Plant materials
Cuscuta campestris seeds were germinated and seedlings were parasitized onto host plants as described previously57. Nicotiana tabacum plants were grown and parasitized by C. campestris as described previously51. The C. campestris-N. tabacum parasitic complexes were grown at 25°C under a 16/8 h light/dark cycle, and flowers and fruits of the C. campestris were harvested. The harvested tissues were frozen in liquid nitrogen and stored at -80°C. Seeds of Sesamum indicum cv. ‘Masekin’ were germinated and grown in soil (Sukoyakabaido, Yanmar, Osaka, Japan) mixed with the same volume of vermiculite (GS30L, Nittai Co., Ltd., Aichi, Japan) under natural sunlight illumination from May to August in 2019. Mature stems of 8- to 9-week-old S. indicum were parasitized by C. campestris.
Chemicals
(+)-Sesamin (Chromadex), (+)-sesaminol (Nagara Science), (+)-pinoresinol (Sigma-Aldrich), and (+)-piperitol, synthesized previously16, were prepared as the reference samples.
LC-MS analysis
Lignans in extracts of flowers and seeds of Cuscuta plants (C. campestris and C. japonica) were analyzed as follows. A 10 mg sample of lyophilized flowers or seeds was homogenized to a fine powder using a TissueLyser II (Qiagen). Next, 1 ml of 70% acetonitrile aqueous solution was added to the homogenized samples and the samples were extracted using an ultrasonic cleaner at room temperature for 2 min. The filtered extracts were analyzed using an ion-trap time-of-flight mass spectrometer (LCMS-IT-TOF, Shimadzu) equipped with a photodiode array detector (Shimadzu). Each component was separated using a YMC Triart C18 column (TA12S03-1503WT, 150 mm × 3 mm I.D., 3 µm P.S.) with mobile phases A, 0.1% HCO2H-H2O; and B, 0.1% HCO2H-MeOH, in a linear gradient elution with 30-50-90-90-30-30% B (0-10-25-32-32.01-40 min) at a flow rate of 0.3 ml/min.
PCR
Genomic DNA from C. campestris (Cc), C. japonica (Cj) and S. indicum (Si) was prepared from stems using the DNeasy Plant Mini kit (Qiagen) according to the manufacturer’s protocol. Total RNA was prepared using the RNeasy Plant Mini kit (Qiagen) from the following tissue following lysis using a TissueLyser II (Qiagen): Cc seeds, Cc seedlings (at 7 days after germination), Si stem-Cc haustorium junction (parasitic interface tissues), Cc stems, Si stems with and without mechanical wounding with a blade, and Si leaves. The RNA samples were treated with DNase Set (Qiagen) to eliminate contaminating genomic DNA. After DNase I treatment, cDNA was synthesized using an oligo dT primer and the PrimeScript RT reagent kit (TAKARA BIO). Reactions were performed according to the manufactures’ instructions. PCR products were amplified using specific primer sets (listed in Supplemental Data 4) as described previously17,58,59,60. Briefly, genomic PCR, qPCR and RT-PCR was conducted using PrimeSTAR Max DNA Polymerase (TAKARA BIO), GoTaq qPCR Master Mix (Promega) and PrimeSTAR GXL DNA Polymerase (TAKARA BIO), respectively.
RNA extraction for RT-PCR and RNA-seq
Total RNA for RT-PCR was prepared from S. indicum (Si) CYP81Q1 in C. campestris (Cc) stems and total RNA for RNA-seq was prepared from Cc seedlings, Si-Cc haustorium junctions (parasitic interface tissues), Si-Cc Si leaves, Cc stems growing on the Si stem, Si stems with and without mechanical wounding with a blade, and Si leaves. Cc seedlings were harvested 7 days after germination, which was grown under 16h light/ 8h dark cycle for 5 days, blue-light illumination for 1h, under dark condition for 23h, and a 16/8 h light/dark cycle 1 day. Si plants were used 4-6 weeks and 12-16 cm height as the parasitized plants and as the plant samples with and without mechanical wounding. After parasitization or mechanical wounding, plant samples were under blue-light illumination for 1h, under dark conditions for 23h, and a 16/8 h light/dark cycle for 5 days. The samples were harvested 6 days later after parasitization or mechanical wounding. Parasitic interface tissues were harvested from 1.5 cm length include the parasitic interface. Si stems with mechanical wounding were harvested from 1.5 cm length including the cutting point. Si stems without mechanical wounding were harvested from a 1.5 cm length of epicotyl. To collect Si leaves, the topmost portions of leaves that had grown to at least 3 cm were chosen. Cc stems were harvested at 10.5 -13.5 cm length from 1.5 cm near the parasitic interface to the Cc stem tip to avoid cross-contamination with the host. Harvested tissues were washed twice with 70% EtOH for 2 min and rinsed with nuclease-free water for 2 min to clean the surface of the tissues. Total RNA was extracted using the RNeasy Plant Mini kit (Qiagen) after lysis with a TissueLyser II (Qiagen) and then treated using the TURBO DNA-free Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Each RNA sample was derived from a single organism.
Genome and transcriptome
We used open source Cuscuta transcriptome and genome data sets. The RNA-seq data for C. campestris were obtained from the DNA Data Bank of Japan Sequenced Read Archive accession number DRA009453 (https://trace.ddbj.nig.ac.jp/dra/index_e.html/DRA009453)30. The assembled genome sequence and annotations for C. campestris were obtained from the plaBi database (http://plabipd.de/portal/cuscuta-campestris) and for C. australis from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA394036). Cc_CPR1, Cc_CYP81Q110, Cc_CYP81Q111, Cc_CYP81AX6, and Ca_CYP81Q111 correspond to Cc043955, Cc046292, Cc015414, Cc047366, and RAL50776 (responsible for C65N0022E0.1), respectively. The DNA-seq data for C. americana was obtained from SRA experiment ERR3569498 of BioProject PRJEB34450 and for C. californica, from SRA experiment ERR3569499. Synteny analysis was performed using BLASTP to search for best hit protein sequences in the databases indicated in Supplementary Table 3. All translation products of the genes listed in Supplementary Table 3 were used as queries to search all of the databases.
Molecular phylogenetic analysis
The nucleotide sequences of Cuscuta CYP81-related genes used in this study are listed in Supplementary Table 4 and Supplementary Data 1. The phylogenetic trees shown in Fig. 1, Supplementary Fig. 2, and Supplementary Fig. 9 were constructed using a maximum likelihood (ML) method in the Seaview software (phyML ln(L)=-23653.0, 1868 sites, GTR 4 rate classes)61. The timetree shown in Supplementary Fig. 5 was inferred by applying the RelTime method31, 62 to a phylogenetic tree whose branch lengths were calculated using the ML method and the Tamura-Nei substitution model. Evolutionary analyses were conducted in MEGA X software63.
Genome comparisons
The 10 kb genomic sequence containing the CYP81Q-related gene in the S. indicum genome was compared to CYP81Q-related genes in Cuscuta genomes using nucmer in the Mummer -v3.1.0 package64. Structural similarities were visualized with dotplots using the DNAnexus Dot browser (available at https://dnanexus.github.io/dot/) and Unipro UGENE software (http://ugene.unipro.ru/) (Supplementary Fig. 6).
Molecular cloning
Cc_CYP81Q110 (Cc15414), Cc_CYP81Q111 (Cc046292), and Cc_CYP81AX6 (Cc047366) were amplified by reverse transcription-polymerase chain reaction (RT-PCR) from cDNA prepared from a mixture of flower buds and ovary tissue from C. campestris with the specific primers described in Supplementary Table 4. Ca_CYP81Q111 was artificially synthetized without codon-optimization (Eurofins Genomics), based on the sequence of RAL50776. All of the P450 genes were cloned into yeast expression vectors and heterologously co-expressed in yeast with a C. campestris cytochrome P450 reductase, Cc_CPR1 (Cc043955), as described previously29.
Biochemical analysis
Biochemical analysis of Cuscuta Cc_CYP81Q110, Cc_CYP81Q111, Cc_CYP81AX6, and Ca_CYP81Q111 was performed basically as described previously29. Briefly, yeast cells expressing Cuscuta CYP genes were pre-cultured overnight at 30°C with rotary shaking at 120 rpm in 3 ml synthetic defined liquid medium containing a set of amino acids appropriate for the designated expression vectors. Next, 50 µl of stationary phase culture was transferred to 1 ml of fresh medium in 24-well plates supplemented with 100 µM of lignans as substrates. The cultures were further incubated for 24 h at 30°C with rotary shaking at 120 rpm. For extraction, the cells were harvested with the medium and disrupted by sonication. The homogenate (50 µl) was mixed with 50 µl acetonitrile and centrifuged at 21,000×g for 10 min. The supernatant was collected, filtered through a Millex-LH syringe filter (Merck Millipore) and subjected to the following HPLC analysis. Briefly, the filtered assay products were separated using a Cortecs UPLC C18+ column (part# 186007401, 2.7 µm, 3 mm × 75 mm, Waters) with mobile phases (A; 0.1% trifluoroacetic acid-H2O, and B; 0.1% trifluoroacetic acid-acetonitrile) in a linear gradient with 30-80-80-30-30% B (0-1.4-1.8-2.0-2.5 min) at a flow rate of 1.25 ml/min, and lignans were detected using a photodiode array detector at 280 nm.
Preparation and imaging of agarose-embedded sections
Parasitic interface tissues were fixed with 4% (w/v) paraformaldehyde in phosphate buffer solution (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), embedded in 8% (w/v) agarose, and cut using a MicroSlicerTM ZERO 1N (DOSAKA, Kyoto, Japan) into 200-μm sections. Histochemical staining of sections was performed using a 0.5% (w/v) solution of Toluidine Blue O (1B-481, Waldeck GmbH & Co., Munster, Germany) in distilled water. Stained slices were observed using a BX53 Biological Microscope (Olympus, Tokyo, Japan).