Marco Bürger

Rafflesiaceae, including Rafflesia, Sapria, and Rhizanthes , are some of the rarest flowering plants in the world, known for producing the largest blooms on Earth. These holoparasites depend exclusively on Tetrastigma (Vitaceae) vines, yet the chemical basis for this host specificity is poorly understood, complicating conservation efforts. Untargeted negative-ion LC- MS metabolomics was used to profile Rafflesiaceae buds and seeds, infected Tetrastigma hosts associated with Rafflesia lagascae, R. speciosa, and Sapria himalayana , uninfected Tetrastigma hosts, and corresponding non-host species from the Philippines and Thailand. Python scripts used for data processing and visualization were developed with generative AI assistance and validated by the authors. Principal component analysis revealed distinct metabolomic profiles across samples, with significant shifts in host metabolites upon infection. Infected Tetrastigma tissues showed nominal enrichment in phenylpropanoid and gibberellin-related metabolites, suggestive of defense and hormonal pathway activation during Rafflesia infection, as well as citric acid, suggesting increased energy demand to support the parasite. Non-host Tetrastigma species showed nominal enrichment in metabolites putatively annotated as stilbenoids, resveratrol diglucosides, and condensed tannins such as procyanidin B2, compounds broadly associated with plant defense. Rafflesia seeds harbored fatty acids/oxylipins, while Rafflesia buds accumulated gallic acid derivatives, compounds also reported in galls. These findings support a chemically mediated model of host compatibility and parasitism, with implications for the ex-situ conservation of these endangered plants.

Rafflesiaceae, known for producing the largest flowers in the world, are obligate parasites that exclusively infect Tetrastigma sp. (Vitaceae). Despite their unique biology, the interactions between parasitic tissues and host roots remain poorly understood, particularly during the flower morphogenesis phase, where parasitic tissue erupts through the host root. Here, we performed dual transcriptome analyses of two Rafflesiaceae species and their respective Tetrastigma hosts: Sapria himalayana with T. cauliflorum and Rafflesia speciosa with T. magnum. Our findings reveal species-specific transcriptional responses in Tetrastigma, suggesting divergent parasitism strategies between Rafflesia and Sapria. Moreover, we identify molecular signatures of parasitism that parallel plant gall formation, particularly in genes governing cell wall modification and host tissue reorganization. Unlike bacterial or insect-induced galls, these mechanisms may involve fungal symbionts, highlighting the unique nature of these interactions. Together, our results demonstrate that Rafflesiaceae parasitism represents a complex tripartite relationship among host, holoparasite, and associated microbes, offering new insights into the hidden biology of these remarkable parasitic plants.

Conopholis americana (American cancer-root) and Epifagus virginiana (beechdrops) are sister genera of holoparasitic plants (Orobanchaceae) native to eastern North America, parasitizing oaks and American beech, respectively. Both have served as models for plastid genome reduction, yet no nuclear genomes exist for either genus or any New World holoparasitic Orobanchaceae. Here we present the first nuclear genome assemblies for both species using PacBio HiFi sequencing. The C. americana assembly totals 1.82 Gb and E. virginiana totals 440 Mb, representing an approximately 4-fold difference in genome size between these sister genera. We observed a BUSCO completeness of 79% to 80% in both species, which is typical of holoparasites. While gene prediction identified 33,889 genes in C. americana and 21,031 in E. virginiana, repeat annotation revealed that LTR retrotransposons account for 78% of the genome size difference. These assemblies reveal contrasting mechanisms of genome evolution in sister holoparasitic genera and provide foundational resources for comparative genomics of parasitic plants.

Castilleja lassenensis, a species of root-parasitic plants endemic to the Mt. Lassen region in northern California, has been subject to taxonomic debate since its first description in 1940. While morphological and ecological distinctions from its close relative Castilleja lemmonii have been documented, many regional floras do not list C. lassenensis or treat it as a synonym of C. lemmonii. We sequenced the nuclear genomes of C. lassenensis and C. lemmonii and performed comparative genomic analyses including gene content, functional enrichment, and phylogenetic placement. The genome of C. lassenensis, although slightly smaller than that of C. lemmonii, shows higher gene content, with different patterns of gene duplication. Functional genomic analysis revealed distinct enrichment patterns in the two species, with metabolic and transport functions enriched in C. lassenensis versus cellular organization in C. lemmonii. The taxa also show divergence in peptidase and CAZyme distributions, as well as in KAI2d proteins involved in strigolactone perception. Population-level validation using diagnostic gene markers across multiple individuals from geographically separate populations confirmed stable genomic differences. Our results support the recognition of C. lassenensis as a distinct species and underscore the need for measures to ensure its conservation.

SUMMARY: Strigolactones (SLs) are a class of plant hormones that play a critical role in the suppression of shoot branching. Furthermore, they are exuded from roots and act as signaling molecules for inter‐organism communication in the rhizosphere. Strigolactones trigger those responses by inducing protein–protein interactions (PPIs) of signaling components and subsequent proteolysis of transcriptional repressors. The sequential event involves SL hydrolysis mediated by SL receptors belonging to an α/β‐hydrolase family, although the physiological role of SL hydrolysis is a subject of debate. To date, SL‐induced PPIs have been analyzed by methods such as yeast‐two hybrid, pull‐down, and AlphaScreen assays. However, the kinetic aspect of PPI profiles has not been well studied. Here, we developed an in vitro method to monitor the formation of the SL signaling complex based on Time‐Resolved Förster Resonance Energy Transfer (TR‐FRET) technology. Our TR‐FRET‐based assay system allows us to analyze the mode of action of SL analogs from kinetic and quantitative perspectives. Notably, our method revealed differences in the intensity and time dependency of PPI signals among different SL analogs with a range of hydrolyzabilities. In addition, we found that tolfenamic acid, an antagonist of the SL receptor, inhibited the SL‐induced PPI but could not disrupt the already‐formed signaling complex. The TR‐FRET system was also used to rapidly and specifically detect naturally occurring SLs from root exudates containing many impurities. This work provides insights into the molecular mechanism of SL perception as well as a powerful tool for activity‐based screening of SL signaling modulators.

Root parasitic plants in the Orobanchaceae family cause widespread and serious damage to crop production, especially in sub-Saharan Africa. The obligate root parasitic plants in this family, such as Striga and Orobanche, germinate in response to host root-derived small molecules called strigolactones (SLs). Facultative root parasitic plants in the same family, such as Phtheirospermum japonicum, do not require SLs for germination. However, they also sense host root-derived SLs for the chemotropic response to grow toward the host. Thus, both the obligate and facultative root parasitic plants can detect the presence of the host by sensing host-derived SLs. SLs are known to act as plant hormones that regulate shoot branching. Therefore it might be possible that root parasitic plants also can produce SLs by themselves. However, whether root parasitic plants can biosynthesize SLs has not been resolved. To address this fundamental question, we searched for SL biosynthetic genes in both obligate (Orobanche minor) and facultative (Phtheirospermum japonicum) parasitic plants and analysed the biochemical function of those gene products. Our results demonstrate that the biochemical functions of SL biosynthetic enzymes (D27, CCD7, CDD8, CYP711A, and CLAMT) in both plant species were conserved compared with the corresponding orthologs in non-parasitic plant species. Therefore, our results strongly suggest that the root parasitic plants can themselves produce SLs.

Parasitic plants in the genus Orobanche display varying levels of host specificity, however, the molecular features determining host range are not fully understood. We sequenced the genomes of three Orobanche species with different host ranges: Orobanche minor (generalist), O. gracilis (Fabaceae-specific), and O. hederae (ivy-specific). Comparative analysis revealed considerable genome size variation but consistent holoparasitic gene loss patterns. While synteny analysis revealed strong collinearity between O. gracilis, O. hederae, and O. minor, we found distinct molecular repertoires among species. O. gracilis showed expanded protein-degrading and carbohydrate-active enzyme families, while putative strigolactone receptor (KAI2d) numbers varied from five in specialists to eleven in the generalist O. minor, suggesting diverse molecular strategies across different host ranges.