The peptide hormone PjCLE1 stimulates haustorium formation in the parasitic plant Phtheirospermum japonicum.

Phtheirospermum japonicum is a hemiparasitic plant of the Orobanchaceae, the largest family of parasitic plants. It extracts water and nutrients from other plants through haustoria along its roots. Haustoriogenesis, the formation of haustoria, is initiated by host-derived haustorium-inducing factors (HIFs). The first step in haustoriogenesis is the development of parasitically inactive protohaustoria. Here, we report that an endogenous peptide hormone, CLAVATA3/Embryo Surrounding Region 1 (PjCLE1), is sufficient to induce protohaustorium formation. PjCLE1 hyperactivated HIF-responses and caused prolific protohaustoria formation. PjCLE1 expression and activation by the subtilisin-type protease PjSBT1.2.3 occur in fully developed, mature haustoria, suggesting that PjCLE1 acts as an internal signal produced by mature haustoria to stimulate additional protohaustorium formation for effective extraction of resources from hosts. PjCLE1 is similar in sequence to CLEs regulating nodulation in legumes and part of a regulatory system for haustoria formation in parasitic plants.

Identification challenges of Castilleja (Orobanchaceae) on iNaturalist.

Citizen science platforms like iNaturalist play a crucial role in biodiversity monitoring. However, the identification of plants from the genus Castilleja (Indian Paintbrush), which comprises about 200 species with often similar features and frequent introgression, presents considerable challenges. Our study examines the agreement between initial identifications (often made by computer vision algorithms), subsequent identifications, and the final Research-Grade identifications for Castilleja species on iNaturalist. We focus on prevalent identification problems within this genus, particularly noting that Castilleja densiflora and Castilleja exserta are most frequently confused. This study highlights the need for improved algorithms to enhance initial species identification accuracy, especially for complex genera like Castilleja. Our findings have implications for the efficiency of the identification process on citizen science platforms and underscore the importance of expert verification in challenging taxonomic groups.

Host shift promotes divergent evolution between closely related holoparasitic species.

Distinct hosts have been hypothesized to possess the potential for affecting species differentiation and genome evolution of parasitic organisms. However, what host shift history is experienced by the closely related parasites and whether disparate evolution of their genomes occur remain largely unknown. Here, we screened horizontal gene transfer (HGT) events in a pair of sister species of holoparasitic Boschniakia (Orobanchaceae) having obligate hosts from distinct families to recall the former host-parasite associations and performed a comparative analysis to investigate the difference of their organelle genomes. Except those from the current hosts (Ericaceae and Betulaceae), we identified a number of HGTs from Rosaceae supporting the occurrence of unexpected ancient host shifts. Different hosts transfer functional genes which changed nuclear genomes of this sister species. Likewise, different donors transferred sequences to their mitogenomes, which vary in size due to foreign and repetitive elements rather than other factors found in other parasites. The plastomes are both severely reduced, and the degree of difference in reduction syndrome reaches the intergeneric level. Our findings provide new insights into the genome evolution of parasites adapting to different hosts and extend the mechanism of host shift promoting species differentiation to parasitic plant lineages.

Interspecies and temporal dynamics of bacterial and fungal microbiomes of pistil stigmas in flowers in holoparasitic plants of the Orobanche series Alsaticae (Orobanchaceae).

Little is known about the microbiomes of flower parts, and even less information is available regarding these microorganisms' colonization of specific niches in parasitic plants. We investigate the temporal interspecies dynamics of the parasitic plants microbiome of flower stigmas in two stages of development: immature stigmas in flower buds and mature stigmas in opened flowers. We compared two related holoparasitic Orobanche species from localities approximately 90 km apart and characterize their bacterial and fungal communities using 16S rRNA gene and ITS sequences, respectively. We identified from 127 to over 228 OTUs per sample for fungi, sequences belonging to genera: Aureobasidium, Cladosporium, Malassezia, Mycosphaerella, and Pleosporales, constituting approximately 53% of the community in total. In the bacterial profile, we recorded 40 to over 68 OTUs per sample consisting of Enterobacteriaceae, and genera Cellulosimicrobium, Pantoea, and Pseudomonas spp., with an approximately 75% frequency. In microbial communities, higher numbers of OTUs colonizing mature stigmas were recorded than in immature. This implies that the dynamics and concurrence of microbial communities were different between O. alsatica and O. bartlingii and underwent significant changes during flower development. To the best of our knowledge, is the first study of the interspecies and temporal dynamics of the bacterial and fungal microbiomes of pistil stigmas in flowers.

Comparing complete organelle genomes of holoparasitic Christisonia kwangtungensis (Orabanchaceae) with its close relatives: how different are they?

BACKGROUND: Orobanchaceae is the only flowering plant family with species from free-living nonparasite, hemi-parasite to holoparasite, making it an ideal system for studying the evolution of parasitism. However, both plastid and mitochondrial genome have been sequenced in only few parasitic species in Orobanchaceae. Therefore, further comparative study is wanted to investigate the impact of holoparasitism on organelle genomes evolution between close relatives. Here, we sequenced organelle genomes and transcriptome of holoparasitic Christisonia kwangtungensis and compared it with its closely related groups to analyze similarities and differences in adaption strategies to the holoparasitic lifestyle. RESULTS: The plastid genome of C. kwangtungensis has undergone extensive pseudogenization and gene loss, but its reduction pattern is different from that of Aeginetia indica, the close relative of C. kwangtungensis. Similarly, the gene expression detected in the photosynthetic pathway of these two genera is different. In Orobanchaceae, holoparasites in Buchnereae have more plastid gene loss than Rhinantheae, which reflects their longer history of holoparasitism. Distinct from severe degradation of the plastome, protein-coding genes in the mitochondrial genome of C. kwangtungensis are relatively conserved. Interestingly, besides intracellularly transferred genes which are still retained in its plastid genome, we also found several horizontally transferred genes of plastid origin from diverse donors other than their current hosts in the mitochondrial genome, which probably indicate historical hosts. CONCLUSION: Even though C. kwangtungensis and A. indica are closely related and share severe degradation of plastome, they adapt organelle genomes to the parasitic lifestyle in different ways. The difference between their gene loss and gene expression shows they ultimately lost photosynthetic genes but through different pathways. Our study exemplifies how parasites part company after achieving holoparasitism.

Structural mutations of small single copy (SSC) region in the plastid genomes of five Cistanche species and inter-species identification.

BACKGROUND: Cistanche is an important genus of Orobanchaceae, with critical medicinal, economic, and desertification control values. However, the phylogenetic relationships of Cistanche genus remained obscure. To date, no effective molecular markers have been reported to discriminate effectively the Cistanche closely related species reported here. In this study, we obtained and characterized the plastomes of four Cistanche species from China, to clarify the phylogenetic relationship within the genus, and to develop molecular markers for species discrimination.  RESULTS: Four Cistanche species (Cistanche deserticola, Cistanche salsa, Cistanche tubulosa and Cistanche sinensis), were deep-sequenced with Illumina. Their plastomes were assembled using SPAdes and annotated using CPGAVAS2. The plastic genomes were analyzed in detail, finding that all showed the conserved quadripartite structure (LSC-IR-SSC-IR) and with full sizes ranging from 75 to 111 Kbp. We observed a significant contraction of small single copy region (SSC, ranging from 0.4-29 Kbp) and expansion of inverted repeat region (IR, ranging from 6-30 Kbp), with C. deserticola and C. salsa showing the smallest SSCs with only one gene (rpl32). Compared with other Orobanchaceae species, Cistanche species showed extremely high rates of gene loss and pseudogenization, as reported for other parasitic Orobanchaceae species. Furthermore, analysis of sequence divergence on protein-coding genes showed the three genes (rpl22, clpP and ycf2) had undergone positive selection in the Cistanche species under study. In addition, by comparison of all available Cistanche plastomes we found 25 highly divergent intergenic spacer (IGS) regions that were used to predict two DNA barcode markers (Cis-mk01 and Cis-mk02 based on IGS region trnR-ACG-trnN-GUU) and eleven specific DNA barcode markers using Ecoprimer software. Experimental validation showed 100% species discrimination success rate with both type of markers. CONCLUSION: Our findings have shown that Cistanche species are an ideal model to investigate the structure variation, gene loss and pseudogenization during the process of plastome evolution in parasitic species, providing new insights into the evolutionary relationships among the Cistanche species. In addition, the developed DNA barcodes markers allow the proper species identification, ensuring the effective and safe use of Cistanche species as medicinal products.

Analysis of the genetic diversity and population structure of Monochasma savatieri Franch. ex Maxim using novel EST-SSR markers.

BACKGROUND: Monochasma savatieri Franch. ex Maxim is a medicinally valuable herb. However, the collection and protection of the wild germplasm resources of M. savatieri are still insufficient, and their genetic diversity and population structure have been poorly studied. RESULTS: We collected and examined 46 M. savatieri individuals from Fujian, Hunan, Jiangxi, and Zhejiang provinces for genetic diversity and population structure, using 33 newly developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. Applying these markers, we detected a total of 208 alleles, with an average of 6.303 alleles per locus. The polymorphic information content varied from 0.138 to 0.884 (average: 0.668), indicating a high level of polymorphism. At the population level, there was a low degree of genetic diversity among populations (I = 0.535, He = 0.342), with Zhejiang individuals showing the highest genetic diversity among the four populations (Fst = 0.497), which indicated little gene flow within the M. savatieri populations (Nm = 0.253). Mantel test analysis revealed a significant positive correlation between geographical and genetic distance among populations (R2 = 0.3304, p < 0.05), and structure and principal coordinate analyses supported classification of populations into three clusters, which was consistent with the findings of cluster analysis. CONCLUSIONS: As a rare medicinal plants, the protection of M. savatieri does not look optimistic, and accordingly, protective efforts should be beefed up on the natural wild populations. This study provided novel tools and insights for designing effective collection and conservation strategies for M. savatieri.

Comparative genomics of orobanchaceous species with different parasitic lifestyles reveals the origin and stepwise evolution of plant parasitism.

Orobanchaceae is the largest family of parasitic plants, containing autotrophic and parasitic plants with all degrees of parasitism. This makes it by far the best family for studying the origin and evolution of plant parasitism. Here we provide three high-quality genomes of orobanchaceous plants, the autotrophic Lindenbergia luchunensis and the holoparasitic plants Phelipanche aegyptiaca and Orobanche cumana. Phylogenomic analysis of these three genomes together with those previously published and the transcriptomes of other orobanchaceous species created a robust phylogenetic framework for Orobanchaceae. We found that an ancient whole-genome duplication (WGD; about 73.48 million years ago), which occurred earlier than the origin of Orobanchaceae, might have contributed to the emergence of parasitism. However, no WGD events occurred in any lineage of orobanchaceous parasites except for Striga after divergence from their autotrophic common ancestor, suggesting that, in contrast with previous speculations, WGD is not associated with the emergence of holoparasitism. We detected evident convergent gene loss in all parasites within Orobanchaceae and between Orobanchaceae and dodder Cuscuta australis. The gene families in the orobanchaceous parasites showed a clear pattern of recent gains and expansions. The expanded gene families are enriched in functions related to the development of the haustorium, suggesting that recent gene family expansions may have facilitated the adaptation of orobanchaceous parasites to different hosts. This study illustrates a stepwise pattern in the evolution of parasitism in the orobanchaceous parasites and will facilitate future studies on parasitism and the control of parasitic plants in agriculture.

Nitrogen represses haustoria formation through abscisic acid in the parasitic plant Phtheirospermum japonicum.

Parasitic plants are globally prevalent pathogens that withdraw nutrients from their host plants using an organ known as the haustorium. The external environment including nutrient availability affects the extent of parasitism and to understand this phenomenon, we investigated the role of nutrients and found that nitrogen is sufficient to repress haustoria formation in the root parasite Phtheirospermum japonicum. Nitrogen increases levels of abscisic acid (ABA) in P. japonicum and prevents the activation of hundreds of genes including cell cycle and xylem development genes. Blocking ABA signaling overcomes nitrogen's inhibitory effects indicating that nitrogen represses haustoria formation by increasing ABA. The effect of nitrogen appears more widespread since nitrogen also inhibits haustoria in the obligate root parasite Striga hermonthica. Together, our data show that nitrogen acts as a haustoria repressing factor and suggests a mechanism whereby parasitic plants use nitrogen availability in the external environment to regulate the extent of parasitism.

A Phelipanche ramosa KAI2 protein perceives strigolactones and isothiocyanates enzymatically.

Phelipanche ramosa is an obligate root-parasitic weed that threatens major crops in central Europe. In order to germinate, it must perceive various structurally divergent host-exuded signals, including isothiocyanates (ITCs) and strigolactones (SLs). However, the receptors involved are still uncharacterized. Here, we identify five putative SL receptors in P. ramosa and show that PrKAI2d3 is involved in the stimulation of seed germination. We demonstrate the high plasticity of PrKAI2d3, which allows it to interact with different chemicals, including ITCs. The SL perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic plants. We provide evidence that PrKAI2d3 enzymatic activity confers hypersensitivity to SLs. Additionally, we demonstrate that methylbutenolide-OH binds PrKAI2d3 and stimulates P. ramosa germination with bioactivity comparable to that of ITCs. This study demonstrates that P. ramosa has extended its signal perception system during evolution, a fact that should be considered for the development of specific and efficient biocontrol methods.

How do holoparasitic plants exploit vitamin K1?

Phylloquinone (vitamin K1) is a thylakoid-embedded electron carrier essential for photosynthesis. Paradoxically, we found that phylloquinone biosynthesis is retained in the nonphotosynthetic holoparasite Phelipanche aegyptiaca (Egyptian broomrape). The phylloquinone pathway genes are preferentially expressed during development of the invasive organ, the haustorium, and exhibit strong coexpression with redox-active proteins known to be involved in parasitism. Unlike in photoautotrophic taxa, the late pathway genes of the holoparasite lack the chloroplast-targeting sequence and their proteins are targeted to the plasma membrane instead. Plasma membrane phylloquinone may enable Phelipanche to sense changes in the redox environment during host interactions. The N-truncated isoforms are conserved in several other Orobanchaceae root holoparasites, and interestingly, in a number of closely related photoautotrophic species as well. This suggests an ancient origin of distinct phylloquinone pathways predating the evolution of parasitic plants in the Orobanchaceae. These findings represent exciting opportunities to probe plasma membrane phylloquinone function and diversification in parasitic and nonparasitic plant responses to external redox chemistry in the rhizosphere.

Complete Plastid and Mitochondrial Genomes of Aeginetia indica Reveal Intracellular Gene Transfer (IGT), Horizontal Gene Transfer (HGT), and Cytoplasmic Male Sterility (CMS).

Orobanchaceae have become a model group for studies on the evolution of parasitic flowering plants, and Aeginetia indica, a holoparasitic plant, is a member of this family. In this study, we assembled the complete chloroplast and mitochondrial genomes of A. indica. The chloroplast and mitochondrial genomes were 56,381 bp and 401,628 bp long, respectively. The chloroplast genome of A. indica shows massive plastid genes and the loss of one IR (inverted repeat). A comparison of the A. indica chloroplast genome sequence with that of a previous study demonstrated that the two chloroplast genomes encode a similar number of proteins (except atpH) but differ greatly in length. The A. indica mitochondrial genome has 53 genes, including 35 protein-coding genes (34 native mitochondrial genes and one chloroplast gene), 15 tRNA (11 native mitochondrial genes and four chloroplast genes) genes, and three rRNA genes. Evidence for intracellular gene transfer (IGT) and horizontal gene transfer (HGT) was obtained for plastid and mitochondrial genomes. ψndhB and ψcemA in the A. indica mitogenome were transferred from the plastid genome of A. indica. The atpH gene in the plastid of A. indica was transferred from another plastid angiosperm plastid and the atpI gene in mitogenome A. indica was transferred from a host plant like Miscanthus siensis. Cox2 (orf43) encodes proteins containing a membrane domain, making ORF (Open Reading Frame) the most likely candidate gene for CMS development in A. indica.

Subtilase activity in intrusive cells mediates haustorium maturation in parasitic plants.

Parasitic plants that infect crops are devastating to agriculture throughout the world. These parasites develop a unique inducible organ called the haustorium that connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically toward the host vasculature. Following this, some of the intrusive cells re-differentiate to form a xylem bridge (XB) that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells remain poorly understood. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the identified intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of SBT inhibitors in intrusive cells inhibited both intrusive cell and XB development and reduced auxin response levels adjacent to the area of XB development. Therefore, we propose that subtilase activity plays an important role in haustorium development in P. japonicum.

WARPP-web application for the research of parasitic plants.

The lifestyle of parasitic plants is associated with peculiar morphological, genetic, and physiological adaptations that existing online plant-specific resources fail to adequately represent. Here, we introduce the Web Application for the Research of Parasitic Plants (WARPP) as an online resource dedicated to advancing research and development of parasitic plant biology. WARPP is a framework to facilitate international efforts by providing a central hub of curated evolutionary, ecological, and genetic data. The first version of WARPP provides a community hub for researchers to test this web application, for which curated data revolving around the economically important Broomrape family (Orobanchaceae) is readily accessible. The initial set of WARPP online tools includes a genome browser that centralizes genomic information for sequenced parasitic plant genomes, an orthogroup summary detailing the presence and absence of orthologous genes in parasites compared with nonparasitic plants, and an ancestral trait explorer showing the evolution of life-history preferences along phylogenies. WARPP represents a project under active development and relies on the scientific community to populate the web app's database and further the development of new analysis tools. The first version of WARPP can be securely accessed at https://parasiticplants.app. The source code is licensed under GNU GPLv2 and is available at https://github.com/wickeLab/WARPP.

Integrated analyses of the transcriptome and small RNA of the hemiparasitic plant Monochasma savatieri before and after establishment of parasite-host association.

BACKGROUND: Monochasma savatieri is a medicinal root hemiparasitic herb that extracts water and nutrients from the host plant via a haustorium. M. savatieri exhibits an enhanced growth after the establishment of parasite-host associations, but little is known about the molecular mechanism responsible. In this study, endogenous hormones, RNA sequencing and small RNA sequencing analysis were performed on M. savatieri before and after establishment of parasite-host associations. RESULTS: When grown with the host, decreased contents of jasmonic acid (JA) and indole-3-acetic acid (IAA) and increased abscisic acid (ABA) content were observed in M. savatieri with the established parasitic relationship. When grown with the host, 46,424 differentially expressed genes (DEGs) and 162 differentially expressed miRNAs (DEmiRs) were identified in the comparison between M. savatieri with the established parasitic relationship and without the established parasitic relationship. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that these DEGs and targets of DEmiRs mostly participated in plant hormone signal transduction, starch and sucrose metabolism, carbohydrate metabolism, cell growth and death, and transport and catabolism. Furthermore, correlation analysis of mRNA and miRNA revealed that 10 miRNA-target pairs from novel_mir65, novel_mir40, novel_mir80, miR397-5p_1, novel_mir36, novel_mir25 and novel_mir17 may have important roles in regulating the parasitic development of M. savatieri. CONCLUSIONS: Our study not only expands the understanding of enhanced growth in M. savatieri after the establishment of parasite-host associations, but also first provides abundant resources for future molecular and genetic studies in M. savatieri.

Orobanchaceae parasite-host interactions.

Parasitic plants in the family Orobanchaceae, such as Striga, Orobanche and Phelipanche, often cause significant damage to agricultural crops. The Orobanchaceae family comprises more than 2000 species in about 100 genera, providing an excellent system for studying the molecular basis of parasitism and its evolution. Notably, the establishment of model Orobanchaceae parasites, such as Triphysaria versicolor and Phtheirospermum japonicum, that can infect the model host Arabidopsis, has greatly facilitated transgenic analyses of genes important for parasitism. In addition, recent genomic and transcriptomic analyses of several Orobanchaceae parasites have revealed fascinating molecular insights into the evolution of parasitism and strategies for adaptation in this family. This review highlights recent progress in understanding how Orobanchaceae parasites attack their hosts and how the hosts mount a defense against the threats.

Overexpression of RgPAL family genes involved in phenolic biosynthesis promotes the replanting disease development in Rehmannia glutinosa.

Rehmannia glutinosa production is affected by the replanting disease, which involves autotoxic harm mediated by specific endogenous allelochemicals in root exudates. Many phenolics that act as allelochemical agents are mostly phenylpropanoid products of secondary metabolism in plants. Phenylalanine ammonia-lyase (PAL) is the first enzyme that catalyses the deamination of l-phenylalanine for entrance into the phenylpropanoid pathway. PAL family genes have been isolated and functionally characterized in many plant species. However, PAL family genes involved in phenolic biosynthesis remain largely uncharacterized in R. glutinosa. Here, we identified and characterized four PAL family genes (RgPAL2 to RgPAL5) in the species whose sequences exhibited highly conserved domains of PALs according to in silico analysis, implying their potential function in phenolic biosynthesis. Overexpression of RgPALs in R. glutinosa enhanced phenolic production, verifying that RgPAL family genes participate in phenolic biosynthesis pathways. Moreover, we found that the release of several allelopathic phenolics from the roots of RgPAL-overexpressing transgenic R. glutinosa increased, implying that the RgPALs positively promote their release. Importantly, under continuous monoculture stress, we found that the RgPAL transgenic plants exhibited more significant autotoxic harm than did non-transgenic (WT) plants by activating the phenolics/phenylpropanoid pathway, indicating that RgPAL family genes function as positive regulators of the replanting disease development in R. glutinosa. This study revealed that RgPAL family genes are involved in the biosynthesis and release of several phenolics and positively control the replanting disease development in R. glutinosa, laying a foundation for further clarification of the molecular mechanisms underlying the disease formation.

Host-parasite tissue adhesion by a secreted type of ß-1,4-glucanase in the parasitic plant Phtheirospermum japonicum.

Tissue adhesion between plant species occurs both naturally and artificially. Parasitic plants establish intimate relationship with host plants by adhering tissues at roots or stems. Plant grafting, on the other hand, is a widely used technique in agriculture to adhere tissues of two stems. Here we found that the model Orobanchaceae parasitic plant Phtheirospermum japonicum can be grafted on to interfamily species. To understand molecular basis of tissue adhesion between distant plant species, we conducted comparative transcriptome analyses on both infection and grafting by P. japonicum on Arabidopsis. Despite different organs, we identified the shared gene expression profile, where cell proliferation- and cell wall modification-related genes are up-regulated. Among genes commonly induced in tissue adhesion between distant species, we showed a gene encoding a secreted type of ß-1,4-glucanase plays an important role for plant parasitism. Our data provide insights into the molecular commonality between parasitism and grafting in plants.

The loss of photosynthesis pathway and genomic locations of the lost plastid genes in a holoparasitic plant Aeginetia indica.

BACKGROUND: With three origins of holoparasitism, Orobanchaceae provides an ideal system to study the evolution of holoparasitic lifestyle in plants. The evolution of holoparasitism can be revealed by plastid genome degradation and coordinated changes in the nuclear genome, since holoparasitic plants lost the capability of photosynthesis. Among the three clades with holoparasitic plants in Orobanchaceae, only Clade VI has no available plastid genome sequences for holoparasitic plants. In this study, we sequenced the plastome and transcriptome of Aeginetia indica, a holoparasitic plant in Clade VI of Orobanchaceae, to study its plastome evolution and the corresponding changes in the nuclear genome as a response of the loss of photosynthetic function. RESULTS: The plastome of A. indica is reduced to 86,212 bp in size, and almost all photosynthesis-related genes were lost. Massive fragments of the lost plastid genes were transferred into the mitochondrial and/or nuclear genomes. These fragments could not be detected in its transcriptomes, suggesting that they were non-functional. Most protein coding genes in the plastome showed the signal of relaxation of purifying selection. Plastome and transcriptome analyses indicated that the photosynthesis pathway is completely lost, and that the porphyrin and chlorophyll metabolism pathway is partially retained, although chlorophyll synthesis is not possible. CONCLUSIONS: Our study suggests the loss of photosynthesis-related functions in A. indica in both the nuclear and plastid genomes. The lost plastid genes are transferred into its nuclear and/or mitochondrial genomes, and exist in very small fragments with no expression and are thus non-functional. The Aeginetia indica plastome also provides a resource for comparative studies on the repeated evolution of holoparasitism in Orobanchaceae.