Evolution of a horizontally acquired legume gene, albumin 1, in the parasitic plant Phelipanche aegyptiaca and related species

Abstract

Background: Parasitic plants, represented by several thousand species of angiosperms, use modified structures known as haustoria to tap into photosynthetic host plants and extract nutrients and water. As a result of their direct plant-plant connections with their host plant, parasitic plants have special opportunities for horizontal gene transfer, the nonsexual transmission of genetic material across species boundaries. There is increasing evidence that parasitic plants have served as recipients and donors of horizontal gene transfer (HGT), but the long-term impacts of eukaryotic HGT in parasitic plants are largely unknown.

Results: Here we show that a gene encoding albumin 1 KNOTTIN-like protein, closely related to the albumin 1 genes only known from papilionoid legumes, where they serve dual roles as food storage and insect toxin, was found in Phelipanche aegyptiaca and related parasitic species of family Orobanchaceae, and was likely acquired by a Phelipanche ancestor via HGT from a legume host based on phylogenetic analyses. The KNOTTINs are well known for their unique "disulfide through disulfide knot" structure and have been extensively studied in various contexts, including drug design. Genomic sequences from nine related parasite species were obtained, and 3D protein structure simulation tests and evolutionary constraint analyses were performed. The parasite gene we identified here retains the intron structure, six highly conserved cysteine residues necessary to form a KNOTTIN protein, and displays levels of purifying selection like those seen in legumes. The albumin 1 xenogene has evolved through >150 speciation events over ca. 16 million years, forming a small family of differentially expressed genes that may confer novel functions in the parasites. Moreover, further data show that a distantly related parasitic plant, Cuscuta, obtained two copies of albumin 1 KNOTTIN-like genes from legumes through a separate HGT event, suggesting that legume KNOTTIN structures have been repeatedly co-opted by parasitic plants.

Conclusions: The HGT-derived albumins in Phelipanche represent a novel example of how plants can acquire genes from other plants via HGT that then go on to duplicate, evolve, and retain the specialized features required to perform a unique host-derived function.

Figure 1

Alignments of 5’ ends of the genomic and inferred CDS sequences of albumin 1 homologs from five Phelipanche species (for 3’ end, see Additional file4: Figure S4). Two genes are identified from P. aegyptiaca unigene 12653 (first five sequences, red bar) and unigene 75797 (yellow bar). Red box indicates the intron region identified by comparison of the genomic DNA and cDNA sequences. Blue box indicates the putative translation start codon.

Figure 2

Maximum likelihood (ML) and Bayesian inference (BI) phylogeny of albumin 1 homologs in broomrape species and legumes. Horizontal acquisition of albumin 1 by an ancestral Phelipanche/Orobanche species was estimated to have occurred ca. 16 million years ago (Mya, with standard errors SE), with Orobanche-Phelipanche speciation ca. 11 Mya, and a gene duplication ca. 5 Mya in the Phelipanche lineage produced xenparalogous genes designated Albumin1-1 (12653) and Albumin1-2 (75797) (see Supplemental Methods). Papilionoid legumes in black, others as indicated. Age estimate of legume node marked by red circle (39 ± 2.4 Mya) taken from Lavin et al. [51]. Unrooted trees have been rooted with Glycine max, in agreement with a prior KNOTTIN phylogeny [30] and phylogenetic relationships of related legume sequences [50]. Tree shown is ML (BI method produced the same tree topology); bootstrap values (if >50%) and posterior probabilities (if >0.60) are shown on internal nodes. The legume clade containing albumin 1 genes is comprised of the Millettioids clade, which contains genera such as Glycine and Phaseolus, as the sister group to the large, temperate Hologalegina clade, which includes Medicago, Pisum, Astragalus and Onobrychis, as well as several other agriculturally important genera such as Cicer, Lens, Vicia, and Trifolium[50]. Legume KNOTTIN sequences were from the KNOTTIN database [31]. For each legume KNOTTIN, tripartite names are given as: species full name-ID from KNOTTIN database-sequence ID from UniProt database. Additional albumin 1 homologs from M. truncatula were retrieved from Medicago truncatula HapMap Project [52] with original sequence IDs. Branches are scaled by number of substitutions. The two albumin 1 genes in Phelipanche aegyptiaca have nt sequence identity 92%.

Figure 3

Amino acid sequence alignment and 3D structure simulation of albumin 1 sequences from Medicago and P. aegyptiaca. (A) Amino acid alignment for the two P. aegyptiaca albumin 1 sequences and a M. truncatula albumin 1 sequence (Q7XZC5, a confirmed KNOTTIN insect toxin protein). Red squares indicate cysteine residues. (B) and (C) show the simulated 3D structures for both Phelipanche sequences. Protein 2D structures are colored from N-terminal to C-terminal with a rainbow color scheme. The three disulfide bonds are shown as colored sticks. The left most and right most sticks open a space that is pierced by the stick in the center. This “disulfide through disulfide knot” is the characteristic structure of KNOTTIN proteins. (D) 3D structure of the KNOTTIN insect toxin protein in M. truncatula. The toxicity of this protein to insect herbivores was confirmed in an earlier report [29]. The PDB file for this 3D structure was obtained from the KNOTTIN database. (E) Predicted albumin 2 (a non-KNOTTIN albumin, PDB ID#3LP9) protein 3D structure in grass pea (Lathyrus sativus).

Figure 4

ML estimate of dN and dS changes, and evolutionary constraint (dN/dS) through the history of albumin 1 sequences in broomrapes and their homologs in three related legume species. Branch lengths scaled by total number of substitutions. Because the total amount of evolutionary change on individual branches for closely related species can be very low (or even zero in some cases), changes have been pooled within several of the specific lineages.

Figure 5

Expression level (log scale) of P. aegyptiaca albumin 1 genes in P. aegyptiaca across eight developmental stages. Normalized expression levels were estimated by RPKM (= count of mapped Reads to this gene Per Kilobase of sequence length per Million library reads). Numerical values in Additional file 6: Table S1; P. aegyptiaca stages are as defined [54] and in Additional file 7: Table S2. Stage 3 (haustorium attached to host root, pre-vascular connection) is the earliest post-attachment stage for this parasite [54].