Introduction

Horizontal gene transfer (HGT) refers to the transfer of genetic material to non-offspring genomes1,2,3. In prokaryotes, it is a vital source of evolutionary novelty. In eukaryotes, the role of HGT in physiological and ecological adaptations has become increasingly clear with examples now known from protists, fungi, animals and plants3,4,5,6,7,8,9,10,11,12. For instance, a neochrome originating in hornworts was transferred horizontally to ferns, where it may have played a role in the diversification of modern ferns5. Most HGTs in plants involve mitochondrial DNA transferred to nuclear or mitochondrial genomes, while HGTs of nuclear and plastid DNA are rare, with the nuclear transfers mostly involving parasitic plants6,7,8,9,10,11,12. Thus, the root parasite Striga hermonthica (Orobanchaceae) has acquired an unknown protein-coding gene from its host Sorghum (Poaceae), apparently via an RNA intermediate10; the root parasite Rafflesia cantleyi (Rafflesiaceae) has acquired several dozen actively transcribed protein-coding genes from its obligate host Tetrastigma rafflesiae (Vitaceae)6; the root parasites Orobanche spp. and Phelipanche spp. (Orobanchaceae) and the shoot parasite Cuscuta pentagona (Convolvulaceae) have acquired albumin genes from legume hosts11 and Phelipanche aegyptiaca (Pers.) Pomel (formerly Orobanche aegyptiaca Pers.) and Cuscuta australis further acquired strictosidine synthase-like (SSL) genes from Brassicaceae hosts12. The frequency of HGT in parasitic plants is attributed to parasites establishing intimate connections with their hosts through haustoria that enable them to take up water, nutrients and macromolecules including mRNAs13 and sometimes also DNA.

Transposable elements (TEs) constitute the main components of most eukaryotic genomes, occupying 3–20% of fungal, 3–45% of metazoan, 50% of human and 80% or more of plant genomes14. They translocate to new positions in the same genome by either a copy-and-paste mechanism involving an RNA intermediate (class I TEs, retrotransposons) or by a cut-and-paste mechanism (class II TEs, transposons)14. Each class is subdivided into many ‘superfamilies’ based on shared structures, sequence homology and transposition mechanisms15. Class I TEs usually cause genome expansion since they can duplicate repeatedly and they are considered important sources of genome size differences among species16,17. Class II TEs exist in the majority of organisms and are the major form of transposable DNA in prokaryotes18. Their transposition is catalyzed by transposases that recognize short terminal inverted repeats (TIRs), excise the DNA transposon and then ligate it into a new target site. In some cases, TEs may lose their transposition ability and are recruited as functional proteins by a process called TE domestication with or without gain of additional domains18.

Due to their mobility and capacity of transposition, transposable elements (TEs) are also involved in HGT. Horizontal transfers of both classes of TEs have been reported in fungi and animals, from which 199 transferred TEs were collected by the HTT-DB database with 1/3 involving class II transposons19. By searching 40 photosynthetic plant genomes, El Baidouri et al.20 recently detected 32 class I LTR retrotransposon transfer events in 26 species, with the transposons in some cases still functional. Some of these transfer events occurred between distantly related lineages, such as palms (Arecaceae) and grapevine (Vitaceae), tomato (Solanaceae) and bean (Fabaceae), or poplar (Salicaceae) and peach (Rosaceae)20. In contrast, only one horizontal transfer event of a class II transposon was identified in plants, involving two grass species21. Here we show that two novel nuclear-encoded transposon genes of class II were transferred from Brassicaceae into Orobanchaceae and are actively transcribed in both genera of Orobanchaceae, although there is little evidence that their homologs in Brassicaceae are transcribed.

Results

Two hAT transposon genes horizontally acquired by the common ancestor of Orobanche and Phelipanche from Brassicaceae

We developed a pipeline to perform transcriptome screening for foreign genes in P. aegyptiaca12. Using this screening pipeline, we found that two sequences in the P. aegyptiaca transcriptomes exhibited high similarities to the A. thaliana hAT-type DNA transposons: (i) one 1121-bp sequence (OrAeBC4_1751) showed 83% and 75% identities with AT3G17260 at the amino acid (AA) and nucleotide (NT) level, respectively, while being highly divergent from the most similar sequence in the Phrymaceae Mimulus guttatus (the highest identity was 28% at the AA level and there was no significant similarity at the NT level); (ii) another 1656-bp fragment (OrAeBC4_65399) had 85% and 65% identities with AT3G17290 at AA and NT level, while showing ~40% identity with the most similar homolog in M. guttatus at the AA level and no significant similarity at the NT level (Fig. 1). We then searched the partial genome sequences of Orobanche austrohispanica, O. densiflora, O. gracilis and O. rapum-genistae22,23 and found that O. austrohispanica and O. gracilis also had these two genes; therefore, other species in Orobanche and Phelipanche probably have them as well.

Figure 1
figure 1

The protein alignment of the two foreign genes from P. aegyptiaca and the most similar proteins from A. thaliana.

The species names of Orobanche and Phelipanche are indicated after the sequence IDs. Dashes indicate the sequences were incomplete or gaps introduced in the alignment. Background colors indicate the degree of conservation of the sites.

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Genomic PCR amplification in seven Orobanche and three Phelipanche species (including P. aegyptiaca) using primers from the conserved regions resulted in amplification of both genes in these 10 species. We then obtained all sequences of Orobanchaceae and submitted to GenBank (Accession numbers: KM037755-6, KT892680-696, KU187277) and multiple sequence alignments showed that the two genes belong to two distinct types and that some gene copies have short indels, indicating that they have evolved into pseudogenes due to frameshift mutations in their coding regions (Fig. 2; Supplementary Fig. S1). All these data indicate that gene transfer events occurred between the common ancestor of Orobanche/Phelipanche and Brassicaceae. The genes were hereafter named BO (Brassicaceae and Orobanchaceae) to reflect their patchy distribution.

Figure 2
figure 2

The protein tree of all the BOs in Brassicaceae and Orobanchaceae.

Numbers at branches show bootstrap support values for maximum likelihood analysis (before the slash) and posterior probability values for MrBayes analysis (after the slash). Dashes indicate values lower than 70% in the maximum likelihood analysis and 0.90 in the MrBayes analysis. Asterisks after the species names indicate putative pseudogenes.

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To gain insight into the direction of the horizontal transfer and its evolutionary time, we investigated the distribution of the BO transposase genes in the genomes of 18 sequenced species (Supplementary Table S1) that span the core Brassicaceae24,25 (320 genera, 3660 species, 49 tribes) as well as the available genomes of other parasitic flowering plants. In most Brassicaceae genome sequences, the BO genes have not been well annotated. We therefore extended the core regions of the BO gene candidates in Brassicaceae species to 5 kb in both directions and annotated the BO genes manually, using different gene prediction software. ORF Finder and GENSCAN successfully predicted the coding regions of most of the genes. To detect pseudogenized BO genes, we used three additional programs, Augustus, GeneSeqer and Transeq in the EMBOSS package and combined multiple sequence alignment with other well-annotated genes for optimal prediction. Except for three species, all analyzed Brassicaceae possess 2 to 11 BO genes (Supplementary Table S1), with half of them apparently pseudogenized, as determined by premature stop codons or indels causing frame shifts (Fig. 2). That we failed to detect the BO genes in three of the species may be due to the incomplete genome sequence data. The same procedure was used to search the genomes of non-Brassicaceae species in the Phytozome database, but no BO genes were found.

To detect BO genes in other parasitic plants, we used the BO genes from A. thaliana and P. aegyptiaca as queries to BLAST-search relevant transcriptome assemblies, including the assemblies of the Convolvulaceae Cuscuta australis and C. pentagona, the assemblies of the Orobanchaceae Striga hermonthica (StHeBC2), Triphysaria versicolor (TrPuRnBC1) and Triphysaria pusilla (TrVeBC2) in PPGP, the Lauraceae Cassytha filiformis and the Apodanthaceae Pilostyles thurberi in the 1KP project (http://onekp.com/project.html). This yielded only sequences with low identities and subsequent phylogenetic analysis indicated that none of them clustered with the BO genes.

A phylogeny of the protein sequences encoded by the BO transposase genes and proteins from other angiosperms whose genomes have been sequenced is shown in Supplementary Fig. S2. The BO proteins and their relatives form two clusters. Cluster I includes the BOs from Brassicaceae and Orobanchaceae and several sequences from peach (Prunus, Rosaceae). Cluster II contains the remaining angiosperms, the A. thaliana DAYSLEEPER gene26,27 and other DAYSLEEPER-like genes in three statistically well-supported groups (Supplementary Fig. S2). Within Cluster I, there are two groups, each containing at least one gene from the Brassicaceae and Orobanchaceae species. A highest posterior probability tree and a maximum likelihood tree obtained from all BO genes of 11 Orobanchaceae and 14 Brassicaceae (the BO genes in Thellungiella parvula were excluded because of their 100% identity with the homologs from T. halophila) both show distinct BO1 and BO2 clades (Fig. 2). The BO1 genes in Brassicaceae have more copies than do the BO2 genes and the BO2 genes further form two clusters, BO2a and BO2b, each containing genes from most Brassicaceae species, suggesting ancient divergence. Notably, each BO gene clustered with homologs from Sisymbrium irio (Fig. 2). This suggests that the two BO genes may have been laterally transferred into the common ancestor of the Orobanche/Phelipanche clade from Sisymbrium, a small Old World genus that is the sole member of the tribe Sisymbrieae24, or from a closely related species.

The BO transposase genes constitute a new member of the hAT transposon superfamily

To gain further insight into the function and evolution of the BO transposase genes, we searched for their domains using InterProScan. Three domains were found: a hAT dimerization, a hAT-like transposase and a ribonuclease H-like domain. The hAT dimerization domain is ~80 AA long and located in the C-terminus; the hAT-like transposase domain has ~100 AA and is located in the medial region; the ribonuclease H-like domain is ~500 AA long and covers the above two domains (Fig. 3; Supplementary Fig. S3). The conserved hAT dimerization domain is a hallmark of the hAT transposons and has been shown to be involved in dimerization as well as additional interaction functions28. The ribonuclease H-like domain is constituted of a three-layer alpha/beta/alpha fold and exists in some transposases, ribonucleases, exonucleases and retroviral integrases29. Notably, the ribonuclease H-like domain in the BOs contains an insertion of 12-71 AA that lies between the hAT-like transposase domain and the hAT dimerization domain (Fig. 3; Supplementary Fig. S3). These results indicate that the BOs belong to a new class of the hAT superfamily.

Figure 3
figure 3

Domain structure comparison of a classic hAT family transposon with the BO transposase gene family in Brassicaceae, Orobanche and Phelipanche.

The ribonuclease H-like domain, the hAT-like transposase domain and the hAT dimerization domain are in purple, orange and red, respectively. The insertion position in the ribonuclease H-like domain of the BOs is displayed as a rectangle.

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Given that the BOs are closely related to the A. thaliana DAYSLEEPER protein (Supplementary Fig. S2), which contains an additional C2H2 type BED-zinc finger domain in the N terminus ahead of the three classic domains, we searched for this domain in other sequences of cluster II and found that most proteins possess this additional domain. Knip et al.30 also identified these genes as DAYSLEEPER-like or SLEEPER. The hAT family members have been categorized as Ac, Buster and BuT2 and the DAYSLEEPER protein together with the Ac and TAM3 belongs to the Ac group. To investigate how the BOs are related to these Ac group members, we first used the two BOs in A. thaliana as representatives to compare with the typical Ac of maize and TAM3 of snapdragon. They showed 33% and 37% sequence identities with Ac of maize (GenBank accession number ACG45782) and 32% and 20% sequence identities with TAM3 of snapdragon (GenBank accession number CAA38906) at AA level. Sequence alignment also showed that the BOs are less similar to Ac and TAM3 than to DAYSLEEPER-like proteins. This supports that the BOs in Orobanche, Phelipanche and Brassicaceae are a new hAT superfamily member, closely related to the DAYSLEEPER-like genes.

BO genes in P. aegyptiaca and O. cumana are actively transcribed

The presence of the two BO genes in the RNA-seq data of P. aegyptiaca indicates that they are actively transcribed. To determine how the expression level changes in different tissues at different developmental stages, we mapped all clean paired-end reads to our own Trinity assembly of the P. aegyptiaca transcriptome. The FPKM values of the BO1 gene ranged from 2.1 to17.4 and those of the BO2 genes were from 6.0 to 26.5. Figure 4a shows the relative expression levels of BO1 and BO2 with actin as the reference gene. Except in flowers, the BO2 gene had higher expression levels than the BO1 gene in all tissues31 (0G, seeds imbibed, pre-germination; 2G, seedling after exposure to haustorial induction factors; 3G, haustoria attached to host root, early penetration stages, pre-vascular connection; 4.1G, early established parasite, parasite vegetative growth after vascular connection; 4.2G, spider stage; 5.1G, pre-emerged leaves and stems; 5.2G, pre-emerged roots; 6.1G, post emergence from soil - vegetative structures, leaves/stems; 6.2G, post emergence from soil - reproductive structures, floral buds) and a comparison among different development stages showed that both BO1 and BO2 exhibited greater expression levels in the 3G and 4.1G stages than in the other stages.

Figure 4
figure 4

Expression levels of the two BO transposase genes in P. aegyptiaca.

(a) Expression levels at different developmental stages (PPGP) estimated from RNA-seq data with ACTIN as the reference gene. (b) Expression levels (±SE, n = 3) of the two BO genes in stems and flowers assessed by qRT-PCR.

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We further assessed the transcription levels of the BO transposase genes in flowers and stems of P. aegyptiaca by quantitative real time-PCR (qRT-PCR), which provides accurate assessments of gene expression. In flowers and stems, the expression levels of the two BO genes were similar to those of the reference gene actin (Ctactin was 24.2 ± 0.09 and 25.2 ± 0.32, CtBO1 was 23.6 ± 0.19 and 24.3 ± 0.04 and CtBO2 was 25.0 ± 0.37 and 24.5 ± 0.28 in flowers and stems, respectively). The BO1 gene presented similar expression levels in stems and flowers, while the expression level of the BO2 gene in stems was more than 1-fold higher than in flowers (Fig. 4b). To determine whether the BO transposase genes are expressed in O. cumana, we performed RT-PCR using the stem and flower tissues with the same primer pair as used in qRT-PCR. It produced a band with the expected size and the identity of which was confirmed by sequencing.

These transcriptional data indicate that both BO genes have relatively high transcript levels in Phelipanche and Orobanche and that different pathways regulate their transcription.

The BO genes in Brassicaceae show low transcriptional activity

The transcriptional levels of the BO genes in Brassicaceae were investigated by searching the microarray dataset of A. thaliana in Genevestigator and the NCBI EST and SRA databases. The Genevestigator analysis indicated that the BOs in A. thaliana are transcribed at low levels in most tissues except that the BO1 gene (AT3G17260) is expressed at medium level in sperm cells (Supplementary Fig. S4). No ESTs corresponding to the two BO genes of A. thaliana were found in the Phytozome database. We further searched multiple RNA-seq datasets of A. thaliana (ecotype Col-0) in the SRA database but found no transcription evidence of the two BO genes, except a BO2 RPKM value of 0.48 in one dataset32. Data-mining in the Phytozome revealed that two BO1 genes in Capsella rubella (Caprub1 and Caprub2), one BO1 gene (Thehal1) and two BO2 genes in Thellungiella halophila (Thehal3 and Thehal4) and two BO1 genes in Brassica rapa (Brarap1 and Brarap2) have very low expression levels. No expression data were available for S. irio and searching the SRA database indicated that except for two BO1 genes in Brassica napus (Branap6 and Branap7) and all four BO genes in Raphanus raphanistrum (all these genes’ RPKM values were <0.5), other genes had no mapped reads in Brassicaceae. The low transcriptional status, together with pseudogenization in half of the BO genes, suggest that the BO genes in Brassicaceae are generally not actively transcribed and unlikely to be able to transpose.

The BO transposase genes and their flanking regions show synteny in Brassicaceae species

We also investigated the chromosomal location of the BO transposase genes and the synteny of their flanking regions in the 15 available Brassicaceae genomes. Most BO genes were arranged in tandem or very close to each other (Fig. 5). Detailed inspection indicated that the ORF directions are the same for all but one BO2 gene in B. oleracea (Fig. 5). As for the synteny of genes flanking the BO genes, we used six conserved genes with transcript activity in the flanking regions of BO1-BO2 in A. thaliana to search against the other 14 Brassicaceae genomes. Of the six genes, two are located in the upstream flanking region of the BO1-BO2 structure; the other four genes are located in the downstream flanking region of the BO1-BO2 structure. This indicates that most flanking genes retained synteny around the BO genes except for a few losses (Fig. 5), such as the two-gene loss in the downstream flanking region of the BO gene in S. irio.

Figure 5
figure 5

The chromosomal location and synteny of Brassicaceae genes encoding the BO family of hAT transposons.

Intergenic regions are indicated above the dash lines. Red rhombuses on the upstream and red rectangles on the downstream flanking regions of the BO genes with letters or numbers indicate the following conserved genes: a, AT3G17205; b, AT3G17240; 1, AT3G17300; 2, AT3G17310; 3, AT3G17320; 4, AT3G17340.

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To further investigate whether the BO genes in Orobanchaceae and Brassicaceae can transpose, we inspected their flanking regions. Active hAT genes have short flanking regions that are converted to the donor sequence upon insertion (target site duplication, TSD) and short terminal inverted repeats (TIRs) (TSD-TIR-transposase-TIR-TSD). The length of the TSD structure is reported to be 8 bp for the hAT superfamily genes33. We failed to obtain the flanking regions of the BO genes in Orobanchaceae and therefore focused on the Brassicaceae BO genes. Transpolator was used to identify the 8-bp TSD and TIR structures in the flanking regions of the Brassicaceae BO genes. Different TSD-TIR structures were found but few had 8 bp-long TSDs, nor could their consensus sequence be obtained, including the “(T/C)A(A/G)NG” consensus sequence proposed by Rubin et al.33. The collinearity of the flanking genes and the absence of the typical TSD-TIR structures suggest that the BO genes in Brassicaceae did not have a history of transposition.

Discussion

The hAT superfamily (named after the families: hobo from Drosophila melanogaster, Activator (Ac) from maize and Tam3 from snapdragon) of class II TEs is widely distributed in fungi, animals and plants34 and phylogenetic analyses suggest that it is ancient, predating the early stages of the divergence of these kingdoms33. In animals, hAT has been horizontally transferred among species of Drosophila35,36,37 and a novel hAT transposon having the 8-bp TSDs with the characteristic of recent transposition has been found in the silkworm, Bombyx mori and in a Triatominae vector of the Chagas parasite, Rhodnius prolixus38. Prior to this study, no transfer events involving the transposase genes of the hAT superfamily had been reported in plants. By HGT screening in P. aegyptiaca transcriptomic data, we discovered two BO genes with high similarities to their homologs in Brassicaceae. Because parasitic plants can take up mRNAs from their hosts39, foreign mRNAs in P. aegyptiaca might be a contamination from Brassicaceae hosts, since P. aegyptiaca sometimes parasitizes Arabidopsis. To exclude this possibility, we carried out BLAST searches against the draft genomes of four Orobanche species and did genomic PCRs in seven Orobanche and three Phelipanche species and this revealed two BO genes in the 11 species. Subsequent multiple sequence alignments clearly showed that the BO genes form two clusters, with each present in all tested Orobanche and Phelipanche (Supplementary Fig. S1).

By gene annotation and homology search, we found that the BO genes are absent from bacteria, viruses and archaea and are unique to Brassicaceae and the Orobanchaceae genera Orobanche and Phelipanche. Based on phylogenetic analysis of species representing the core Brassicaceae25,40, it appears that the two BO genes in Orobanche and Phelipanche are likely to be derived from Sisymbrium, a small Old World genus that is the sole member of Sisymbrieae24,25, or certain closely related species. Future genome sequences of species from the tribes Thelypodieae (26 genera, 224 species) and Isatideae (5 genera, 90 species), which are the closest relatives of Sisymbrieae, will further clarify the origin of the BO genes in Orobanchaceae. It is unlikely that the BO genes were instead transferred from Orobanchaceae to Brassicaceae, given that the BO transcripts were not found in Striga hermonthica (Orobanchaceae) and that the BOs spread from Sisymbrieae (or a close relative) to all the other core Brassicaceae species is implausible.

A scenario explaining the two foreign BO genes in Orobanche and Phelipanche is given in Fig. 6. It assumes a single gene transfer event involving the BO1-BO2 DNA fragment from an ancestor, such as S. irio, to the common ancestor of minimally Orobanche and Phelipanche (our sampling of eight Orobanche and three Phelipanche is insufficient to exclude an earlier uptake). Orobanchaceae are a mostly temperate zone family of ca. 2000 species in 89 to 99 genera and the earliest lineages of Orobanchaceae are not parasitic41. Attempts to confirm whether the BO1 and BO2 genes in Orobanchaceae are arranged in tandem failed, suggesting either long intervals between the BO1 and BO2 genes or that they are located on different chromosomes. Given the possibility of genome structure rearrangement in the recipients, different chromosomal locations or a distant placement on the same chromosome do not rule out an initial simultaneous acquisition.

Figure 6
figure 6

Scenario for the evolutionary history of the BO transposase genes in Brassicaceae and their transfer from an S. irio-like plant to Orobanche and Phelipanche.

The genes BO1, BO2a and BO2b are indicated as blocks in different colors. The a and b in tribe Camelineae indicate the gene duplication and subsequent divergence of the BO1 gene. The lineage specific gene duplication of the BO1 gene in the tribe Eutremeae and the tribes Brassiceae and Sisymbrieae is shown in the same color. The two small unsampled tribes closest to Sisymbrieae are discussed in the text.

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The gene arrangement BO1-BO2a-BO2b likely was present in the ancestor of core Brassicaceae, judging from the analyzed genomes, which represent eight of their 49 tribes of the family and span the root of core Brassicaceae25,40. The BO2b gene was lost in the Cardamineae; gene duplication and divergence of BO1 occurred in the Camelineae; and gene loss in A. thaliana, A. halleri and Capsella, with further gene duplication in C. sativa; lineage-specific gene duplication of the BO1 gene also occurred before the split of Raphanus and Brassica.

The HGT event of BO genes from Brassicaceae to Orobanche and Phelipanche must have occurred before the divergence of Orobanche and Phelipanche. Based on molecular-clock dating, Orobanche and Phelipanche diverged from each other 38 million years ago (Ma), with a 95% confidence interval of 16–45 Ma23. In our BO gene phylogeny, Orobanchaceae genes cluster with Sisymbrium with a stem age of 18 to 25 million years40 (Fig. 2; Supplementary Fig. S2). The related tribes Thelypodieae and Isatideae, from whose species genome information is not yet available and who could also have been the donor of the BO genes, shared a common ancestor with Sisymbrieae around 32 to 33 Ma40. The HGT might thus have happened between 16 and 33 Ma.

Parasite/host plant systems, which all involve distantly related species, are prone to HGT, because of the tight physical connections in the haustoria, which in the case of endoparasites, such as Rafflesia and Pilostyles, involve a network of parenchyma cells living inside the host42,43. DNA fragments are therefore transported between parasites and hosts and occasionally become integrated into parasite genomes (Introduction), with end-joining, homologous recombination and virus-mediated integration as possible mechanisms1,3,4. In line with this, the horizontal transfer of TEs has been documented20,21, but so far TE-mediated integration of laterally transferred genes has not be found. Such transfer probably is facilitated by the transposons’ ability to incorporate into a recipient genome. This could have involved translation in the recipient cells of transferred BO genes contained in a DNA fragment. In plants, DNA transferred to stem tissue can be passed to the next generation because floral meristems can develop in leaf axils.

The BO genes in Brassicaceae likely lost their transposition ability just after their origin as evidenced by: 1) their flanking regions still being syntenic in almost all the investigated Brassicaceae species, indicating that they did not transpose at all; 2) their lack of the classic TSD border structure required for the recognition of transposases; 3) their closest relatives being the SLEEPER genes (Fig. S2), which are domesticated hAT transposons that are involved in the normal plant growth of both A. thaliana26,27 and rice30; 4) both BO genes in A. thaliana being considered domesticated44; and lastly by 5) many brassicaceous BO genes being pseudogenes (Fig. 2) and all BOs having very little transcriptional activity. Thus, the transfer of these BO genes from Brassicaceae to Orobanchaceae mostly likely did not involve any TE transposition, but instead another mechanism. The relatively high transcript levels of the BO genes in Orobanchaceae could either result from a location in the Orobanchaceae genomes, where transcription is especially active, or from them having gained new functions.

Methods

Data sources

All transcriptome assemblies of species of Phelipanche (the sole available Phelipanche species in the Parasitic Plant Genome Project (PPGP) is P. aegyptiaca, erroneously called Orobanche aegyptiaca), Striga and Triphysaria and the pair-end RNA-seq data from P. aegyptiaca were retrieved from the PPGP website (http://ppgp.huck.psu.edu). The protein database used by AlienG45 for BLAST searches was the NCBI non-redundant (nr) database (Sep., 2012) and included all predicted proteins from 22 plant genomes available in the Phytozome database version 9.046. The nucleotide database used was the NCBI nucleotide (nt) collection with all predicted transcripts of 22 plant genomes from Phytozome. The databases in the 1 KP project (http://www.onekp.com/project.html), including the transcriptome assemblies from Cuscuta pentagona Englm., Cassytha filiformis L. and Pilostyles thurberi A. Gray), PlantGDB (http://www.plantgdb.org) and SOL Genomics Network (http://solgenomics.net), were searched online. Our own RNAseq assembly of Cuscuta australis R.Br.12 was searched locally. The genomic sources of the 18 Brassicaceae species are provided in Supplementary Table S1.

Transcriptome screening for foreign genes in Phelipanche aegyptiaca

Transcriptome screening for the transferred genes was performed according to the procedure described previously12. Briefly, the contamination from the host transcripts was first excluded by only choosing the genes expressed in seedlings not yet attached to the host; second, the coding regions were predicted and only sequences with a contiguous amino acid length >100 AA were kept for AlienG45 analysis; finally, after filtering out those with highest similarities to the genes in Mimulus guttatus at the nucleotide acid level, the candidates predicted by AlienG with score ratios of the first non-Lamiales hit to the first Lamiales hit >1.2 were retained for manual identification of horizontally transferred genes.

Identification of the foreign hAT transposon in Orobanchaceae and the closest homologs in Brassicaceae and other plants

The foreign genes detected in P. aegyptiaca by searching the PPGP data were two partial gene fragments. To obtain longer sequences, we downloaded the paired-end data from PPGP website and reassembled them using the Trinity software47 following the standard procedure. We also searched (BLASTn and tBLASTn) the partial draft genomes from other species of Orobanche, O. densiflora Salz. ex Reut. O. austrohispanica M.J.Y.Foley, O. rapum-genistae Thuill. and O. gracilis Sm.23. Hits with identity values >30% were extracted and distantly divergent sequences in the phylogeny tree were removed from further analysis. The closest homologs in the genomes of 15 Brassicaceae species were originally found by obtaining the sequence segments using the two BO transposase genes of A. thaliana (AT3G17260 and AT3G17290) as queries, which were extended in both directions to 5 kb for analysis in the next step.

Four types of software were used to identify the genes, including ORF Finder (NCBI)48, GENSCAN49 (http://genes.mit.edu/GENSCAN.html), Augustus50 (http://bioinf.uni-greifswald.de/augustus/submission), Transeq (http://www.ebi.ac.uk/Tools/st/emboss_transeq/) and GeneSeqer51 (http://www.plantgdb.org/cgi-bin/GeneSeqer/index.cgi), with Arabidopsis amino acid sequences as references when necessary. The pseudogenes were determined by the premature stop codons or indels causing coding frame shifts. The same procedure was used to identify the closest homologs with the two A. thaliana BO transposase genes in other genomes of relevant species.

Phylogenetic analyses of the new hAT transposases

The protein homologs are extracted from representative species genomes in the Phytozome database and our own assembly of P. aegyptiaca by BLAST searching using the two BO transposase genes of A. thaliana as queries with the score value threshold set to 40. The sequences were aligned with ClustalX v2.152, visually inspected and manually refined. Gaps and ambiguously sites were removed from the alignment. ModelGenerator (v_851) was used to find the best-fitting model of protein substitution53. Phylogenetic trees were inferred under maximum likelihood optimization using PHYML v3.054 and under Bayesian optimization using MrBayes v3.2.555. Markov chain Monte Carlo chains were run with the default four chains and using two million generations, sampling trees every 200 generations. After discarding the first 5,000 trees (that is, half the trees) as burn-in, a consensus tree with the posterior probability support values for all clades was calculated from the remaining trees. Trees were viewed and edited using NJplot56.

Expression level estimation of hAT-like genes in Phelipanche aegyptiaca, Arabidopsis thaliana and other Brassicaceae

The expression levels of the two foreign hAT transposons in P. aegyptiaca, BO1 and BO2, in different tissues and at different developmental stages were estimated using RSEM57 with actin as the reference gene. We mapped all clean reads to the Trinity assembly and to obtain normalized expression levels we used the fragments per kilo base of exon per million fragments mapped (FPKM) values of the sequences. The actin gene was found with nine transcript isoforms, of which two contained the primer sequences Actin-F/Actin-R (below) and the sum of their FPKM values was used as the reference. Web-based expression analysis of the two BO transposase genes in A. thaliana (AT3G17260 and AT3G17290) in different tissues, different development stages and under various treatments was performed using GENEVESTIGATOR v3 (https://www.genevestigator.com/gv/)58. Estimation of RNAseq-based transcriptional levels was also performed in other Brassicaceae by blast searches against randomly selected SRA datasets.

Genomic PCR amplification of the two BO transposase genes in seven Orobanche and three Phelipanche species

Seeds of P. aegyptiaca and Orobanche cumana Wallr. were inoculated near the roots of 50 days-old tobacco for establishing parasite infection. Two-month-old P. aegyptiaca and O. cumana were sampled and stored at −80 °C. Samples of the following Orobanchaceae, O. amethystea Thuill., O. crenata Forssk., O. salvia F.W.Schultz, O. hederae Duby, O. lucorum A.Braun ex F.W.Schultz, O. gracilis Sm., Phelipanche purpurea (Jacq.) Soják and P. ramosa (L.) Pomel were collected in the Munich Botanical Garden. Total DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method. For genomic PCR, all BO1 genes were obtained with OhAT-AF2/OhAT-AR primer pair, while the BO2 genes were amplified by using multiple primer pairs depending on species (Supplementary Table S2). Two primer pairs B2F/B2R and C3F/C3R, were used to get two overlapping regions of the BO2 in O. cumana. PCR primer sequences and the GenBank accession numbers of the BO genes in the 10 Orobanchaceae species with the herbarium voucher information are indicated in Supplementary Table S2. These primers were designed according to the multiple sequence alignment of the BO genes extracted from the transcriptome and draft genomic data above. Amplification products with expected sizes were sequenced directly or after cloning.

qRT-PCR of the BO genes in Phelipanche aegyptiaca and RT-PCR in Orobanche cumana

Fresh tissues of stems and flowers were collected from P. aegyptiaca. Total RNA from each sample was extracted with RNAiso Plus (TaKaRa) following the manufacturer’s instructions. RNA concentrations were quantified and 500 ng of each RNA sample was reverse-transcribed using oligo_(dT) 18 and RevertAidTM H Minus Reverse Transcriptase (Fermentas) in a total volume of 10 μL. The obtained cDNA samples were diluted to 25 μL. Specific primer pairs, P.aeg-bo1F/P.aeg-bo1R (5′-AGAACCAAGTCTGTGATGGAACC-3′/5′-TCATTTGTAACACCCGAGCCTAT-3′) for the BO1 gene and bo2F/bo2R (5′-AGTGCCGATACTGATTACTTCCG-3′/5′-GAGGGTTCGTCACAGACTTGGTT-3′) for the BO2 gene in P. aegyptiaca were designed according to their DNA sequences. Actin was again selected as the reference gene for normalizing cDNA concentration variations59 and the primer pair Actin-F/Actin-R (5′-CGTGAGAAGATGACGCAGATT-3′/5′-GAACAGCCTGGATAGCAACATAC-3′) was designed accordingly. Quantitative real time-PCR (qRT-PCR) was carried out on an CFX ConnectTM Real-Time System (BIO-RAD) using iTaqTM Universal SYBR Green Supermix (BIO-RAD) following the manufacturer’s instructions. The expression levels were calculated using the comparative CT method. Three biological replicates were run for each gene in stems and flowers. Reverse transcription PCR (RT-PCR) was performed to check the expression of the BO transposase genes in O. cumana with the primer pair Actin-F/Actin-R.

TSD-TIR structure search and synteny analysis of genes flanking the BO transposase genes in Brassicaceae

To detect the TSD-TIR-transposase-TIR-TSD structure, genomic sequences covering 5000 bp in each direction of the BO genes in Brassicaceae species or covering the intergenic segments between two tandem BO genes were retrieved. Within these regions, TIRs with 8–23 bp flanked by 8 bp TSDs were searched by using an updated version of the program Transpolator33, with a single imperfect nucleotide in the first base of TIRs allowed and TIRs composed of only two types of nucleotides not considered so as to avoid simple repeats. For the colinearity analysis, six A. thaliana genes flanking the two BO genes with transcriptional activity were selected to search their most similar homologs in other Brassicaceae species.

Additional Information

Accession codes: Sequence data for the Orobancheceae PCR amplification genes have been deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/) under accession number KM037755, KM037756, KT892680-698 and KU187277.

How to cite this article: Sun, T. et al. Two hAT transposon genes were transferred from Brassicaceae to broomrapes and are actively expressed in some recipients. Sci. Rep. 6, 30192; doi: 10.1038/srep30192 (2016).