthesis. In Agrobacterium transformed T 0 transgenic line, we found editing events in the MAX1 targeted locus in one (Line 1) out of five lines (Fig. 1). The heritability of the mutation and the generation of transgene-free plants are major concern, when using the CRISPR/Cas9 system 40,41 . To avoid the somatic nature of editing events, T 0 line 1 was self-pollinated to generate T 1 transgenic plants. In the T 1 generation, we observed that the mutations induced in T 0 line 1 are stably inherited by the T 1 generation, without any new mutations (Fig. 2). www.nature.com/scientificreports/ Using sanger DNA sequencing of potential off-target sites with mismatches of fewer than 4nt with MAX1-sgRNA, we did not identify any off-target mutation ( Fig. S1 and Table S2). The specificity of Cas9 is greatly influenced by various factors and non-specific off-target site cleavage is a major challenge in the use of the CRISPR system 42 . In higher plants, undesirable mutations resulting from the CRISPR/Cas9 system are generally rare, however, non-specific undesired mutations can be avoided using more specific sgRNAs [43][44][45][46][47] .
SLs play a major role in controlling plant architecture, regulating shoot branching and to influence lateral and adventitious root formation 48 . Recent studies on the morphology of tomato max1-mutant plants by gene silencing have shown an increase in shoot branching, reduced plant height and increased adventitious roots formation 49 . Interestingly, in our study MAX1-Cas9 heterozygous tomato plants shown intermediate phenotype with respect to homozygous and wild type plants. These heterozygous plants displayed reduced plant height, increased number of axillary branches, nodes and adventitious roots compared to the wild-type plants (Figs. 3,  4). However, morphological phenotypic changes associated with heterozygous lines were mild in nature than homozygous plants.
In the view of above, an argument can be raised that transgenic plants bearing Cas9 expression cassette in their genome constitutively express Cas9-sgRNA throughout their life and mutation could be accumulated during growth that will affect phenotype in heterozygous plants. To support our results, we showed that homozygous plants generated from the same line showed no off-target effects. Moreover, our MAX1 heterozygous plant shows a characteristic phenotype of SL defective mutant though it is mild in nature. These results strongly support our data and provides an explanation of phenotypic defect in MAX1 heterozygous. Another explanation for this could be the heterozygous mutants (in-frame 9nt deletion) due to the absence of amino acid Lys252-Arg253-Iso254 in the mutated protein behaving as semi-dominant negative or it could be a dosage gene effect, where one normal www.nature.com/scientificreports/ copy of gene exists and one copy mutated, that could affect the normal growth of plants if the protein has a role in important cellular function however these explanations need to be thoroughly investigated.
To evaluate host resistance to P. aegyptiaca, we used a pot system containing soil infested with P. aegyptiaca seeds as described previously 50 . In the current study, coincidently with the suppression of SL content, a significant decrease in the number of total germinated parasite tubercles and shoots was observed in MAX1 mutated plants as relative to the wild type plants. In contrast, the wild-type plants were highly susceptible to the parasite infestation (Fig. 5a). Since orobanchol is a major SL in tomato root exudates 51 , and acts as a specific germination inducer for P. aegyptiaca, hence we determine the orobanchol content in the root extract of the MAX1-Cas9 mutated lines. Orobanchol content was found to be significantly reduced in the MAX1-mutated lines relative to the wild type (Fig. 5b).
Previously it has been reported that SLs are able to modulate local auxin levels, and the action of SL is dependent on the auxin status of the plant, suggesting that specific SL content influences the expression of genes involved in auxin biosynthesis 52 . Another study reported that an increase in transcript level of SL biosynthetic gene D27 and CCD8 in tomato roots occurs after parasitic weed infection, suggesting SL-biosynthesis pathway gets activated after parasitic infection 53 . Several studies reported that SL-biosynthetic pathways are strictly www.nature.com/scientificreports/ regulated by negative-feedback inhibition such as D10 transcript levels are elevated in the dwarf mutants (d10-1) of rice, similarly transcript level of RMS1 are enhanced in ramosus mutants of pea and application of synthetic SL GR24 restored the transcripts level [54][55][56] . To evaluate the role of MAX1 in feedback regulation and mechanism of resistance, we analyzed the transcript level of CCD8, MAX1 and ABCG45 (Fig. 5c). However, no alteration in transcript level was observed suggesting that possibly MAX1 did not involve in the control of feedback inhibition of SL pathway or carlactone accumulation has no role in modulation of SL biosynthetic pathway. Moreover, the expression of SL exporter ABCG45 was also unaffected. Interestingly in our study MAX1-mutated homozygous lines have increased levels of a carotenoid biosynthetic gene PDS1 and total carotenoids (Fig. 5d). These results indicate that block in the SL biosynthesis pathway positively affects carotenoid biosynthesis possibly due to interconnection between SL production and carotenoid biosynthesis.
Previous studies from our laboratories demonstrated the movement of mobile exogenous siRNA from the host to the root parasites 50 , this provides an insight that, CRISPR/Cas9 genome editing technique could be used against the parasite itself by indirectly transforming parasite-specific sgRNA in the hosts, if the host gene does not share sufficient homology with the targeted sequences of the parasite. The unwanted cleavage due to the nonspecificity of the sgRNA within the genome exerts significant limitations to the CRISPR/Cas9 system which can alter the function of a gene or induce genomic instability. Currently, several naturally occurring and genetically modified Cas9 enzymes and specific sgRNA designing tools have been developed to enhance site specific target cleavage [57][58][59] , however, the off-target effect is still considered as a major limiting factor 60 . In the current study, we demonstrate that genetic resistance to root parasitic weeds can be obtained using CRISPR/Cas9 mediated targeted mutagenesis of the MAX1, a SL biosynthetic gene in tomato. A similar strategy could be effectively used against other parasitic weeds to generate host resistance.

Experimental procedures
Materials and growth conditions. Tomato (Solanum lycopersicum) cultivar MP-1 was chosen for generation of transgenic plants. Tomato seeds were surface-sterilized using 1% bleach with tween-20 and 70% ethanol and grown on half strength Murashige and Skoog (MS) basal medium (CAISSON Laboratories, USA) containing 1.5% sucrose (Sigma), pH 5.8 and 7 g/L phytagel (Sigma). The P. aegyptiaca seeds were collected from infected field in Northern Israel and used to infest tomato host plants.
sgRNA design and Cas9 vector construction. The tomato MAX1 (Solyc08g062950) gene was chosen as the target and sgRNA sequence of 20 nucleotides was designed using the CRISPR-P webtool and cloned into the plant binary vector. The binary vector contains nptII gene as kanamycin selection marker, under the control of the NOS promoter. sgRNA sequence together with the guide scaffold, was amplified using forward primer containing SalI site as part of the U6 Arabidopsis promoter and a reverse primer of the Pol III-terminator sequence that contained a HindIII site and pRCS35S:Cas9-AtU6:sgRNA-PDS was used as a template. The amplified DNAs (130 bp) were cloned into SalI and HindIII digested pRCS-35S:Cas9-AtU6:sgRNA-PDS vector [61][62][63] . The verification of positive clones was done using diagnostic PCR and sequencing. The construct was transformed into tomato cultivar MP-1 using Agrobacterium tumefaciens strain EHA105.
Agrobacterium-mediated transformation of tomato plants. The transformation of tomato was conducted as previously described, with slight modifications 64 . The cotyledons were pre-cultured for 2 day in dark with Murashige and Skoog (MS) medium containing 100 μM Acetosyringone, 0.1 mg/L IAA, 1 mg/L Zeatin and 0.7% phytagel subsequently infected with the Agrobacterium strain EHA105 (optical density at 600 nm less than 0.5) by immersion for 20 min. The explants were co-cultivated with Agrobacterium strain EHA105 for two days in dark and then transferred to the MS medium supplemented with 0.1 mg/L IAA, 1 mg/L Zeatin, 100 µg/ml Kanamycin, 300 µg/ml timentin and 0.7% phytagel for a week. Kanamycin-resistant shoots regenerated were transferred to tissue culture bottles containing the subculture medium with 0.1 mg/L Zeatin. Developed vegetarians were transferred to the half strength MS rooting substrate with the addition of 2 mg/L IBA. After three months, Kanamycin-resistant plantlets were obtained and used for subsequent analysis.
Genotyping of transgenic plant. For genotyping, tomato plant leaves or roots genomic DNA was extracted using plant genomic DNA extraction kit and the genomic DNA flanks containing the sgRNA target sites were amplified using the specific primers SlMAX1-Int-F & SlMAX1-Int-R and then run on a 2% agarose gel using electrophoresis. Image was acquired using DNR Mini Lumi with UV light system. Direct sequencing of PCR products was done using appropriate primer. For PCR product cloning, pGEM-T kit from Promega were used.
Analysis of off-target mutations. The potential off-target sites associated with sgRNA target sequence were analyzed with the CRISPR-P program 65 . Three off target sites with the highest probability score were selected. The genomic region flanks upstream and downstream to the off-target sites (300-400 bp) was amplified and sequenced with specific primers using sanger DNA sequencing (Table S3).
Evaluation of P. aegyptiaca resistance assay. Resistance analysis of MAX1-Cas9 mutant tomato lines to the parasite was done as reported earlier 66 . For infection one-month-old tomato seedlings were transferred into pots containing soil with a peat moss to perlite mixture ratio of 3:1, infested with seeds of P. aegyptiaca ( www.nature.com/scientificreports/ exposure to the P. aegyptiaca seeds. The total number of viable P. aegyptiaca tubercles larger than 2 mm diameter attached to host roots were counted.

RNA isolation and quantitative real-time PCR.
Total RNA from tomato roots was extracted using spectrum plant total RNA kit (Sigma-STRN50-1KT) according to the manufacturer's protocol. 500 ng of total RNA was used to obtained cDNA according to the protocol of Quanta Bioscience cDNA Synthesis Kit. Quantitative real-time PCR (qRT-PCR) was performed in a volume of 10 μl using PerfeCTa SYBR Green FastMix ROX (Quanta biosciences) with 5 times diluted cDNA as template. Tomato elongation factor 1-α was used as an internal control gene. Specificity of the primers was confirmed by melting curve analysis. The generated Ct values of target genes were normalized to the Ct value of internal reference EF1-α gene. Relative expression was calculated using 2 −ΔΔCt method and expressed as fold increase with respect to control 67 .
SL extraction and analysis using HPLC-MS/MS. The SL extraction and quantification were performed as described previously 68 . Lyophilized tomato roots were grounded into fine powders using liquid nitrogen and extracted with the ethyl acetate extraction method. The tissues were transferred to a 4-10 times volume of ethyl acetate. The flask was treated with an ultra-sonic bath for a few minutes and then placed in a cool place (4 °C) for 2-3 days. The tissues are filtered off and washed well with ethyl acetate. The combined ethyl acetate was washed with 0.2 M K 2 HPO 4 or saturated NaHCO 3 to remove acidic compounds. The extract was dried over anhydrous MgSO 4 or Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure. Samples were dissolved in 2 ml acetonitrile or methanol: water (25:75 v/v) and the quantification of orobanchol in extracts was performed using LC-MS/MS using orobanchol as standard.
Carotenoid extract analysis. Carotenoid extract analysis was performed as reported earlier 69 . In brief, 1.5 g of fine grinded roots was used for extraction in presence of 8 ml hexane: acetone: ethanol (50:25:25 v/v), followed by 5 min of saponification in 1 ml of 8% (w/v) KOH. After addition of 1 ml of NaCl (25%), the saponified material was extracted twice with hexane, which was then evaporated in speed vacuum. The solid pellet was resuspended in 400 μl of ACN: MeOH: DCM (45:5:50 v/v) and passed through a 0.2 μm Nylon filter before HPLC analyses. Two independent biological samples from each line were pooled for carotenoid analysis.
Statistical analysis. For statistical analysis, experiments were performed independently at least three times using three biological repeats and the results are expressed as mean ± SD. Statistical significance difference was analyzed by Student's t-test using JMP Pro 14 software. A p-value of < 0.05 was used as a cutoff for statistical significance that is indicated with different letters above the bar.