ABA signaling components in Phelipanche aegyptiaca

Obligate root holoparasite Phelipanche aegyptiaca is an agricultural pest, which infests its hosts and feeds on the sap, subsequently damaging crop yield and quality. Its notoriously viable seed bank may serve as an ideal pest control target. The phytohormone abscisic acid (ABA) was shown to regulate P. aegyptiaca seed dormancy following strigolactones germination stimulus. Transcription analysis of signaling components revealed five ABA receptors and two co-receptors (PP2C). Transcription of lower ABA-affinity subfamily III receptors was absent in all tested stages of P. aegyptiaca development and parasitism stages. P. aegyptiaca ABA receptors interacted with the PP2Cs, and inhibited their activity in an ABA-dependent manner. Moreover, sequence analysis revealed multiple alleles in two P. aegyptiaca ABA receptors, with many non-synonymous mutations. Functional analysis of selected receptor alleles identified a variant with substantially decreased inhibitory effect of PP2Cs activity in-vitro. These results provide evidence that P. aegyptiaca is capable of biochemically perceiving ABA. In light of the possible involvement of ABA in parasitic activities, the discovery of active ABA receptors and PP2Cs could provide a new biochemical target for the agricultural management of P. aegyptiaca. Furthermore, the potential genetic loss of subfamily III receptors in this species, could position P. aegyptiaca as a valuable model in the ABA perception research field.

www.nature.com/scientificreports www.nature.com/scientificreports/ leaves, have not been identified in obligate holoparasitic plants, such as P. aegyptiaca that rely completely on their host for continuous supply of water and nutrients. The reduction in ABA-related functions in holoparasitic plants might correspond with some degree of degeneration in the ABA signal transduction pathway. This hypothesis is strengthened by the recent identification and classification of ABA receptors and co-receptors in the hemiparasitic Orobanchaceae species Striga hermonthica, which transcribes four ABA co-receptors, including one which is mutated in such way that it effectively blocks ABA signaling 9 .
In this study, we explored ABA perception in P. aegyptiaca and provide early insights into genetic variance and its functionality in a wild species. Alongside the potential evolutionary implications of such discoveries, the insights may also illuminate new approaches for agrotechnical control of this pest.

Results
P. aegyptiaca transcribes core ABA signaling components. The basis of the biochemical response to ABA is facilitated by an interaction between ABA and its receptor, followed by the ABA-receptor inhibitory effect on a co-receptor (PP2CA). Identification of these components in P. aegyptiaca was based on sequence homology with Arabidopsis ABA receptor PYR1 and ABA co-receptor ABI1. In the absence of a publically available sequenced genome, the Parasitic Plant Genome Project (PPGP) EST database is currently the most extensive source of information about P. aegyptiaca genetics. The database contains cDNA sequences obtained from P. aegyptiaca and two other parasitic plant species at specific developmental stages and from different tissues 16 .
In-silico analysis of P. aegyptiaca transcript data revealed five putative ABA receptors (PaPYL4-8) and five putative ABA PP2C co-receptors (Figs 1 and S1). Regions predicted to be key to receptor functionality 13,17 in the amino acid sequences of the putative ABA receptors were found to be highly similar to those of the Arabidopsis ABA receptors (Fig. 1). One exception was PaPYL5, which varied from both Arabidopsis and the rest of the P. aegyptiaca receptors in a highly conserved region, which includes the "latch" loop (PYR1 H 115 , R 116 and L 117 ) (Fig. 1). The latch is one of two surface loops that bind ABA and co-receptors. A change in this region may therefore affect ABA receptor function.
The individual transcription pattern of the putative ABA receptors and co-receptors was analyzed using the publically available PPGP transcriptome data, which are categorized by developmental stage and tissue type (summarized on Fig. 2). Results showed that at least two ABA receptors are transcribed at any given stage of P. aegyptiaca life cycle, which can be an indication of active ABA perception. PaPYL6 and the ABI-like 2 co-receptor (PaABIL2) were only transcribed during seed germination and early established parasite stage. PaABIL2 was also transcribed during "post-emergence from soil" stage.

None of the transcribed P. aegyptiaca ABA receptors classify as a subfamily III ABA receptor.
In-silico phylogenetic analysis clustered PaPYL4-6 with subfamily II of A. thaliana ABA receptors, and PaPYL7 and 8 with subfamily I (Fig. 3). None of the putative P. aegyptiaca ABA receptors clustered with subfamily III, an unusual finding as compared to ABA receptor expression analyses in other higher plants 9,18-23 . Thus we decided to number the receptors in accordance to Arabidopsis subfamily clustering. Subfamily II receptors were named PaPYL4, 5 and 6 and Subfamily I receptors were named PaPYL7 and PaPYL8.
A functional analysis was then performed to confirm the computational phylogenetic classification of putative P. aegyptiaca ABA receptors into subfamilies I and II. To this end, the five receptors were cloned from plant samples collected in Israel. The cloned receptors were highly similar to the PPGP database sequences, with the exception of PaPYL5. The latch loop of this variant, unlike its PPGP counterpart, was found to be conserved as compared to other functional receptors. This version was named PaPYL5 JV after the source of the sample -Jezreel Valley.
The interactions between ABA receptors and A. thaliana ABA co-receptors ABI1 and its mutant ABI1 G180D (encoded by abi1-1) in a yeast two-hybrid assay, can be used as an indication of a subfamily affiliation 22 . PaPYL4 and PaPYL5 interacted with ABI1 in an ABA-independent manner, while the interaction with ABI1 G180D was ABA-dependent ( Fig. 4), which coincided with the characteristics of the A. thaliana ABA receptor subfamily II. PaPYL6-8 interacted with both ABI1 and ABI1 G180D in an ABA-independent manner, in accordance with the characteristics of the A. thaliana ABA receptor subfamily I.
In order to determine whether the absence of subfamily III transcription is a common feature of parasitic plants, sequences encoding putative ABA receptors of the following species were analyzed in-silico: obligate root  Figure 1. High sequence similarity between the ABA receptors of P. aegyptiaca and of A. thaliana in regions key to functionality. Alignment of residues which interact with ABA (black asterisks) or HAB1 (red asterisks), according to the crystal structures of PYL2-ABA 13 and PYR1-HAB1 17 . Amino acid sequences are color-coded according to side chain characteristics. PaPYL5 JV is the variant which was amplified from a sample obtained in the Jezreel Valley (Israel).
www.nature.com/scientificreports www.nature.com/scientificreports/ hemiparasite Striga hermonthica 9 , facultative root hemiparasite Triphysaria versicolor (Orobanchaceae, EST libraries available in the PPGP website) and obligate stem holoparasites Cuscuta pentagona and Cuscuta suaveolens 24 (EST libraries available in the GenBank TSA database). In all tested species, unlike in P. aegyptiaca, at least one putative ABA receptor clustered with the subfamily III ABA receptor family (Fig. 3). P. aegyptiaca ABA receptors interact with P. aegyptiaca ABA co-receptors and inhibit their activity in an ABA-dependent manner. Of the five putative ABA co-receptors identified in the in-silico analysis of the P. aegyptiaca transcriptome, only two were shown to interact with P. aegyptiaca ABA receptors in the yeast two-hybrid assay (Fig. 5). Furthermore, only these two co-receptors interacted with Arabidopsis SnRK (Fig. S1). The interaction between P. aegyptiaca ABI like 1 (PaABIL1) and PaPYL4-8 was ABA-independent. PaABIL2 only interacted with PaPYL6. The other three putative P. aegyptiaca clade A subfamily of type II C protein phosphatases like 1-3 (PaPP2CAL1-3) ABA co-receptors did not interact with any of the receptors. A receptor-mediated phosphatase activity assay performed to further investigate the interaction between recombinant PaABIL1 and PaPYL4-8 showed that PaPYL4, PaPYL5, PaPYL7 and PaPYL8 inhibited the de-phosphorylation activity of PaABIL1 in an ABA dose-dependent manner (Fig. 5). The young parasite continues to develop underground until it is ready to reproduce; then, the flowering shoots emerge from the surface. (6) Each flower can produce around 500 seeds by cross-pollination, selfpollination and apomixis. In table: orange boxes represent highly similar (>95% nucleotide identity in pairwise sequence alignment) matches in the Parasitic Plants Genome Project database. To eliminate the possibility of the matches that were the product of a contaminated genetic material (host tissues), the uniqueness of each match was verified by basic local alignment search (BLAST) in the NCBI database.
www.nature.com/scientificreports www.nature.com/scientificreports/ Allelic variations in PaPYL4 affect its interaction with co-receptors. As part of the characterization of P. aegyptiaca ABA receptors, multiple genes encoding PaPYL4-5 originating from the Jezreel Valley (32° 35′ 47″N, 35°14′31″E region) population, were cloned and sequenced. In-silico analysis revealed that PaPYL4 and PaPYL5 presented both synonymous and non-synonymous mutations. Amongst PaPYL4 clones, 35 different alleles were discovered, 6 of which had nucleotide insertions or deletions resulting in a frame-shift. PaPYL5 clones included 12 different alleles, one with a frame-shift and three with nonsense mutations. Assessment of the interaction between the 11 PaPYL4 alleles with complete open reading frames and ABA co-receptors (PaABIL1, PaABIL2, HAB1, ABI1, ABI1 G180D , ABI2 and ABI2 G168D ) in a yeast two-hybrid assay (Fig. S2), showed that the allelic variation did not affect this interaction, regardless of ABA concentration. However, variant PaPYL4.2 showed a substantially decreased interaction with all the ABA co-receptors, manifested by higher ABA concentration requirements, and failure to interact with the mutated co-receptors at any tested concentration of ABA. In comparison to the normally interacting receptor, PaPYL4.1, PaPYL4.2 displayed no in-vitro PP2C inhibition activity, even in the presence of 5 µM of ABA (Fig. S3).

Discussion
This work presented evidence of the capacity of an obligate holoparasitic plant, Phelipanche aegyptiaca, to biochemically perceive ABA signaling, by the apex components of the ABA signal transduction pathway. Through characterization of the plant's ABA receptors and co-receptors and comparison with homologous autotrophic angiosperm genes, we propose a possible deterioration of the P. aegyptiaca ABA perception mechanism. This might be the result of the evolutionary transition of this species from self-dependence to parasitism, in which loss of redundancies in once critical traits can occur without decreasing fitness.
The first and most prominent element hinting to a reduction in P. aegyptiaca ABA perception, was the absence of subfamily III ABA receptor transcription. This was unique as compared to other species of higher plants, which consistently expressed receptors of all three ABA receptor subfamilies 9,18-23 . Arabidopsis ABA receptor subfamily III is comprised of dimeric receptors 15,25 , which, recent data suggest, are main mediators of the downstream transcription effect of ABA 26 . Activation of dimeric receptors requires higher levels of ABA in comparison to monomeric receptors, suggestive of an advanced, modular response mechanism. Evidence of subfamily III receptor transcription in Orobanchaceae hemiparasitic species and in other holoparasitic plants, suggests that the absence of transcription might be limited to Orobanche species, or perhaps only to P. aegyptiaca. This possibility could be explored pending release of the sequenced genomes of P. aegyptiaca and other Orobanche www.nature.com/scientificreports www.nature.com/scientificreports/ species, which will allow us to unequivocally determine whether subfamily III genes are present, lost or merely not transcribed. Loss of the gene expression would coincide with previous evidence of key autotrophic genes which are also not transcriptionally active in the Orobanchaceae family 27,28 . Secondly, nearly a quarter of the discovered PaPYL4 alleles are likely to encode incomplete proteins caused by small insertions or deletions. Amongst the twelve different alleles with a full coding sequence and evaluated for interaction with co-receptors in the presence of a range of ABA concentrations, only PaPYL4 exhibited reduced affinity to ABA co-receptors. The presence of numerous inactive alleles, together with the high proportion of alleles which most probably encode non-functioning proteins, is a strong indication of a relaxed selection of PaPYL4. As with PaPYL4, the PaPYL5 coding sequence obtained from the PPGP database, also seemed to be the product of low selective pressure, which enabled vast mutation of a highly conserved region, including the "latch", one of two surface loops that bind ABA and co-receptors.
Nonetheless, ABA clearly plays a major regulatory role in P. aegyptiaca seed dormancy, and likely in other parts of the life cycle, as could be deduced from ABA receptor and co-receptor transcription throughout multiple developmental stages. This, together with transcription of ABA biosynthesis components, signifies the prominence of ABA even in an obligate holoparasitic plant. However, many aspects of the role of ABA in P. aegyptiaca are yet to be understood, especially in the parasitism dynamics with the host plant. The new information gained here could provide a basis for further exploration of ABA involvement in parasitism mechanisms in plants, in general, and of Orobanche physiology, in particular. Moreover, structural data of functional P. aegyptiaca ABA receptors might serve as a scaffold to engineer selective agonists that differentially affect P. aegyptiaca without harming the host. As ABA inhibits germination and growth, such agonists can provide a new strategy for pest management. www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/

Methods
Identification of putative ABA receptor and ABA co-receptor sequences. The PYR1 and ABI1 amino acid sequences were obtained from The Arabidopsis Information Resource (Loci AT4G17870.1 and AT4G26080 respectively). These sequences were used to query (TBLASTN) the Parasitic Plant Genome Project database 16 for homologous nucleic acid sequences in P. aegyptiaca, T. versicolor and S. hermonthica, and the GenBank TSA database for C. pentagona and C. suaveolens sequences. Expressed sequence tag (EST) sequences overcoming the e-value 1.0 −10 threshold, were imported to Geneious ® 7.1.9 (https://www.geneious.com) and were assembled using the De Novo Assemble tool (default settings). Open reading frames in the assembled sequences were predicted, translated to amino acids (Standard Code/transl_table 1) and aligned (pairwise MUSCLE alignment, default settings) to PYR1 or ABI1 using Geneious ® 7.1.9. Since, in some cases, the genetic material was extracted from tissue connected to the host plant, a basic local alignment search (Standard Nucleotide BLAST) of the PYR1 and ABI1 homologous sequences was conducted using the NCBI database. Cases with high identity with the host species were excluded. In order to identify the tissues and developmental stages in which any ABA receptor or co-receptor were likely to be transcribed, the newly identified sequences were used to query (BLASTN) the PPGP database for homologous nucleic acid sequences in P. aegyptiaca. In some cases, highly similar sequences were identified via the database nucleotide sequence pairwise alignments, yet some variation (no greater than 5% of the entire sequence) was present. We attributed this to the large allelic variation we observed in our own in-vitro experiments, and decided to include these less than perfect matches in the transcription pattern in the presented results. Growth conditions of P. aegyptiaca. P. aegyptiaca seeds were mixed in soil (8 g seeds per 1 L soil) and transferred to 4 L pots, into which two-week-old tomato cultivar Solanum Lycopersicum M82 sp were planted. The inoculated plants were grown under greenhouse conditions (natural day length, 25 °C/20 °C day/night temperature). The first P. aegyptiaca flowers broke soil during the third month of the growing period. Tissue samples of the flowers and the stems were collected during the following month, and stored at −80 °C. DNA extraction and amplification. P. aegyptiaca tissue samples were ground to powder using a TissueLyser II (QIAGEN). Samples were mixed with 600 µl DNA extraction buffer and incubated at 65 °C, for 30 min. Chloroform (600 µl) was then added to the samples, which were then centrifuged at 20,000 RCF, for 2 min. The upper phase was isolated and mixed with 600 µl chloroform and centrifuged at 20,000 RCF, for 2 min. The upper phase was isolated again, mixed with isopropanol at a 2:3 ratio, and stored for a least 30 min, at −20 °C. The samples were then centrifuged at 20,000 RCF, for 30 min. The supernatant was discarded and the pellet was washed (not resuspended) with 600 µl cold (−20 °C) 70% ethanol. The samples were then centrifuged at 20,000 RCF, for 5 min, and the supernatant was discarded. In order to remove residual ethanol, the samples were incubated at 60 °C, until the pellet fully dried. The pellet was then resuspended in water.
Selected genes were amplified using Phusion ® High-Fidelity DNA Polymerase (New England BioLabs, catalog number M0530L), according to the manufacturer's instructions. All gene primers were designed using Primer3 version 2.3.4, via Geneious ® 7.1.9, and are listed in Table S1.